RU2795161C1 - L-amino acid producing microorganism and method for producing l-amino acid with its use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: microorganism that produces L-amino acid and its precursor are proposed, the microorganism is modified by incorporating a protein containing the amino acid sequence of SEQ ID NO: 1. Also a method for producing L-amino acid or L-amino acid precursor using the said microorganism, a composition for producing L-amino acid or L-amino acid precursor, using the said microorganism for production of L-amino acid or L-amino acid precursor are proposed.

EFFECT: invention provides production of L-amino acid or L-amino acid precursor in high yields.

17 cl, 29 tbl, 9 ex

Description

FIELD OF TECHNOLOGY

The description of the present invention relates to a microorganism producing L-amino acid or its precursor, and to a method for producing L-amino acid or its precursor using this microorganism.

PRIOR ART

L-amino acids, the main constituents of a protein unit, are used as main raw materials in medicines, food additives, animal feed, nutritional supplements, pesticides, sterilizers, and the like. Extensive research has been carried out to develop microorganisms and fermentation methods to obtain L-amino acids and other beneficial substances in high yields. For example, target-specific approaches such as a method for increasing the expression of a gene encoding an enzyme involved in L-lysine biosynthesis and a method for removing a gene not necessary for biosynthesis have been mainly used (Korean Patent No. 10-0838038).

At the same time, strains of the genus Corynebacterium, in particular Corynebacterium glutamicum, are gram-positive microorganisms widely used to obtain L-amino acids and other useful substances. Intensive research has been carried out to develop microorganisms and fermentation methods to obtain amino acids in high yields. For example, target-specific approaches such as a method for increasing the expression of a gene encoding an enzyme involved in amino acid biosynthesis or a method for removing a gene not essential for biosynthesis in strains of the genus Corynebacterium have been widely used (Korean Patent Publication Nos. 10-0924065 and 10-1208480). In addition to these methods, a method for deleting a gene not involved in amino acid production and a method for deleting a gene whose specific functions in terms of amino acid production are unknown have also been used. However, there is still a need to research methods for the efficient production of L-amino acids in high yields.

DESCRIPTION OF IMPLEMENTATIONS OF THE INVENTION TECHNICAL PROBLEM

The present inventors have made strenuous efforts to develop a microorganism capable of producing L-amino acids in high yields, and have found that the productivity of L-amino acids can be improved by incorporating a protein originating from another microorganism into it, and thus, it has been accomplished the present invention.

THE SOLUTION OF THE PROBLEM

The object of the present invention is to provide a microorganism producing an L-amino acid or its precursor modified to express a protein containing the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof.

Another object of the present invention is to provide a composition for producing an L-amino acid or its precursor, containing a microorganism modified to express a protein containing the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof, or said protein.

Another object of the present invention is to provide a method for producing an L-amino acid or a precursor thereof, comprising: culturing said microorganism in a culture medium; and isolating the L-amino acid or its precursor from the cultured microorganism or from the culture medium.

Another object of the present invention is to provide the use of a protein containing the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof, to increase the production of an L-amino acid or its precursor.

PREFERRED EFFECTS OF THE DESCRIPTION OF THE INVENTION

The microorganism according to the present invention producing L-amino acid or its precursor, where the microorganism is modified to express a protein containing the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof, can produce L-serine, L-tryptophan, L-histidine, L-methionine, L-cysteine and/or O-phosphoserine.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail.

However, each description and embodiment of the invention presented in the description of the present invention can be applied in this document to other descriptions and embodiments. In other words, all combinations of various components disclosed in the description of the present invention are included in the scope of the description of the present invention. In addition, the scope of the description of the present invention should not be limited to the following detailed descriptions.

Additionally, those skilled in the art, using routine experimentation, can recognize or confirm many equivalents to specific aspects of the present invention. Such equivalents are intended to be included within the scope of the present invention.

To achieve the above objects, an aspect of the present invention provides an L-amino acid-producing microorganism or a precursor thereof, wherein said microorganism is modified to express a protein comprising the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof.

A protein containing the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof, may be a protein having D-3-phosphoglycerate dehydrogenase activity.

In the description of the present invention, "D-3-phosphoglycerate dehydrogenase" is an enzyme that predominantly catalyzes the chemical reactions described below.

3-phospho-D-glycerate + NAD ⁺ (nicotinamide adenine dinucleotide) ↔ 3-phosphonooxypyruvate + NADH + H ⁺

2-hydroxyglutarate + NAD ⁺ ↔2-oxoglutarate + NADH + H ⁺

For the purpose of describing the present invention, D-3-phosphoglycerate dehydrogenase may be SerA and its sequence may be identified in the well-known Genbank NCBI (National Institute for Biotechnology Information) database. Additionally, you can also, without limitation, use any other protein having an equivalent activity and isolated from microorganisms that are different from the microorganism described above, producing L-amino acid or its precursor and including this protein.

Specifically, the protein may be a protein comprising the amino acid sequence of SEQ ID NO: 1, and may, without limitation, be used interchangeably with a protein comprising the amino acid sequence of SEQ ID NO: 1, a protein consisting of the amino acid sequence of SEQ ID NO: 1 , or a protein having the amino acid sequence of SEQ ID NO: 1.

The protein may have the amino acid sequence of SEQ ID NO: 1 and/or at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with the amino acid sequence of SEQ ID NO: 1. It will further be apparent that any accessory protein having an amino acid sequence comprising a deletion, modification, substitution, or addition of one or more amino acids is within the scope of the present invention so long as that amino acid sequence retains the homology or identity described above and effect, equivalent to the effect of said protein.

In addition, any polypeptide having D-3-phosphoglycerate dehydrogenase activity and encoded by a polynucleotide that hybridizes under stringent conditions to a probe designed using known genetic sequences, such as a nucleotide sequence fully or partially complementary to the nucleotide sequence, may also be used without limitation. encoding a polypeptide.

Additionally, for the purposes of describing the present invention, the protein may be derived from other microorganisms other than the microorganism described above, producing L-amino acid or its precursor and including the protein, and this particular protein may, without limitation, be a protein derived from the genus Azotobacter, a protein identical to a protein derived from the genus Azotobacter, or any protein capable of increasing the production of L-amino acid or its precursor. More specifically, the microorganism of the genus Azotobacter may be Azotobacter agilis, Azotobacter armeniacus, Azotobacter beijerinckii, Azotobacter chroococcum, Azotobacter sp.DCU26, Azotobacter sp.FA8, Azotobacter nigricans, Azotobacter paspali, Azotobacter salinestris, Azotobacter tropicalis, or Azotobacter vinelandii, and in the embodiment of the present invention may be a protein derived from Azotobacter vinelandii, but the microorganism is not limited to this.

As used herein, the term "functional fragment" refers to an amino acid sequence having an effect equivalent to that of the specified protein, and it is clear that any protein having an amino acid sequence that includes the deletion, modification, substitution or addition of one or more amino acids, and maintaining the effect, equivalent to the effect of said protein is within the scope of the present invention and may be considered as a functional fragment for purposes of describing the present invention.

Although the expression "a protein or polypeptide comprising the amino acid sequence of a particular SEQ ID NO:", "a protein or polypeptide consisting of the amino acid sequence of a particular SEQ ID NO:", or "a protein or polypeptide having the amino acid sequence of a particular SEQ ID NO:" is used herein. ”, it is obvious that any protein having an amino acid sequence including a deletion, modification, substitution, conservative substitution or addition of one or more amino acids can also be used in the description of the present invention, if this protein has an activity identical or equivalent to a polypeptide consisting of an amino acid sequence specific SEQ ID NO:. For example, a protein may have a sequence addition at the N-terminus and/or C-terminus of an amino acid sequence that causes no change in protein function, natural mutation, silent mutation, or conservative substitution.

The term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and/or chemical properties. Such an amino acid substitution can generally occur based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residue. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

Another aspect of the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 1.

As used herein, the term "polynucleotide" has a broad meaning to include DNA and RNA molecules, and a nucleotide that is the main structural unit in a polynucleotide may include not only a natural nucleotide, but also an analog in which the sugar or nitrogenous base is modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

A polynucleotide encoding a protein containing the amino acid sequence of SEQ ID NO: 1 may have, without limitation, any sequence capable of encoding a protein having D-3-phosphoglycerate dehydrogenase activity derived from Azotobacter vinelandii. Alternatively, the polynucleotide may have, without limitation, any sequence encoding a protein having the activity of increasing the production of L-amino acid or its precursor, containing the amino acid sequence of SEQ ID NO: 1.

The polynucleotide may, for example, be a polynucleotide encoding a polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% homology with the amino acid sequence of SEQ ID NO: 1. Specifically, for example, a polynucleotide encoding a protein containing the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence, having at least 70% homology or identity with the amino acid sequence of SEQ ID NO: 1, may be a polynucleotide sequence of SEQ ID NO: 95 or a polynucleotide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or sequence identity with the nucleotide sequence of SEQ ID NO: 95 .

In addition, it is clear that the polynucleotide can also be a polynucleotide that can be translated into a protein containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70% homology or identity with the amino acid sequence of SEQ ID NO: 1, or into a protein that has homology or identity with it due to codon degeneracy. Alternatively, a polynucleotide may have, without limitation, a nucleotide sequence that can hybridize to a probe designed using known genetic sequences, such as a nucleotide sequence fully or partially complementary to that nucleotide sequence, under stringent conditions, to encode a protein containing an amino acid sequence having 70% identity with SEQ ID NO: 1. The term "stringent conditions" refers to conditions that allow for specific hybridization between polynucleotides. Such conditions are described in detail in known documents (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). For example, stringent conditions may include conditions in which genes having high homology or identity, such as at least 70%, 80%, specifically 85%, specifically 90%, more specifically 95%, more specifically 97%, or even more specifically 99 % homology or identity hybridize with each other, while genes having lower homology or identity than described above do not hybridize with each other; or conditions in which washing is carried out once, specifically two or three times, under normal washing conditions for Southern hybridization at a salt concentration and temperature corresponding to 60°C, 1×SSC (buffer saline-sodium citrate), 0, 1% SDS (sodium dodecyl sulfate), specifically 60°C, 0.1×SSC, 0.1% SDS, and more specifically 68°C, 0.1×SSC, 0.1% SDS. Hybridization requires two polynucleotides to have complementary sequences, although due to stringent hybridization conditions, the nitrogenous bases may not match. The term "complementary" is used to describe the ratio between the nitrogenous bases of nucleotides that can hybridize with each other. For example, in relation to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Thus, the present invention may include not only substantially similar polynucleotide sequences, but also their isolated polynucleotide fragment, complementary to the full-length sequence.

Specifically, a polynucleotide having homology or identity can be detected using the hybridization conditions described above, including a hybridization process with a Tm value (melting point) of 55°C. Additionally, the value of Tm may be 60°C, 63°C or 65°C, but is not limited to them, and can be appropriately adjusted by experts in the art in accordance with the task.

"Homology" and "identity" refer to the degree of correspondence between two amino acid sequences or nucleotide sequences and may be expressed as a percentage.

The terms "homology" and "identity" can often be used interchangeably.

Homology or sequence identity of conserved polynucleotides or polypeptides can be determined using a standard alignment algorithm, and program default gap penalties can be used. Substantially homologous or identical sequences can typically hybridize to each other over the entire length of the sequence, or at least 50%, 60%, 70%, 80%, or 90% of the full length sequence under conditions of medium or high stringency. In hybridizable polynucleotides, polynucleotides including a degenerate codon instead of a codon may also be considered.

Homology or identity between polypeptides or polynucleotide sequences can be determined using any algorithm known in the art, such as BLAST (see: Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90, 5873, (1993)) or FASTA introduced by Pearson (see: Methods Enzymol., 183, 63, 1990). Based on the BLAST algorithm, programs known as BLASTN or BLASTX have been developed (see: http://www.ncbi.nlm.nih.gov). In addition, the presence of homology, similarity or identity between amino acid or polynucleotide sequences can be confirmed by comparing these sequences through Southern hybridization experiments under certain stringent conditions, and these certain stringent hybridization conditions are within the scope of the technology of the subject invention and can be determined by the method, known to one of ordinary skill in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

The term used in this document

"expressed/expressed" in relation to a protein means a state in which the target protein is introduced into a microorganism, or in the case of the presence of this protein in the microorganism, the activity of this protein is enhanced compared to its endogenous activity or activity before modification.

Specifically, the term "introduction of a protein" refers to providing the activity of a particular protein in a microorganism that does not originally have that protein, or to enhancing the activity of a protein compared to its endogenous activity or activity before modification. For example, the introduction of a protein may refer to the introduction of a polynucleotide encoding a particular protein into a chromosome, or the introduction into a microorganism of a fragment or vector comprising a polynucleotide encoding a particular protein, thereby capable of expressing the activity of that protein. "Endogenous activity" refers to the activity of the protein, which initially has the parental strain of the microorganism before transformation, if the microorganism is transformed through genetic modification caused by a natural or artificial factor.

As used herein, the term "amino acid or precursor thereof" refers to an amino acid or precursor thereof that can be produced using said protein and may include serine, tryptophan, histidine, methionine, cysteine, L-cystathionine, L-homocysteine, O- acetylhomoserine, O-succinylhomoserine, L-homoserine and/or O-phosphoserine, without limitation. In the description of the present invention, an amino acid may be an L-amino acid, specifically L-serine, L-tryptophan, L-histidine, L-methionine or L-cysteine, but may include all L-amino acids produced by microorganisms from various carbon sources through metabolic processes . The precursor may be O-acetylhomoserine or O-succinylhomoserine, which is a precursor converted to methionine by O-acetylhomoserine sulfhydrylase (KR10-1048593); L-homoserine, L-homocysteine or L-cystathionine, which is a precursor of methionine; and acetylserine, which is a precursor of L-cysteine; and/or O-phosphoserine, which is a precursor converted to cysteine by O-phosphoserine sulfhydrylase, without limitation. More specifically, the amino acid or its precursor may be, but are not limited to, L-serine, L-tryptophan, L-histidine, L-methionine, O-phosphoserine, or L-cysteine.

To enhance the biosynthesis of L-amino acids or their precursors, you can use a protein containing the amino acid sequence of SEQ ID NO: 1 or its functional fragment in accordance with the description of the present invention. For example, to enhance the biosynthesis of L-serine, L-tryptophan, L-histidine, L-methionine, L-cysteine, L-homocysteine, L-cystathionine, acetylserine, O-acetylhomocysteine, O-succinylhomoserine, L-homoserine and / or O -fospherin microorganism can be modified to express a protein containing the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof, in accordance with the description of the present invention. As a specific example, you can enter a protein containing the amino acid sequence of SEQ ID NO: 1, or you can enhance the activity of the specified protein. Additionally, the ability to produce an L-amino acid or its precursor can be further enhanced by additional introduction or enhancement of the activity of a particular protein, or inactivation of the activity of a particular protein.

Specifically, the microorganism can produce L-amino acids or their precursors by further incorporating (1) phosphoserine phosphatase having attenuated activity, (2) 3-phosphoserine aminotransferase having enhanced activity, or (3) both phosphoserine phosphatase having attenuated activity and 3-phosphoserine aminotransferase having enhanced activity, but not limited to them.

The microorganism can be, without limitation, further modified by enhancing the trp (tryptophan) operon, inactivating tryptophanase (TnaA), inactivating the membrane protein Mtr (Mtr), or any combination thereof.

Specifically, the microorganism can be, without limitation, further modified by enhancing the trp operon by inactivating TrpR, which inhibits the expression of genes (trpEDCBA) associated with the biosynthesis of L-tryptophan involved in the production of L-tryptophan, by inactivating tryptophanase (TnaA), which plays role in the introduction of extracellular L-tryptophan into the cell, and by inactivation of the membrane protein Mtr, which plays a role in the breakdown of intracellular L-tryptophan and water molecules to indole, pyruvate and ammonia (NH ₃ ).

Additionally, for purposes of describing the present invention, the microorganism can be, without limitation, further modified by amplifying the his (histidine) operon.

Specifically, biosynthetic genes divided into a total of 4 operons can be introduced into a microorganism in the form of a cluster in which the promoter has been changed to enhance the L-histidine biosynthetic pathway, and the L-histidine biosynthetic cluster is divided into a total of 4 operons (hisE -hisG, hisA-impA-hisF-hisI, hisD-hisC-hisB and cg0911-hisN). The His operon can be enhanced, without limitation, using a vector that can simultaneously introduce biosynthetic genes into a microorganism.

Additionally, for purposes of describing the present invention, the microorganism can be, without limitation, further modified by inactivating a transcription regulator (McbR), enhancing methionine synthase (meth), enhancing the sulfite reductase [NADPH] hemoprotein beta component (cysI), or any combination thereof.

Specifically, the microorganism can be, without limitation, further modified by inactivating McbR, which is a methionine/cysteine transcriptional regulator, enhancing methionine synthase (Meth), enhancing the sulfite reductase [NADPH] beta component of the hemoprotein, or any combination thereof.

The introduction, enhancement or inactivation of the activity of a particular protein and/or gene can be performed using any suitable method known in the art.

Used in this document, the term "enhancement" of the activity of the protein means that the activity of the protein is introduced or increased in comparison with its endogenous activity. "Introduction" of activity means that the microorganism acquires the activity of a particular polypeptide, which the microorganism did not possess either naturally or artificially.

As used herein, the term "enhancing" the activity of a protein relative to its endogenous activity means that the activity of a protein incorporated in a microorganism is enhanced compared to the endogenous activity of the protein or its activity before modification. The term "endogenous activity" refers to the activity of the protein, which originally had the parent strain of the microorganism or unmodified microorganism before transformation, when the microorganism is transformed through genetic modification caused by a natural or artificial factor. "Endogenous activity" can also be used interchangeably with "activity before modification". An increase in activity may include both the introduction of a foreign protein and an increase in the endogenous activity of the protein.

Increasing/enhancing the activity of a protein can be achieved by increasing/enhancing gene expression.

Specifically, the increase in activity of a protein according to the present invention can be achieved by one of the following methods, without limitation:

(1) a method for increasing the copy number of a protein-encoding polynucleotide,

(2) a method for modifying an expression control sequence to increase the expression of a polynucleotide,

(3) a method for modifying a polynucleotide sequence in a chromosome to enhance the activity of a protein,

(4) a method of introducing a foreign polynucleotide encoding an active protein, or a codon optimization modified polynucleotide encoding an active protein, or

(5) a method of enhancing activity through any combination thereof.

The method for increasing the copy number of a polynucleotide described in (1) above is not particularly limited, but may be in a form operably linked to a vector or integrated in a chromosome of a host cell. Specifically, this method can be performed by introducing into the host cell a vector that replicates and functions independently of the host cell and is operably linked to a polynucleotide encoding a protein according to the description of the present invention; or by introducing into the host cell a vector that inserts the polynucleotide into the host cell's chromosome and is operably linked to the polynucleotide, thereby increasing the copy number of the polynucleotide on the host cell's chromosome.

The following method for modifying an expression control sequence to increase the expression of a polynucleotide described in (2) above can be performed, without limitation, by inducing modification of the nucleic acid sequence by deletion, insertion, non-conservative substitution, conservative substitution, or any combination thereof, to further enhance the activity of the expression control sequence, or by replacing the nucleotide sequence with a nucleotide sequence having a stronger activity. An expression control sequence may include, without limitation, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a transcription and translation termination regulation sequence.

A strong heterologous promoter may be linked upstream of the expression unit of a polynucleotide instead of a self promoter, and examples of this strong promoter may include, without limitation, the CJ1-CJ7 promoters (Korean Patent No. 0620092 and International Publication No. WO 2006/065095), the lysCP1 promoter (International Publication No. WO 2009/096689), EF-Tu promoter, groEL promoter, aceA promoter, aceB promoter, lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, P _L promoter, tet promoter, gapA promoter, SPL1 promoter, SPL7 or SPL13 (Korean Patent No. 10-1783170) or O2 promoter (Korean Patent No. 10-1632642). In addition, the method for modifying a polynucleotide sequence in a chromosome described in (3) above can be performed, without limitation, by inducing variation in an expression control sequence by deletion, insertion, non-conservative substitution, conservative substitution, or any combination thereof, so that further enhance the activity of the polynucleotide sequence, or by replacing the nucleotide sequence with a nucleotide sequence modified to have stronger activity.

In addition, the method of introducing a foreign polynucleotide sequence described in (4) above can be performed by introducing into a host cell a foreign polynucleotide encoding a protein having an activity identical/similar to that of said protein, or its codon-optimized polynucleotide option. This foreign polynucleotide can be any polynucleotide, without limitation, encoding a protein having identical/similar activity to said protein. In addition, its codon-optimized variant can be introduced into the host cell to perform optimized transcription and translation of the foreign polynucleotide introduced into the host cell. This introduction can be performed by any known method of transformation, suitably chosen by those skilled in the art. When the introduced polynucleotide is expressed in the host cell, the protein is produced and its activity can be increased.

Finally, the method of enhancing activity by any combination of methods (1) to (4) described in paragraph (5) above can be performed by combining at least one of the methods of increasing the copy number of a protein-encoding polynucleotide, modifying an expression control sequence to increase its expression, modification of the polynucleotide sequence in the chromosome, the introduction of a foreign polynucleotide encoding a protein with activity, or its codon-modified polynucleotide variant.

Used in this document, the term "attenuation" of the activity of the protein is a concept that includes both the reduction and removal of activity compared to endogenous activity.

Reducing the activity of a protein can be achieved by a number of methods well known in the art. Examples of this method may include: a method of deleting a portion or full length of a gene encoding a protein on a chromosome, including the case of removing this activity; a method for replacing a gene encoding a protein on a chromosome with a gene mutated to reduce the activity of the protein; a method for introducing a mutation into an expression control sequence of a gene encoding a protein; replacing a gene expression control sequence with a sequence with less or no activity (eg, replacing an endogenous gene promoter with a weaker promoter); a method for deleting a portion or full length of a gene encoding a protein on a chromosome; a method of introducing an antisense oligonucleotide (eg, antisense RNA) that binds complementarily to a gene transcript on a chromosome to inhibit translation of mRNA to protein; a method for artificially adding a sequence complementary to an SD (Shine-Dalgarno) sequence upstream of the SD sequence of a gene encoding a protein to form a secondary structure thereby inhibiting ribosome binding thereto, and a method for inserting a promoter into the 3'end of an open reading frame (ORF) with induction of reverse transcription (reverse transcription engineering (RTE)), or any combination thereof, without limitation.

Specifically, a method for deleting a portion or full-length gene encoding a protein can be performed by replacing a polynucleotide encoding an endogenous target protein with a full-length nucleotide encoding an endogenous target protein within a chromosome, wherein the polynucleotide or marker gene has a partial deletion of the nucleic acid sequence, using an insertion vector into the chromosome of a microorganism. For example, a method of deleting a portion or full length of a gene through homologous recombination can be used without limitation. In addition, the term "part", although it may vary according to the type of polynucleotide and can be appropriately defined by one of ordinary skill in the art, refers to from 1 nucleotide to 300 nucleotides, specifically from 1 nucleotide to 100 nucleotides, and more specifically from 1 nucleotide to 50 nucleotides, respectively, without limitation.

Additionally, the method of modifying the expression control sequence can be carried out by inducing modification of the expression control sequence by deletion, insertion, conservative substitution or non-conservative substitution, or any combination thereof, to further weaken the activity of the expression control sequence, or by replacing the expression control sequence with a nucleotide sequence, less active. An expression control sequence may include, without limitation, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence for regulating transcription and translation termination.

In addition, the method of modifying the sequence of a gene in a chromosome can be carried out, without limitation, by inducing a modification by deletion, insertion, conservative substitution or non-conservative substitution in the sequence, or any combination thereof, to further reduce the activity of the protein, or carried out by replacing the gene sequence a gene sequence modified to have less or no activity.

As used herein, "a microorganism producing an L-amino acid or a precursor thereof" refers to a microorganism capable of producing an L-amino acid or its precursor in large quantities from carbon sources contained in the culture medium, compared to wild-type or unmodified microorganisms. Additionally, "microorganism" may refer to a microorganism naturally having the ability to produce an L-amino acid or its precursor, or a microorganism obtained by providing the ability to produce an L-amino acid or its precursor in a parent strain of a microorganism that is unable to produce an L-amino acid or its predecessor. Specifically, this microorganism may, without limitation, be a microorganism modified to express a protein containing the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof, to produce an L-amino acid or its precursor.

Additionally, "a microorganism producing an L-amino acid or its precursor" includes both wild-type microorganisms and microorganisms in which natural or artificial genetic modification has occurred, such as microorganisms in which a particular mechanism is attenuated or enhanced by introducing an exogenous gene, amplification or inactivation an endogenous gene, and so on, and in which a genetic modification has occurred or an activity has been enhanced to produce a target L-amino acid or a precursor thereof. In particular, the types of microorganisms are not particularly limited as long as the microorganism is capable of producing L-amino acid or its precursor, but the microorganism may belong to the Enterobacier genus, Escherichia genus, Erwinia genus, Serratia genus, Providencia genus, Corynebacterium genus, or Brevibacterium genus. More specifically, the microorganism may be any microorganism belonging to the genus Corynebacterium or the genus Escherichia. The microorganism of the genus Corynebacterium may be, but is not limited to, Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium thermoaminogenes, Corynebacterium efficiens, Corynebacterium stationis and the like. More specifically, the microorganism of the genus Escherichia may be Escherichia coli, and the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum, without limitation.

For purposes of describing the present invention, a microorganism can be any microorganism that includes a protein and is thus capable of producing an L-amino acid or a precursor thereof.

As used herein, "a microorganism capable of producing an L-amino acid or a precursor thereof" may be used interchangeably with the expressions "a microorganism producing an L-amino acid or a precursor thereof" and "a microorganism having the ability to produce an L-amino acid or its precursor".

Another aspect of the present invention provides a composition for producing an L-amino acid or its precursor, containing a microorganism modified to express a protein containing the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof, or said protein.

A composition for producing an L-amino acid or its precursor refers to a composition capable of producing an L-amino acid or its precursor via a protein according to the present invention. The composition may, without limitation, contain the specified protein, its functional fragment, or any components used to work with the protein.

Another aspect of the present invention provides a method for producing an L-amino acid or a precursor thereof, comprising culturing said microorganism in a culture medium.

The method may further include isolating the L-amino acid or its precursor from their culture medium or culture.

In the above-described method, culturing the microorganism can be carried out, without limitation, by batch culture, continuous culture, fed-batch culture, and the like, as is known in the art. In this regard, the culture conditions are not particularly limited, but the optimum pH (for example, pH 5 to 9, specifically pH 6 to 8, and most specifically pH 6.8) can be maintained by using a basic compound (for example, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (eg phosphoric acid or sulfuric acid). Additionally, aerobic conditions can be maintained by adding oxygen or an oxygen-containing gas mixture to the culture. The culturing temperature can be maintained at 20°C-45°C, specifically 25°C-40°C, and culturing can be carried out for about 10 hours to about 160 hours without limitation. Amino acids produced during cultivation can be released into the culture medium or remain in the cells.

Examples of a carbon source to be contained in the culture medium may include, without limitation, saccharides and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose), oils, and fats (e.g., soybean oil, sunflower oil, peanut oil, coconut oil), fatty acids (eg palmitic acid, stearic acid and linoleic acid), alcohols (eg glycerol and ethanol) and organic acids (acetic acid), which can be used alone or in combination, and the like. Examples of a nitrogen source to be contained in the culture medium include, without limitation, a nitrogen-containing organic compound (e.g., peptone, yeast extract, meat juice, malt extract, corn syrup, soy flour, and urea), an inorganic compound (e.g., ammonium sulfate , ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate), which can be used alone or in combination, and the like. As a source of phosphorus, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and their respective sodium salts can be used alone or in combination without limitation. In addition, the culture medium may include, without limitation, necessary growth promoting substances such as metal salts (eg, sodium sulfate and ferrous sulfate), amino acids, and vitamins.

The amino acids produced in the above-described culture step of the present invention can be recovered by harvesting the target amino acids from the culture solution using any known method selected according to the culture method. For example, centrifugation, filtration, anion exchange chromatography, crystallization, and high performance liquid chromatography (HPLC) can be used, and the target amino acids can be isolated from the culture medium or microorganism using any suitable method in the art, without limitation.

Additionally, the extraction step may include a purification process, which can be carried out using any suitable method well known in the art. Thus, the isolated amino acid may be a purified amino acid or a microorganism fermentation broth comprising the amino acid (Introduction to Biotechnology and Genetic Engineering, A. J. Nair., 2008).

In addition, for purposes of describing the present invention, in the case of a microorganism modified to express D-3-phosphoglycerate dehydrogenase derived from the genus Azotobacter, the yields of L-amino acids and their precursors, including serine, tryptophan, histidine, methionine, and O-phosphoserine, are increased. It is important that the modified microorganism increases the yields of L-amino acids and their precursors, while wild-type strains of the genus Corynebacterium are unable to produce L-amino acids or their precursors or are able to produce them in very small quantities.

Another aspect of the present invention provides a method for producing an L-amino acid or its precursor using a composition that contains a microorganism modified to express a protein containing the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof, or the specified protein.

The microorganism modified to express a protein containing the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof, and the microorganism comprising it are as described above.

Another aspect of the present invention provides the use of a protein containing the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof, to increase the production of L-amino acid or its precursor.

SEQ ID NO: 1 or a functional fragment thereof, L-amino acids and its precursor are as described above.

EXAMPLES OF CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. At the same time, the technical essence not disclosed in this description can be sufficiently understood and easily performed by specialists in the field of technology to which the present application relates, or in a similar field of technology.

Example 1 Preparation of a D-3-Phosphoglycerate Dehydrogenase (serA(Avn)) Gene Overexpression Vector Originating from Azotobacter

In order to identify whether the ability to produce serine and O-phosphoserine (OPS) is improved by enhancing D-3-phosphoglycerate dehydrogenase (hereinafter referred to as SerA(Avn)) originating from Azotobacter vinelandii, an expression vector was obtained.

The pCL1920 vector (GenBank No. AB236930) was used to express the serA(Avn) gene (SEQ ID NO: 1) encoding SerA(Avn) and the trc (Ptrc) promoter was used as an expression promoter, resulting in the construction of a vector in the form of pCL -Ptrc-serA(Avn).

As a control, a vector comprising D-3-phosphoglycerate dehydrogenase originating from E. coli, in which feedback inhibition by serine is eliminated, was obtained and named pCL-Ptrc-serA*(G336V). The primer sequences used to generate the vectors are shown in Table 1 below.

PCR (polymerase chain reaction) for Ptrc, which was used in the preparation of both vectors, was performed using primers of SEQ ID NO: 2 and 3. Specifically, PCR for the foreign gene serA(Avn) was performed using primers of SEQ ID NO: 4 and 5, a PCR for serA*(G336V) was performed using primers of SEQ ID NOs: 6 and 7. Amplified fragments of Ptrc and serA(Avn) and serA*(G336V) of the respective genes were cloned into pCL1920 vector treated with Sma1 restriction enzyme. , by the Gibson assembly method, respectively, resulting in the construction of pCL-Ptrc-serA(Avn) and pCL-Ptrc-serA*(G336V).

Example 2 Preparation of a strain by introducing serA(Avn) originating from Azotobacter into wild-type E. coli and evaluating the ability to produce serine

Using the wild-type E. coli W3110 strain as the carrier strain, strains were obtained by introducing each of the two types of plasmids obtained in Example 1 into the W3110 strain, and then their serine-producing ability was evaluated.

Each of the strains was sown on a solid medium LB (Luria - Bertani) and cultivated overnight in an incubator at 33°C. The strain that was cultured overnight on LB solid medium was inoculated with 25 ml of titration medium as shown in Table 2 below, and cultured in an incubator at 34.5° C. at 200 rpm for 40 hours. The results are shown in Table 3 below.

As shown in Table 3, the W3110/pCL-Ptrc-serA*(G336V) strain, which eliminated feedback inhibition by serine and enhanced serA activity, showed a 60% increase in serine production compared to the wild-type strain. For comparison, it was confirmed that the W3110/pCL-Ptrc-serA(Avn) strain, including serA(Avn) originating from Azotobacter, showed a 160% increase in serine production compared to the W3110 wild-type strain, and also showed an increase by 62.5% compared with strain W3110/pCL-Ptrc-serA*(G336V).

Example 3 Preparation of a strain in which serB activity is attenuated and introduced foreign serA(Avn) gene originating from Azotobacter and evaluation of the ability of this strain to produce OPS

An O-phosphoserine (OPS)-producing microorganism was obtained by attenuating endogenous phosphoserine phosphatase (SerB) in wild-type E. coli strain W3110 (also named "CA07-0012", accession number: KCCM11212P, described in Korean Patent No. 10-1381048 and Publication Application for US patent No. 2012-0190081).

Each of the two types of plasmids obtained in Example 1 was introduced into CA07-0012 and the ability of the obtained strains to produce OPS was evaluated.

Each of the strains was sown on solid LB medium and cultivated overnight in an incubator at 33°C. The strain that was cultured overnight on LB solid medium was inoculated with 25 ml of titration medium as shown in Table 4 below, and cultured in an incubator at 34.5° C. at 200 rpm for 40 hours. The results are shown in Table 5 below.

As shown in Table 5 above, CA07-0012/pCL-Ptrc-serA*(G336V), which eliminated serine feedback inhibition and enhanced serA activity, showed a 57% increase in OPS production compared to the wild-type strain. It was confirmed that the CA07-0012/pCL-Ptrc-serA(Avn) strain including serA(Avn) originating from Azotobacter showed a 107% increase in OPS production compared to the wild-type strain, and also showed a 32% increase compared with strain CA07-0012/pCL-Ptrc-serA*(G336V).

Example 4: Preparation of a vector for co-overexpression of serA(Avn) originating from Azotobacter and serC originating from E. coli

In order to identify whether serine and OPS production capacity is further improved by introducing the serA(Avn) gene into a strain overexpressing E. coli-derived 3-phosphoserine aminotransferase (serC), a vector in the form of pCL-Ptrc-serA was generated. (Avn)-(RBS)serC for expression of serA(Avn) and serC as operons.

As its positive control, the vector pCL-Ptrc-serA*(G336V)-(RBS)serC was constructed to obtain a microorganism co-expressing the serA*(G336V) and serC genes originating from E. coli. The primer sequences used to generate the vectors are shown in Table 6 below.

PCR for Ptrc_serA(Avn) was performed using as template pCL-Ptrc-serA(Avn) obtained in Example 1 and primers of SEQ ID NOs: 2 and 8, and PCR for Ptrc_serA*(G336V) was performed using as pCL-Ptrc-serA*(G336V) templates and primers of SEQ ID NOs: 2 and 11. E. coli-derived (RBS)serC used in both vectors was obtained by PCR performed using w3110 DNA as template and primers with SEQ ID NO: 9 and 10.

Amplified Ptrc_serA(Avn) and (RBS)serC fragments and Ptrc_serA*(G336V) and (RBS)serC fragments were cloned into pCL1920 vector treated with SmaI restriction enzyme using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6, NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix), respectively, resulting in the construction of pCL-Ptrc-serA(Avn)-(RBS)serC and pCL-Ptrc-serA*(G336V)-(RBS)serC vectors .

Example 5 Preparation of a strain in which serC activity is enhanced and the serA(Avn) gene originating from Azotobacter is introduced and evaluation of the serine-producing ability of this strain

In order to evaluate the serine-producing ability by introducing the serA(Avn) gene originating from Azotobacter into a strain in which serC was overexpressed, two types of plasmids obtained in Example 4 were introduced into W3110, respectively.

Each of the strains was sown on solid LB medium and cultivated overnight in an incubator at 33°C. The strain that was cultured overnight on LB solid medium was inoculated with 25 ml of titration medium as shown in Table 7 below, and cultured in an incubator at 34.5° C. at 200 rpm for 40 hours. The results are shown in Table 8 below.

As shown in Table 8 above, it was confirmed that the w3110/pCL-Ptrc-serA(Avn)-(RBS)serC strain including the serA(Avn) gene originating from Azotobacter showed an increase in L-serine production compared to with strain w3110/pCL-Ptrc-serA*(G336V)-(RBS)serC, including serA*(G336V). Thus, it was confirmed that the ability to produce L-serine was further improved by incorporating the serA(Avn) gene originating from Azotobacter into a strain in which the ability to produce L-serine was improved.

The w3110/pCL-Ptrc-serA(Avn)-(RBS)serC strain was named CA07-4383 and deposited with the Korea Microorganism Culture Center (KCCM) under the Budapest Treaty and was assigned accession number KCCM12381P on November 9, 2018.

Example 6 Preparation of a strain in which the activity of the serB gene is attenuated, the activity of the serC gene is increased, and the serA(Avn) gene originating from Azotobacter is introduced, and evaluation of the ability of this strain to produce QPS

In order to evaluate the serine-producing ability when the serA(Avn) gene originating from Azotobacter was introduced into a strain with attenuated serB activity and overexpressing serC, two types of plasmids obtained in Example 4, respectively, were introduced into strain CA07-0012, and the ability was evaluated. these strains produce OPS.

Each of the strains was sown on solid LB medium and cultivated overnight in an incubator at 33°C. The strain that was cultured overnight on LB solid medium was inoculated with 25 ml of titration medium as shown in Table 9 below, and cultured in an incubator at 34.5° C. at 200 rpm for 40 hours. The results are shown in Table 10 below.

As shown in Table 10 above, it was confirmed that the CA07-0012/pCL-Ptrc-serA(Avn)-(RBS)serC strain including the serA(Avn) gene originating from Azotobacter was confirmed to have higher OPS production, than strain CA07-0012/pCL-Ptrc-serA*(G336V)-(RBS)serC including serA*(G336V). Thus, it was confirmed that the OPS-producing ability was further improved by incorporating the serA(Avn) gene originating from Azotobacter into a strain in which the OPS-producing ability was improved.

Example 7 Preparation of a strain of the genus Escherichia into which the serA(Avn) gene derived from Azotobacter has been introduced and evaluation of the ability of this strain to produce tryptophan

Example 7-1 Preparation of an L-Tryptophan Producing Escherichia Microorganism

An L-tryptophan producing strain of the genus Escherichia was developed from wild-type E. coli W3110. In order to identify whether the ability to produce L-tryptophan is significantly increased as a result of the modification to express a protein having the ability to export L-tryptophan, a strain obtained for producing L-tryptophan was used as a parent strain. Specifically, TrpR inhibits the expression of L-tryptophan biosynthesis genes (trpEDCBA), which are involved in the production of L-tryptophan from chorismate. Thus, the trpR gene encoding TrpR has been deleted. In addition, to eliminate feedback inhibition by the TrpE polypeptide in accordance with increased production of L-tryptophan, proline, which is the 21st amino acid from the N-terminus of TrpE, was replaced by serine (J. Biochem. Mol. Biol. 32, 20-24 (1999)).

The membrane protein Mtr plays a role in the transport of extracellular L-tryptophan into the cell, and the TnaA protein plays a role in the degradation of intracellular L-tryptophan and water molecules to indole, pyruvate and ammonia (NH ₃ ). Thus, the mtr and tnaA genes, which inhibit the production of L-tryptophan and carry out the degradation of L-tryptophan, were deleted.

To remove these genes, the λ-red (lambda-red) recombination method was used (One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Datsenko KA, Wanner BL., Proc Natl Acad Sci USA. 2000 Jun 6; 97(12):6640-5). To remove the mtr gene, PCR was performed using the pKD4 vector as a template and primers of SEQ ID NOs: 12 and 13 to obtain a gene fragment (1580 bp (base pairs)) in which the FRT-kanamycin-FRT cassette and homologous 50 bp base pairs flanking the mtr gene, where chromosomal homologous recombination occurs between them. The pKD4 vector kanamycin antibiotic marker was used to confirm the deletion of the target gene and the insertion of the antibiotic resistance gene, and the FRT region plays a role in the deletion of the antibiotic marker after the deletion of the target gene. Solg™TM Pfu-X DNA polymerase was used as the polymerase and PCR was performed under the following amplification conditions: denaturation at 95°C for 2 minutes; 27 cycles of denaturation at 95°C for 20 seconds, annealing at 62°C for 40 seconds and polymerization at 72°C for 1 minute; and polymerization at 72°C for 5 minutes.

E. coli strain W3110 was transformed with the pKD46 vector, which expresses the λ-red recombinase (gam, bet and exo), by electroporation and plated on LB solid medium containing 50 mg/l kanamycin. In E. coli strain W3110 with confirmed transformation with the pKD46 vector, expression of the recombinant enzyme was induced by adding 10 mM L-arabinose to it when reaching an OD600 (optical density at 600 nm) of approximately 0.1 at 30°C.

When an OD600 of approximately 0.6 was reached, this strain was prepared as competent cells and transformed by electroporation with a linear gene fragment obtained in the process described above, in which the FRT-kanamycin-FRT cassette and the 50 bp homologous base pairs flanking the gene were linked mtr. For colonies grown on LB solid medium containing 25 mg/l kanamycin, colony PCR was performed using primers of SEQ ID NOs: 14 and 15, and colonies were selected where a 782 bp gene fragment was obtained.

The strain from which the mtr gene had been removed by homologous recombination was prepared as competent cells to remove the antibiotic marker kanamycin and then transformed with the pCP20 vector by electroporation. The pCP20 vector is designed to recognize FRT sites flanking the kanamycin antibiotic resistance gene and bind to them on the chromosome via FLP protein expression, whereby it removes the antibiotic marker between the FRT sites. The strain transformed with pCP20 vector and grown on solid LB medium containing 100 mg/l ampicillin and 25 mg/l chloramphenicol was cultured in liquid LB medium at 30°C for 1 hour, further cultivated at 42°C for 15 hours, and were sown on solid medium LB. The grown colonies were cultured on solid LB medium containing 100 mg/l ampicillin and 25 mg/l chloramphenicol, solid LB medium containing 12.5 mg/l kanamycin, and solid LB medium containing no antibiotic. Only colonies grown on solid LB medium containing no antibiotic were selected. Finally, deletion of the mtr gene was confirmed by genome sequencing and the strain was named CA04-9300.

To remove the tnaA gene, genetic manipulations were performed in the same manner as described above. PCR was performed using the pKD4 vector as template and primers of SEQ ID NO: 16 and 17 to obtain a gene fragment (1580 bp) in which the FRT-kanamycin-FRT cassette and 50 bp homologous base pairs are linked, flanking the tnaA gene, where chromosomal homologous recombination occurs between them. Solg™ Pfu-X DNA polymerase was used as polymerase and PCR was performed under the following amplification conditions: denaturation at 95°C for 2 minutes; 27 cycles of denaturation at 95°C for 20 seconds, annealing at 62°C for 40 seconds and polymerization at 72°C for 1 minute; and polymerization at 72°C for 5 minutes.

Transformation with the pKD46 vector was confirmed, and the CA04-9300 strain, in which the recombinases were expressed by the addition of 10 mM L-arabinose, was transformed by electroporation with a linear gene fragment in which the FRT-kanamycin-FRT cassette and 50 bp homologous base pairs flanking tnaA gene. For colonies grown on LB solid medium containing 25 mg/l kanamycin, colony PCR was performed using primers of SEQ ID NOS: 14 and 18, and colonies were selected where a 787 bp gene fragment was obtained.

The strain from which the tnaA gene was deleted by homologous recombination was prepared as competent cells and transformed with pCP20 vector to remove the kanamycin antibiotic marker, and a strain from which the antibiotic kanamycin resistance marker was deleted was obtained by expressing the FLP protein. Finally, the deletion of the tnaA gene was confirmed by genome sequencing and the strain was named CA04-9301.

To remove the trpR gene, PCR was performed using the pKD4 vector as a template and primers of SEQ ID NO: 19 and 20 to obtain a gene fragment (1580 bp) in which the FRT-kanamycin-FRT cassette and homologous base pairs 50 were linked bp flanking the trpR gene, where chromosomal homologous recombination occurs between them. Solg™ Pfu-X DNA polymerase was used as the polymerase and PCR was performed under the following amplification conditions: denaturation at 95° C. for 2 minutes; 27 cycles of denaturation at 95°C for 20 seconds, annealing at 62°C for 40 seconds and polymerization at 72°C for 1 minute; and polymerization at 72°C for 5 minutes.

Transformation with the pKD46 vector was confirmed, and the CA04-9301 strain, in which recombinases were expressed by adding 10 mM L-arabinose, was transformed by electroporation with a linear gene fragment obtained in the above process, in which the FRT-kanamycin-FRT cassette and homologous base pairs 50 are linked. b.p. flanking the trpR gene. For colonies grown on LB solid medium containing 25 mg/l kanamycin, colony PCR was performed using primers of SEQ ID NOS: 14 and 21, and colonies were selected where an 838 bp gene fragment was obtained.

The strain from which the trpR gene was deleted by homologous recombination was prepared as competent cells and transformed with pCP20 vector to remove the kanamycin antibiotic marker, and a strain from which the antibiotic kanamycin resistance marker was deleted was obtained by expressing the FLP protein. Finally, the deletion of the trpR gene was confirmed by genome sequencing and the strain was named CA04-9307.

To provide strain CA04-9307 with resistance to trpE inhibition in a feedback manner, PCR was performed using E. coli W3110 gDNA (genomic DNA) as template and primers of SEQ ID NOS: 22 and 23 containing the EcoRI restriction enzyme site, resulting in resulting in a trpE gene fragment containing the EcoRI sequence (1575 bp). Solg™ Pfu-X DNA polymerase was used as the polymerase and PCR was performed under the following amplification conditions: denaturation at 95° C. for 2 minutes; 27 cycles of denaturation at 95°C for 20 seconds, annealing at 62°C for 1 minute and polymerization at 72°C for 1 minute; and polymerization at 72°C for 5 minutes.

The trpE gene obtained as described above and the pSG76-C plasmid (JOURNAL OF BACTERIOLOGY, July 1997, p. 4426-4428) were treated with EcoRI restriction enzyme and cloned. E. coli DH5α was transformed with the cloned plasmid by electroporation, and the transformed E. coli DH5α strain was selected from an LB medium plate containing 25 μg/ml chloramphenicol to obtain plasmid pSG76-C-trpE.

Site-directed mutagenesis (Stratagene, USA) was performed using the obtained plasmid pSG76-C-trpE and primers with SEQ ID NOS: 24 and 25 to obtain pSG76-C-trpE(P21S).

Strain CA04-9307 was transformed with plasmid pSG76-C-trpE(P21S) and cultured in LB-Cm medium (10 g/l yeast extract, 5 g/l NaCl, 10 g/l tryptone and 25 μg/l chloramphenicol) and selected colonies resistant to chloramphenicol. Selected transformants are strains in which the plasmid pSG76-C-trpE(P21S) is included in the trpE region of the genome as a result of the first insertion. The strain into which the resulting trpE(P21S) gene was inserted was transformed with pAScep plasmid (Journal of Bacteriology, July 1997, p. and a strain growing in LB-Ap medium (10 g/l yeast extract, 5 g/l NaCl, 10 g/l tryptone and 100 μg/l ampicillin) was selected. The trpE gene was amplified in the selected strain using primers of SEQ ID NOs: 22 and 23, and the replacement of the trpE(P21S) gene was confirmed by sequencing. The resulting strain was named CA04-4303.

Example 7-2 Preparation of a microorganism of the genus Escherichia into which the serA(Avn) gene derived from Azotobacter has been introduced and evaluation of the ability of this microorganism to produce tryptophan

Vector pCL-Ptrc-serA(Avn) obtained in Example 1 and vector pCL1920 as a control were introduced into CA04-4303 obtained in Example 1, respectively, to obtain strains CA04-4303/pCL1920 and CA04-4303/pCL-Ptrc -serA(Avn). To study the production of L-tryptophan by strains CA04-4303/pCL1920 and CA04-4303/pCL-Ptrc-serA(Avn), these two strains were cultured in liquid LB medium containing 50 mg/l spectinomycin for 12 hours. Subsequently, each of the strains was inoculated into a 250 ml baffled flask containing 25 ml of production medium so that the initial OD600 value reached 0.01, and cultured at 37° C. for 48 hours with shaking at 200 rpm. After completion of cultivation, the amount of L-tryptophan production was measured by HPLC.

The results of the production of L-tryptophan by strains CA04-4303/pCL1920 and CA04-4303/pCL-Ptrc-serA(Avn) in culture medium are shown in Table 17 below. The CA04-4303/pCL1920 strain showed production of L-tryptophan at 1.2 g/l and accumulation of indole, which is an intermediate, at 37 mg/l. However, the strain in which serA(Avn) was introduced showed L-tryptophan production at 1.7 g/L without indole accumulation.

<Production medium (pH 7.0)>

70 g glucose, 20 g (NH ₄ ) ₂ SO ₄ , 1 g MgSO ₄ ⋅7H ₂ O, 2 g KH ₂ PO ₄ , 2.5 g yeast extract, 5 g Na-citrate, 1 g NaCl and 40 g CaCO ₃ (based on 1 liter of distilled water).

As can be seen from the above results, it was found that the supply of L-serine was sufficient due to the introduction of serA(Avn), and it was confirmed that the yield of L-tryptophan was increased without the accumulation of indole, which is an intermediate product, at the final stage of L- biosynthesis. tryptophan.

Example 7-3 Preparation of Tryptophan Producing Corynebacterium glutamicum Strain Into Which Alien Gene serA(Avn) Derived from Azotobacter Is Introduced

In order to identify the effect of the serA(Avn) gene originating from Azotobacter on the tryptophan producing strain of Corynebacterium genus, KCCM12218P (Korean Patent Publication No. 2018-0089329) was used as the L-tryptophan producing strain of Corynebacterium genus.

This strain was obtained by replacing the Corynebacterium glutamicum serA gene (hereinafter referred to as serA(Cgl)) with the Azotobacter-derived serA(Avn) gene to be expressed under the action of the gapA promoter.

For these genetic manipulations, an upstream promoter region and a downstream serA (Cgl) ORF region, where chromosomal homologous recombination occurs, were first obtained. Specifically, the gene fragment of the upstream promoter region was obtained by performing PCR using Corynebacterium glutamicum chromosomal DNA as a template and primers of SEQ ID NOs: 26 and 27, and the gene fragment of the downstream region was obtained by performing PCR using primers of SEQ ID NOS: 28 and 29. Additionally, the gapA promoter region was obtained by performing PCR using Corynebacterium glutamicum chromosomal DNA as a template and primers of SEQ ID NOS: 30 and 31.

Solg™ Pfu-X DNA polymerase was used as the polymerase and PCR was performed under the following amplification conditions: denaturation at 95° C. for 5 minutes; 30 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds and polymerization at 72°C for 60 seconds; and polymerization at 72°C for 5 minutes.

The serA(Avn) gene region originating from Azotobacter was obtained by performing PCR using the pCL-Ptrc-serA(Avn) vector obtained in Example 1 as template and primers of SEQ ID NOS: 32 and 33.

Solg™ Pfu-X DNA polymerase was used as the polymerase and PCR was performed under the following amplification conditions: denaturation at 95° C. for 5 minutes; 30 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds and polymerization at 72°C for 30 seconds; and polymerization at 72°C for 5 minutes.

A recombinant plasmid was obtained by cloning using amplified upstream and downstream regions for chromosomal homologous recombination, the gapA promoter, the serA(Avn) gene originating from Azotobacter, and the pDZ chromosomal transformation vector cleaved with the restriction enzyme SmaI Gibson assembly method and named it pDZ-PgapA-serA(Avn). Cloning was performed by mixing the Gibson assembly reagent and the calculated number of moles of each of the gene fragments, followed by incubation at 50° C. for 1 hour.

The Corynebacterium glutamicum KCCM12218P strain producing L-tryptophan was transformed with the obtained vector pDZ-PgapA-serA(Avn) by electroporation and subjected to a second crossing-over process to obtain a strain in which the serA(cgl) gene was replaced by the serA gene of Azotobacter expressed under the action of the gapA promoter . These genetic manipulations were confirmed by PCR and genomic sequencing using primers of SEQ ID NO: 34 and 35, respectively, amplifying the outer regions of the upstream and downstream homologous recombination regions into which the gene was inserted, and the resulting strain was named KCCM12218P-PgapA-serA(Avn).

The primer sequences used in this example are shown in Table 18 below.

Example 7-4 Evaluation of tryptophan-producing ability of a strain of Corynebacterium glutamicum into which the serA(Avn) gene derived from Azotobacter has been introduced

The KCCM12218P-PgapA-serA(Avn) strain obtained in Example 7-3 and the parent KCCM12218P strain were cultured as follows to identify their tryptophan production. Each of the strains was inoculated into a 250 ml baffled flask containing 25 ml of seed medium and cultured at 30° C. for 20 hours with shaking at 200 rpm. Then, 1 ml of seed medium was inoculated into a 250 ml baffled flask containing 25 ml of production medium, and cultured at 30° C. for 24 hours with shaking at 200 rpm. After completion of cultivation, L-tryptophan production was measured for each strain by HPLC.

<Inoculation Medium (pH 7.0)>

20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH ₂ PO ₄ , 8 g K ₂ HPO ₄ , 0.5 g MgSO ₄ ⋅7H ₂ O, 100 µg biotin, 1000 µg thiamine HCl, 2000 mcg of calcium pantothenate and 2000 mcg of nicotinamide (per 1 liter of distilled water).

<Production medium (pH 7.0)>

30 g glucose, 15 g (NH ₄ ) ₂ SO ₄ , 1.2 g MgSO ₄ ⋅7H ₂ O, 1 g KH ₂ PO ₄ , 5 g yeast extract, 900 µg biotin, 4500 µg thiamine HCl, 4500 µg calcium pantothenate and 30 g CaCO ₃ (per 1 liter of distilled water)

The results of the evaluation of L-tryptophan production by strains KCCM12218P and KCCM12218P-PgapA-serA(Avn) are shown in Table 19 above.

While the parental strain KCCM12218P produced 2.5 g/L L-tryptophan and accumulated 59 mg/L indole intermediate, the serA(Avn) treated strain produced 3.1 g/L L-tryptophan without indole accumulation.

Based on these results, it was determined that the supply of L-serine was also sufficient by introducing serA(Avn) originating from Azotobacter into Corynebacterium glutamicum producing L-tryptophan, and it was confirmed that the yield of L-tryptophan was also increased without accumulation indole, which is an intermediate product at the final stage of L-tryptophan biosynthesis. Thus, it can be seen that the synergistic effects on tryptophan production are improved while improving the production of the precursor.

The KCCM12218P-PgapA-serA(Avn) strain was named CM05-8935 and deposited with the Korea Microorganism Culture Center (KCCM) under the Budapest Treaty and was assigned accession number KCCM12414P on November 27, 2018.

Example 8 Preparation of a strain of Corynebacterium glutamicum into which the serA(Avn) gene derived from Azotobacter has been introduced and evaluation of the ability of this strain to produce histidine

Example 8-1 Preparation of a Histidine-Producing Corynebacterium glutamicum Strain

The L-histidine producing strain of Corynebacterium glutamicum was developed from the wild type strain ATCC13032. To eliminate feedback inhibition by the HisG polypeptide, which is the first enzyme of the L-histidine biosynthetic pathway, the glycine at position 233 from the N-terminus of HisG was replaced by histidine, and the threonine at position 235 from the N-terminus was simultaneously replaced by glutamine. (SEQ ID NO: 88) (ACS Synth. Biol., 2014, 3 (1), pp 21-29). Additionally, in order to enhance the L-histidine biosynthetic pathway, the biosynthetic genes (hisD-hisC-hisB-hisN) divided into 4 operons in total were obtained in the form of a cluster where the promoter was changed and introduced into a strain (SEQ ID NO: 89).

For these genetic manipulations, the upstream and downstream regions of modifications of the 233rd and 235th amino acids of the hisG gene were first obtained, where chromosomal homologous recombination occurs. Specifically, the gene fragment of the upstream and downstream hisG modification regions of amino acids 233 and 235 was obtained by performing PCR using Corynebacterium glutamicum ATCC13032 chromosomal DNA as template and primers of SEQ ID NO: 36 and 37 and a gene fragment of the upstream and downstream hisG amino acid 233 and 235 modification regions was obtained by performing PCR using primers of SEQ ID NOS: 38 and 39.

Solg™ Pfu-X DNA polymerase was used as polymerase and PCR was performed under the following amplification conditions: denaturation at 95°C for 5 minutes; 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds and polymerization at 72°C for 60 seconds; and polymerization at 72°C for 5 minutes.

The recombinant plasmid was obtained by cloning using amplified upstream and downstream modification regions of the 233rd and 235th amino acids of hisG and pDZ vector (Korean Patent No. 10-0924065) for chromosomal transformation cleaved with SmaI restriction enzyme , by the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6, NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) and named pDZ-hisG(G233H, T235Q). Cloning was performed by mixing the Gibson assembly reagent and the calculated number of moles of each of the gene fragments, followed by incubation at 50° C. for 1 hour.

The wild-type Corynebacterium glutamicum strain ATCC13032 was transformed with the obtained vector pDZ-hisG(G233H, T235Q) by electroporation and subjected to a second crossover process to obtain a strain having HisG amino acid substitutions from glycine to histidine at position 233 and from threonine to glutamine at position 235 position on the chromosome (SEQ ID NO: 88). These genetic manipulations were confirmed by PCR and genome sequencing using primers of SEQ ID NO: 40 and 41, respectively, amplifying the outer regions of the upstream and downstream homologous recombination regions into which the gene was inserted, and the resulting strain was named CA14-0011.

The primer sequences used in this example are shown in Table 20 below.

Additionally, to enhance the L-histidine biosynthetic pathway, the biosynthetic genes divided into 4 operons in total were introduced in the form of a cluster where the promoter was replaced. Specifically, the L-histidine biosynthesis cluster was divided into four operons in total (hisE-hisG, hisA-impA-hisF-hisI, hisD-hisC-hisB, and cg0911-hisN), and a vector was obtained that simultaneously introduced these biosynthesis genes into microorganism.

In addition, the Ncgl1108 gene encoding gamma-aminobutyrate permease (Microb Biotechnol. 2014 Jan; 7 (1): 5-25)) was used as the biosynthesis cassette insertion site.

For these genetic manipulations, the upstream and downstream regions of the Ncgl1108 gene, where chromosomal homologous recombination occurs, were first obtained. Specifically, the gene fragment of the upstream region of the Ncgl1108 gene was obtained by performing PCR using Corynebacterium glutamicum ATCC13032 chromosomal DNA as a template and primers of SEQ ID NOs: 42 and 43, and the gene fragment of the downstream region of the Ncgl1108 gene was obtained by performing PCR using primers of SEQ ID NOS: 44 and 45.

Solg™ Pfu-X DNA polymerase was used as polymerase and PCR was performed under the following amplification conditions: denaturation at 95°C for 5 minutes; 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds and polymerization at 72°C for 60 seconds; and polymerization at 72°C for 5 minutes.

The recombinant plasmid was obtained by cloning using amplified upstream and downstream regions of the NCgl1108 gene and pDZ vector (Korean Patent No. 10-0924065) for chromosomal transformation digested with SmaI restriction enzyme by the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6, NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) and named pDZ-ΔNcgl1108. Cloning was performed by mixing the Gibson assembly reagent and the calculated number of mol of gene fragments, followed by incubation at 50° C. for 1 hour.

The CA14-0011 strain was transformed with the resulting pDZ-ΔNcgl1108 vector by electroporation and subjected to a second crossover process to obtain a strain in which the Ncg1108 gene was disrupted. These genetic manipulations were confirmed by PCR and genomic sequencing using primers of SEQ ID NO: 46 and 47, respectively, amplifying the outer regions of the upstream and downstream regions of homologous recombination where the gene was disrupted, and the resulting as a result, the strain was named CA14-0736.

The primer sequences used in this example are shown in Table 21 below.

Additionally, to enhance the biosynthesis cluster, a promoter region was obtained to replace with a group of 4 operon genes. Enhanced lysC promoter region (hereinafter referred to as lysCP1, Korean Patent No. 10-0930203) and hisE-hisG region, gapA promoter region and hisA-impA-hisF-hisI region, synthesized SPL13 promoter region (Korean Patent No. 10-1783170) ) and the hisD-hisC-hisB region and the synthesized CJ7 promoter region (Korean Patent No. 10-0620092 and WO 2006/065095) and the cg0911-hisN region. Specifically, PCR was performed using the chromosome of strain KCCM10919P (Korean Patent No. 10-0930203) as a template and primers of SEQ ID NOs: 48 and 49. PfuUltraTM (Stratagene) high fidelity DNA polymerase (Stratagene) was used as the polymerase for PCR, and thus amplified PCR products were purified using QIAGEN's PCR Purification Kit to obtain the lysCP1 promoter region. The hisE-hisG gene fragment was obtained by performing PCR using Corynebacterium glutamicum CA14-0011 chromosomal DNA as template and primers of SEQ ID NOS: 50 and 51. The gapA promoter region gene fragment was obtained by performing PCR using primers of SEQ ID NO : 52 and 53, and the gene fragment of the hisA-impA-hisF-hisI region was obtained by performing PCR using primers of SEQ ID NOs: 54 and 55. Additionally, PCR was performed using the synthesized SPL13 promoter as a template and primers of SEQ ID NOs. : 56 and 57, and the gene fragment of the hisD-hisC-hisB region was obtained by performing PCR using Corynebacterium glutamicum CA14-0011 chromosomal DNA as a template and primers with SEQ ID NOs: 58 and 59. Then, PCR was performed using the synthesized CJ7 promoter as a template and primers with SEQ ID NOS: 60 and 61, and the gene fragment of the cg0911-hisN region was obtained by performing PCR using Corynebacterium glutamicum CA14-0011 chromosomal DNA as a template and primers with SEQ ID NOS: 62 and 63.

The primer sequences used in this example are shown in Table 22 below.

Solg™ Pfu-X DNA polymerase was used as polymerase and PCR was performed under the following amplification conditions: denaturation at 95°C for 5 minutes; 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds and polymerization at 72°C for 180 seconds; and polymerization at 72°C for 5 minutes.

The recombinant plasmid was obtained by cloning using amplified: lysCP1 region and hisE-hisG region, gapA promoter region and hisA-impA-hisF-hisI region, synthesized SPL13 promoter region and hisD-hisC-hisB region, synthesized CJ7 promoter region and cg0911 region -hisN and pDZ-vector ΔNCgl1108 for chromosomal transformation digested with Seal restriction enzyme, Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6, NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) and was named pDZ-ΔNCgl1108::lysCP1_hisEG-PgapA_hisA-impA-hisFI-SPL13_HisDCB-CJ7_cg0911-hisN. Cloning was performed by mixing the Gibson assembly reagent and the calculated number of moles of each of the gene fragments, followed by incubation at 50° C. for 1 hour.

The CA14-0011 strain was transformed with the obtained vector pDZ-ΔNcgl1108::PlysCm1_hisEG-PgapA_hisA-impA-hisFI-SPL13_HisDCB-CJ7_cg0911-hisN by electroporation and subjected to a second crossover process to obtain a strain in which the biosynthesis genes were inserted. These genetic manipulations were confirmed by performing PCR and genomic sequencing using primers of SEQ ID NO: 46 and 47, respectively, amplifying the outer regions of the upstream and downstream homologous recombination regions into which the gene was inserted, and the transformed strain was named CA14-0737.

Strain CA14-0737 was deposited with the Korea Microorganism Culture Center (KCCM) under the Budapest Treaty and was assigned accession number KCCM 12411P on November 27, 2018.

Example 8-2 Preparation of a His-Producing Corynebacterium glutamicum Strain Introduced with a Foreign serA(Avn) Gene Derived from Azotobacter

To identify the effect of the Azotobacter-derived serA(Avn) gene on increasing L-histidine production, strain CA14-0737 was used.

The strain was obtained by replacing the serA(Cgl) gene with the Azotobacter-derived serA(Avn) gene for expression under the gapA promoter using pDZ-PgapA-serA(Avn) obtained in Example 7-3.

The L-histidine-producing Corynebacterium glutamicum CA14-0737 strain was transformed with the pDZ-PgapA-serA(Avn) vector by electroporation and subjected to a second crossing-over process to obtain a strain in which the serA(Cgl) gene was replaced by the serA gene of Azotobacter expressed under strong gapA promoter. These genetic manipulations were confirmed by performing PCRh genomic sequencing using primers of SEQ ID NO: 34 and 35, respectively, amplifying the outer regions of the upstream and downstream homologous recombination regions into which the gene was inserted, and the resulting as a result, the strain was named CA14-0738.

Example 8-3 Evaluation of an L-Histidine Producing Strain of Corynebacterium glutamicum Introduced with a Foreign Gene serA(Avn) Derived from Azotobacter

The strains CA14-0011, CA14-0736, CA14-0737 and CA14-0738 obtained in Examples 8-1 and 8-2 above were cultured according to the method described below to identify the ability to produce L-histidine. Each of the strains was inoculated into a 250 ml baffled flask containing 25 ml of seed medium and cultured at 30° C. for 20 hours with shaking at 200 rpm. Then, 1 ml of seed medium was inoculated into a 250 ml baffled flask containing 25 ml of production medium, and cultured at 30° C. for 24 hours with shaking at 200 rpm. After completion of cultivation, L-histidine production was measured by HPLC.

<Inoculation Medium (pH 7.0)>

20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH ₂ PO ₄ , 8 g K ₂ HPO ₄ , 0.5 g MgSO ₄ ⋅7H ₂ O, 100 µg biotin, 1000 µg thiamine HCl, 2000 mcg of calcium pantothenate and 2000 mcg of nicotinamide (per 1 liter of distilled water).

<Production medium (pH 7.0)>

100 g glucose, 40 g (NH ₄ ) ₂ SO ₄ , 3 g yeast extract, 1 g KH ₂ PO ₄ , 1 g MgSO ₄ ⋅7H ₂ O, 0.01 g FeSO ₄ ⋅7H ₂ O, 50 μg biotin, 100 μg of thiamine and 30 g of CaCO ₃ (per 1 liter of distilled water).

The results of evaluation of L-histidine production by L-histidine producing strains of Corynebacterium glutamicum are shown in Table 24 above.

Whereas the parental strain CA14-0737, which has enhanced histidine production capacity, produced L-histidine 4.09 g/L, strain CA14-0738, in which serA(Avn) was introduced, produced L-histidine 5.07 g/l, which indicates an increase in the production of L-histidine by 20% compared with the parental strain CA14-0737.

Based on these results, it was confirmed that the ability to produce L-histidine was enhanced by the administration of serA(Avn) originating from Azotobacter. Strain CA14-0738 was deposited with the Korea Microorganism Culture Center (KCCM) under the Budapest Treaty and was assigned accession number KCCM 12412P on November 27, 2018.

Example 9 Preparation and Evaluation of a Methionine (Met) Producing Strain Introduced with serA Azotobacter

Example 9-1: Preparation of a recombinant mcbR gene deletion vector

To obtain a methionine-producing strain, the ATCC13032 strain was used to obtain a vector for inactivating the mcbR gene encoding a methionine/cysteine transcription regulator (J. Biotechnol. 103:51 65, 2003).

In particular, for deleting the mcbR gene from the chromosome of Corynebacterium glutamicum strain ATCC13032, a recombinant plasmid vector was obtained in accordance with the method described below. Based on the nucleotide sequences deposited in the GenBank of the US National Institutes of Health (NIH), the mcbR gene and flanking sequences of Corynebacterium glutamicum (SEQ ID NO: 91) were obtained.

To obtain the deleted mcbR gene, PCR was performed using Corynebacterium glutamicum ATCC13032 chromosomal DNA as template and primers of SEQ ID NOS: 64, 65, 66 and 67. PCR was performed under the following amplification conditions: denaturation at 95° C. for 5 minutes; 30 cycles of denaturation at 95°C for 30 seconds, annealing at 53°C for 30 seconds and polymerization at 72°C for 30 seconds; and polymerization at 72°C for 7 minutes. As a result, DNA fragments of 700 bp were obtained. respectively.

The pDZ vector (Korean Patent No. 10-0924065) unable to replicate in Corynebacterium glutamicum and the amplified mcbR gene fragments were treated with SmaI restriction enzyme to introduce into the chromosome and ligated using DNA ligase. E. coli DH5α was transformed with this vector and plated on LB solid medium containing 25 mg/l kanamycin. Colonies were selected that were transformed with a vector into which a fragment having a deletion of the target gene was inserted. A plasmid was then obtained by plasmid isolation and named pDZ-ΔmcbR.

The primer sequences used in this example are shown in Table 24 below.

Example 9-2: Preparation of a recombinant vector in which metH and cysI are both enhanced

To obtain a methionine-producing strain, the ATCC13032 strain was used to obtain a vector in which both the metH gene (Ncgl1450) encoding methionine synthase and the cysI gene (Ncgl2718) encoding sulfite reductase, well known in the art, were amplified.

Specifically, for additional insertion of the metH and cysI genes into the chromosome of Corynebacterium glutamicum strain ATCC13032, a recombinant plasmid vector was obtained in accordance with the method described below. Based on the nucleotide sequences deposited with the US National Institutes of Health (NIH) GenBank, the metH gene and flanking sequences (SEQ ID NO: 92) and the cysI gene and flanking sequences (SEQ ID NO: 93) of Corynebacterium glutamicum were obtained.

First, a vector for the removal of Ncgl1021 (transposase) was obtained to insert these genes. Based on the nucleotide sequences deposited in the GenBank of the US National Institutes of Health (NIH), the Ncgl1021 gene and flanking sequences (SEQ ID NO: 94) of Corynebacterium glutamicum were obtained. To obtain the deleted Ncgl1021 gene, PCR was performed using Corynebacterium glutamicum ATCC13032 chromosomal DNA as template and primers of SEQ ID NOS: 68, 69, 70, and 71. PCR was performed under the following amplification conditions: denaturation at 95°C for 5 minutes; 30 cycles of denaturation at 95°C for 30 seconds, annealing at 53°C for 30 seconds and polymerization at 72°C for 30 seconds; and polymerization at 72°C for 7 minutes. As a result, DNA fragments were obtained. The pDZ vector unable to replicate in Corynebacterium glutamicum (Korean Patent No. 10-0924065) and amplified Ncgl1021 gene fragments were treated with XbaI restriction enzyme to introduce into the chromosome and cloned by the Gibson assembly method. E. coli DH5α was transformed with this vector and plated on LB solid medium containing 25 mg/l kanamycin. Colonies were selected that were transformed with a vector into which a fragment having a deletion of the target gene was inserted. A plasmid was then obtained by plasmid isolation and named pDZ-ΔNcgl1021.

Subsequently, to obtain the metH and cysI genes, PCR was performed using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template and primers with SEQ ID NOs: 72, 73, 74 and 75. Additionally, the Pcj7 promoter was used to enhance the expression of the metH gene, and to enhance the expression cysI gene, the Pspl1 promoter was used. To obtain these genes, the Pcj7 promoter was first obtained by performing PCR using Corynebacterium ammoniagenes chromosomal DNA as template ATCC 6872 and primers of SEQ ID NOs: 76 and 77, and the Pspl1 promoter was obtained by performing PCR using spl1 vector DNA as template. -GFP known in the art (Korean Patent No. 10-1783170) and primers of SEQ ID NOs: 78 and 79. PCR was performed under the following amplification conditions: denaturation at 95°C for 5 minutes; 30 cycles of denaturation at 95°C for 30 seconds, annealing at 53°C for 30 seconds and polymerization at 72°C for 30 seconds; and polymerization at 72°C for 7 minutes. As a result, DNA fragments of the metH gene, cysI gene, Pcj7 promoter, and Pspl1 promoter were obtained.

After that, the vector pDZ-ΔNcgl1021, unable to replicate in Corynebacterium glutamicum, was treated with the restriction enzyme SeaI and 4 amplified DNA fragments were treated with the restriction enzyme SeaI and cloned by the Gibson assembly method. E. coli DH5α was transformed with this vector and plated on LB solid medium containing 25 mg/l kanamycin. Colonies were selected that were transformed with a vector into which a fragment having a deletion of the target gene was inserted. Then a plasmid was obtained by plasmid isolation and named pDZ-ΔNcgl1021-Pcj7metH-Pspl1cysI.

The primer sequences used in this example are shown in Table 25 below.

Example 9-3: Development of an L-methionine producing strain and production of L-methionine using this strain

The ATCC13032 strain was transformed with each of the pDC-ΔmcBR, pDZ-ΔNcgl1021 and pDZ-ΔNcgl1021-Pcj7metH-Pspl1cysI vectors obtained as described above by electroporation via chromosomal homologous recombination (van der Rest et al., Appl Microbiol Biotechnol 52:541 545 , 1999). Then a second recombination was performed on a solid medium containing sucrose. After completion of the second recombination, the transformed strain of Corynebacterium glutamicum having the deletion of the mcBR gene was identified by performing PCR using primers of SEQ ID NOs: 80 and 81, and the transformed strain having the deletion of the Ncgl1021 gene and the insertion of the Pcj7-metH-Pspl1cysI gene at the Ncgl1021 site, identified by performing PCR using primers of SEQ ID NOS: 82 and 83. Each of these recombinant strains was named - Corynebacterium glutamicum 13032/ΔmcbR, 13032/ΔNcgl1021 and 13032/ΔNcgl102l-Pcj7metH-Pspl1cysI.

The primer sequences used in this example are shown in Table 26 below.

To assess the ability to produce L-methionine obtained strains 13032/ΔmcbR, 13032/ΔNcgl1021 and CJP13032/ΔNcgl1021-Pcj7metH-Pspl1cysI these strains and the parent strain Corynebacterium glutamicum ATCC13032 were cultivated in accordance with the following method.

Each of the strains of Corynebacterium glutamicum ATCC13032 and Corynebacterium glutamicum 13032/ΔmcbR, 13032/ΔNcgl1021 and 13032/ΔNcgl1021-Pcj7metH-Pspl1cysI no of the present invention was inoculated into a 250 ml baffled flask, containing 25 ml of the seed medium described below and cultured at 30°C for 20 hours with shaking at 200 rpm. Then, 1 ml of inoculum was inoculated into a 250 ml baffled flask containing 24 ml of production medium, and cultured at 30° C. for 48 hours with shaking at 200 rpm. The seed and production medium compositions are described below.

<Inoculation Medium (pH 7.0)>

20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH ₂ PO ₄ , 8 g K ₂ HPO ₄ , 0.5 g MgSO ₄ ⋅7H ₂ O, 100 µg biotin, 1000 µg thiamine HCl, 2000 mcg of calcium pantothenate and 2000 mcg of nicotinamide (per 1 liter of distilled water).

<Production medium (pH 8.0)>

50 g glucose, 12 g (NH ₄ ) ₂ SO ₂ , 5 g yeast extract, 1 g KH ₂ PO ₄ , 1.2 g MgSO ₄ ⋅7H ₂ O, 100 µg biotin, 1000 µg thiamine HCl, 2000 µg calcium pantothenate , 3000 μg of nicotinamide and 30 g of CaCO ₃ (per 1 liter of distilled water).

The concentrations of L-methionine contained in the cultures obtained by culturing strains in accordance with the method described above were analyzed and shown in Table 27 below.

As a result, it was confirmed that the strain in which the mcbR gene was deleted alone showed the production of L-methionine 0.12 g/L, indicating an increase compared to the control strain. In addition, the strain with overexpression of the metH and cysI genes without deletion of mcBR showed production of L-methionine 0.18 g/l, indicating an increase compared to the control strain.

Example 9-4 Preparation of a Vector Overexpressing the Azotobacter-Derived D-3-Phosphoglycerate Dehydrogenase Gene (serA(Avn))

An expression vector was generated to identify whether the ability to produce methionine is improved by amplifying the Azotobacter-derived D-3-phosphoglycerate dehydrogenase gene (hereinafter referred to as serA(Avn)).

The pECCG117 shuttle vector (Biotechnology letters vol 13, No. 10, p.721-726 1991 or Korean Patent Publication No. 92-7401) was used to express the serA(Avn) gene (SEQ ID NO: 1) encoding SerA(Avn) available for transformation by Corynebacterium glutamicum. The spl1 promoter (hereinafter Pspl1) was used as an expression promoter to obtain the pECCG117-Pspl1-serA(Avn) vector. PCR for Pspl1 was performed using primers SEQ ID NOS: 84 and 85, and PCR for foreign serA(Avn) was performed using primers SEQ ID NOS: 86 and 87. Amplified Pspl1 and serA(Avn) gene fragments were cloned by the Gibson assembly method using the pECCG117 vector treated with EcoRV restriction enzyme, resulting in pECCG117-Pspl1-serA(Avn).

The primer sequences used in this example are shown in Table 28 below.

Example 9-5 Preparation of an L-methionine-producing strain in which serA(Avn) originating from Azotobacter has been introduced using a wild-type E. coli strain and evaluation of the ability to produce L-methionine

Strains 13032/ΔmcbR and 13032/ΔNcgl1021-Pcj7metH-Pspl1cysI were transformed with the above-described pECCG117-Pspl1-serA(Avn) vector by electroporation, respectively (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). The recombinant strains were named Corynebacterium glutamicum 13032/ΔmcbR (pECCG117-Pspl1-serA(Avn)) and 13032/ΔNcgl1021-Pcj7metH-Pspl1cysI (pECCG117-Pspl1-serA(Avn)), respectively.

To evaluate the ability of the obtained recombinant strains 13032/ΔMCBR (PeccG117-PSPL1-Sera (AVN)) and 13032/Δncgl1021-PCJ7MEMEMETH-PSPL1CYSI (PECCG117-PSPL1-SERA (AVN)) to produce L-METION, these strains and their parental strains (13032 /ΔmcbR and 13032/ΔNcgl1021-Pcj7metH-Pspl1cysI) were cultured according to the following method.

Each of the strains of Corynebacterium glutamicum ATCC13032 and the resulting strains of Corynebacterium glutamicum 13032/ΔmcbR, 13032/ΔNcgl1021 and 13032/ΔNcgl1021-Pcj7metH-Pspl1cysI, in accordance with the description of the present invention, was inoculated into a flask with a corner baffle with a volume 250 ml containing 25 ml of the inoculum described below and cultured at 30°C for 20 hours with shaking at 200 rpm. Then, 1 ml of inoculum was inoculated into a 250 ml baffled flask containing 24 ml of production medium, and cultured at 30° C. for 48 hours with shaking at 200 rpm. In particular, the strains in which the vector was included were cultivated after additional addition of kanamycin (25 mg/l) to them. The seed and production medium compositions are described below.

<Inoculation Medium (pH 7.0)>

20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH ₂ PO ₄ , 8 g K ₂ HPO ₄ , 0.5 g MgSO ₄ ⋅7H ₂ O, 100 µg biotin, 1000 µg thiamine HCl, 2000 mcg of calcium pantothenate and 2000 mcg of nicotinamide (per 1 liter of distilled water).

<Production medium (pH 8.0)>

50 g glucose, 12 g (NH ₄ ) ₂ SO ₂ , 5 g yeast extract, 1 g KH ₂ PO ₄ , 1.2 g MgSO ₄ ⋅7H ₂ O, 100 µg biotin, 1000 µg thiamine HCl, 2000 µg calcium pantothenate , 3000 μg of nicotinamide and 30 g of CaCO ₃ (per 1 liter of distilled water).

The concentrations of L-methionine contained in the culture obtained by culturing strains in accordance with the method described above were analyzed and shown in Table 29 below.

As a result, it was confirmed that both strains transformed with pECCG117-Pspl1-serA(Avn) showed an increase in L-methionine production compared to the control strain. In addition, strain 13032/ΔmcbR (pECCG117-Pspl1-serA(Avn)) showed an increase in L-methionine production by 83% compared to the control strain, and strain 13032/ΔNcgl1021-Pcj7metH-Pspl1cysI pECCG117-Pspl1-serA(Avn) ) showed a 78% increase in L-methionine production compared to the control strain. Thus, according to this example, it was confirmed that the ability of microorganisms to produce L-methionine was improved by introducing serA(Avn) originating from Azotobacter into them.

Strain 13032/ΔmcbR was named CM02-0618 and deposited with the Korea Center for Culture of Microorganisms (KCCM) under the Budapest Treaty and was assigned accession number KCCM12425P on January 4, 2019. In addition, strain 13032/ΔmcbR (pECCG117-Pspl1-serA( Avn)) was named CM02-0693 and deposited with the Korea Culture Center for Microorganisms (KCCM) under the Budapest Treaty and was assigned accession number KCCM12413P on November 27, 2018.

Although the description of the present invention has been made with reference to specific illustrative embodiments, those skilled in the art to which the description of the present invention pertains will appreciate that the present invention may be embodied in other specific forms without deviating from the technical spirit or essential characteristics of the present invention. Thus, the embodiments described above are considered to be illustrative and in all respects non-limiting. Furthermore, the scope of the present invention is to be determined by the appended claims and not the detailed description thereof, and it is to be understood that all modifications or variations derived from the meaning and scope of the present invention and their equivalents are included within the scope of the present invention.

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<110> CJ CheilJedang Corporation

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Gly Thr Val Val Asp Ile Asp Ala Leu Ala Ala Ala Leu Arg Asp Lys

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His Leu Asn Gly Ala Ala Ile Asp Val Phe Pro Val Glu Pro Arg Ser

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<213> Artificial Sequence

<220>

<223> primer

<400> 6

aatttcacac aggaaagata tcatggcaaa ggtatcgctg 40

<210> 7

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 7

gtgaattcga gctcggtacc cttagtacag cagacgggcg 40

<210> 8

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 8

cctcaccacg ttgcgtctcg agtcagaaca gaacccgtga 40

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 9

ctcgagacgc aacgtggtga 20

<210> 10

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 10

agtgaattcg agctcggtac ccttaaccgt gacggcgttc 40

<210> 11

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 11

ctcaccacgt tgcgtctcga gttagtacag cagacgggcg 40

<210> 12

<211> 70

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 12

tgcaatgcat aacaacgcag tcgcactatt tttcactgga gagaagccct gtgtaggctg 60

gagctgcttc 70

<210> 13

<211> 70

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 13

tgcaatgcat aacaacgcag tcgcactatt tttcactgga gagaagccct gtccatatga 60

atatcctcct 70

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 14

gggcaggatc tcctgtcatc 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 15

aaatgtcgga taaggcaccg 20

<210> 16

<211> 70

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 16

tgtaatattc acagggatca ctgtaattaa aataaatgaa ggattatgta gtgtaggctg 60

gagctgcttc 70

<210> 17

<211> 70

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 17

tgtagggtaa gagagtggct aacatcctta tagccactct gtagtattaa gtccatatga 60

atatcctcct 70

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 18

acatccttat agccactctg 20

<210> 19

<211> 70

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 19

tacaaccggg ggaggcattt tgcttccccc gctaacaatg gcgacatatt gtgtaggctg 60

gagctgcttc 70

<210> 20

<211> 70

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 20

gcattcggtg cacgatgcct gatgcgccac gtcttatcag gcctacaaaa gtccatatga 60

atatcctcct 70

<210> 21

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 21

aggacggata aggcgttcac 20

<210> 22

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 22

gaattcatgc aaacacaaaa accgac 26

<210> 23

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 23

gaattctcag aaagtctcct gtgca 25

<210> 24

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 24

cgcttatcgc gacaattcca ccgcgctttt tcaccag 37

<210> 25

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 25

ctggtgaaaa agcgcggtgg aattgtcgcg ataagcg 37

<210> 26

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 26

tcgagctcgg taccgggaag atctagtcgg atacg 35

<210> 27

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 27

tcgtttttag gcctccgact actttgggca atcct 35

<210> 28

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 28

tctgttctga ttagagatcc atttgcttga ac 32

<210> 29

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 29

ctctagagga tcccctcacc cagctcaaag ctgat 35

<210> 30

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 30

tgcccaaagt agtcgggaggc ctaaaaacga ccgag 35

<210> 31

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 31

tcttactcat gttgtgtctc ctctaaag 28

<210> 32

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 32

gagacacaac atgagtaaga cctccctg 28

<210> 33

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 33

ggatctctaa tcagaacaga acccgtgag 29

<210> 34

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 34

accaagagtt cgaagaccag 20

<210> 35

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 35

ttcagtggct tccacatcgc 20

<210> 36

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 36

tcgagctcgg tacccatcgc catctacgtt gctgg 35

<210> 37

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 37

gtgccagtgg ggatacctgt gggtgggata agcctggggt tactg 45

<210> 38

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 38

aaccccaggc ttatcccacc cacaggtatc cccactggca cgcga 45

<210> 39

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 39

ctctagagga tccccgggac gtggttgatg gtggt 35

<210> 40

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 40

atggaaatcc tcgccgaagc 20

<210> 41

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 41

atcgatgggg aactgatcca 20

<210> 42

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 42

tcgagctcgg tacccatcgc catctacgtt gctgg 35

<210> 43

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 43

gagtctagaa gtactcgaga tgctgacctc gtttc 35

<210> 44

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 44

agcatctcga gtacttctag actcgcacga aaaag 35

<210> 45

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 45

ctctagagga tccccttttgg gcagagctca aattc 35

<210> 46

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 46

agtttcgtaa cccaccttgc 20

<210> 47

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 47

cgcttctcaa tctgatgaga 20

<210> 48

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 48

gtcagcatct cgagtgctcc ttagggagcc atctt 35

<210> 49

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 49

gtcaaatgtc ttcacatgtg tgcacctttc gatct 35

<210> 50

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 50

gaaaggtgca cacatgtgaa gacatttgac tcgct 35

<210> 51

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 51

tcgtttttag gcctcctaga tgcgggcgat gcgga 35

<210> 52

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 52

atcgcccgca tctaggaggc ctaaaaacga ccgag 35

<210> 53

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 53

gacagttttg gtcatgttgt gtctcctcta aagat 35

<210> 54

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 54

tagaggagac acaacatgac caaaactgtc gccct 35

<210> 55

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 55

tgaagcgccg gtaccgctta cagcaaaacg tcatt 35

<210> 56

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 56

cgttttgctg taagcggtac cggcgcttca tgtca 35

<210> 57

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 57

agtgacattc aacattgttt tgatctcctc caata 35

<210> 58

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 58

gaggagatca aaacaatgtt gaatgtcact gacct 35

<210> 59

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 59

cgctgggatg ttttctctaga gcgctccctt agtgg 35

<210> 60

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 60

aagggagcgc tctagagaaa catcccagcg ctact 35

<210> 61

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 61

agtcatgcct tccatgagtg tttccttttcg ttggg 35

<210> 62

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 62

cgaaaggaaa cactcatgga aggcatgact aatcc 35

<210> 63

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 63

cgagtctaga agtgcctatt ttaaacgatc cagcg 35

<210> 64

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 64

tcgagctcgg tacccctgcc tggtttgtct tgta 34

<210> 65

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 65

cggaaaatga agaaagttcg gccacgtcct ttcgg 35

<210> 66

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 66

aggacgtggc cgaactttct tcattttccg aaggg 35

<210> 67

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 67

ctctagagga tccccgtttc gatgcccact gagca 35

<210> 68

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 68

acccggggat cctctagaat gtttgtgatg cgcag 35

<210> 69

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 69

gtcagagagt acttacgctg atcgggaggg aaagc 35

<210> 70

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 70

atcagcgtaa gtactctctg actagcgtca ccctc 35

<210> 71

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 71

ctgcaggtcg actctagaaa agggattgga gtgtt 35

<210> 72

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 72

caacgaaagg aaacaatgtc tacttcagtt acttc 35

<210> 73

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 73

tcgagctcgg tacccctgcg acagcatgga actc 34

<210> 74

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 74

atcaaaacag atatcatgac aacaaccacc ggaag 35

<210> 75

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 75

cgctagtcag agagttcaca ccaaatcttc ctcag 35

<210> 76

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 76

ccgatcagcg taagtagaaa catcccagcg ctact 35

<210> 77

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 77

aactgaagta gacattgttt cctttcgttg ggtac 35

<210> 78

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 78

tactttaacg tctaaggtac cggcgcttca tgtca 35

<210> 79

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 79

ggtggttgtt gtcatgatat ctgttttgat ctcct 35

<210> 80

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 80

aatctggatt tccgccaggt 20

<210> 81

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 81

cttcctaact cctgaggaag 20

<210> 82

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 82

atccccatcg gcatctttat 20

<210> 83

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 83

cgatcacact gggctgatct 20

<210> 84

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 84

atcgataagc ttgatggtac cggcgcttca tgtca 35

<210> 85

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 85

ggaggtctta ctcatgatat ctgttttgat ctcct 35

<210> 86

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 86

atcaaaacag atatcatgag taagacctcc ctgga 35

<210> 87

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 87

ctgcaggaat tcgattcaga acagaacccg tgagc 35

<210> 88

<211> 281

<212> PRT

<213> Unknown

<220>

<223> polypeptide

<400> 88

Met Leu Lys Ile Ala Val Pro Asn Lys Gly Ser Leu Ser Glu Arg Ala

1 5 10 15

Met Glu Ile Leu Ala Glu Ala Gly Tyr Ala Gly Arg Gly Asp Ser Lys

20 25 30

Ser Leu Asn Val Phe Asp Glu Ala Asn Asn Val Glu Phe Phe Phe Leu

35 40 45

Arg Pro Lys Asp Ile Ala Ile Tyr Val Ala Gly Gly Gln Leu Asp Leu

50 55 60

Gly Ile Thr Gly Arg Asp Leu Ala Arg Asp Ser Gln Ala Asp Val His

65 70 75 80

Glu Val Leu Ser Leu Gly Phe Gly Ser Ser Thr Phe Arg Tyr Ala Ala

85 90 95

Pro Ala Asp Glu Glu Trp Ser Ile Glu Lys Leu Asp Gly Lys Arg Ile

100 105 110

Ala Thr Ser Tyr Pro Asn Leu Val Arg Asp Asp Leu Ala Ala Arg Gly

115 120 125

Leu Ser Ala Glu Val Leu Arg Leu Asp Gly Ala Val Glu Val Ser Ile

130 135 140

Lys Leu Gly Val Ala Asp Ala Ile Ala Asp Val Val Ser Thr Gly Arg

145 150 155 160

Thr Leu Arg Gln Gln Gly Leu Ala Pro Phe Gly Glu Val Leu Cys Thr

165 170 175

Ser Glu Ala Val Ile Val Gly Arg Lys Asp Glu Lys Val Thr Pro Glu

180 185 190

Gln Gln Ile Leu Leu Arg Arg Ile Gln Gly Ile Leu His Ala Gln Asn

195 200 205

Phe Leu Met Leu Asp Tyr Asn Val Asp Arg Asp Asn Leu Asp Ala Ala

210 215 220

Thr Ala Val Thr Pro Gly Leu Ser His Pro Gln Val Ser Pro Leu Ala

225 230 235 240

Arg Asp Asn Trp Val Ala Val Arg Ala Met Val Pro Arg Arg Ser Ala

245 250 255

Asn Ala Ile Met Asp Lys Leu Ala Gly Leu Gly Ala Glu Ala Ile Leu

260 265 270

Ala Ser Glu Ile Arg Ile Ala Arg Ile

275 280

<210> 89

<211> 10676

<212> DNA

<213> Unknown

<220>

<223> polynucleotide

<400> 89

gctccttagg gagccatctt ttggggtgcg gagcgcgatc cggtgtctga cccacggtgcc 60

ccatgcgatt gttaatgccg atgctagggc gaaaagcacg gcgagcagat tgctttgcac 120

ttgattcagg gtagttgact aaagagttgc tcgcgaagta gcacctgtca cttttgtctc 180

aaatattaaa tcgaatatca atatatggtc tgtttattgg aacgcgtccc agtggctgag 240

acgcatccgc taaagcccca ggaaccctgt gcagaaagaa aacactcctc tggctaggta 300

gacacagttt attgtggtag agttgagcgg gtaactgtca gcacgtagat cgaaaggtgc 360

acacatgtga agacatttga ctcgctgtac gaagaacttc ttaaccgtgc tcagacccgc 420

cctgaagggt ctggaaccgt ggccgccttg gataaaggca tccatcatct aggtaagaag 480

gtcatcgaag aagccggaga ggtctggatt gcagccgagt atgagaccga tgaagagcta 540

gccggagaaa tctcccagct catttattgg acccaggtca tcatggttgc tcgcggcctg 600

aagccagaag atatctacaa gaacctgtag gagttttaaa gcaatcatgt tgaaaatcgc 660

tgtcccaaac aaaggctcgc tgtccgagcg cgccatggaa atcctcgccg aagcaggcta 720

cgcaggccgt ggagattcca aatccctcaa cgtttttgat gaagcaaaca acgttgaatt 780

cttcttcctt cgccctaaag atatcgccat ctacgttgct ggtggccagc tcgatttggg 840

tatcaccggc cgcgaccttg ctcgcgattc ccaggctgat gtccacgaag ttctttccct 900

cggcttcggt tcctccactt tccgttacgc agcaccagct gatgaagagt ggagcatcga 960

aaagctcgac ggcaagcgca tcgctacctc ttaccccaac cttgttcgcg atgacctcgc 1020

agcacgtggg ctttccgctg aggtgctccg cctcgacggt gcagtagagg tatccatcaa 1080

gcttggtgtc gcagatgcca tcgccgatgt tgtatccacc ggccgcacgc tgcgtcagca 1140

aggtcttgca cctttcggcg aggttctgtg cacctctgag gctgtcattg ttggccgcaa 1200

ggatgaaaag gtcaccccag agcagcagat cctgcttcgc cgcatccagg gaattttgca 1260

cgcgcagaac ttcctcatgc tggattacaa cgtcgaccgc gacaacctgg acgctgccac 1320

tgcagtaacc ccaggcttat cccacccaca ggtatcccca ctggcacgcg acaactgggt 1380

tgctgtacgc gccatggtgc cacgcaggtc agctaacgcc atcatggata agcttgctgg 1440

actcggcgct gaagccatcc tggcttctga aatccgcatc gcccgcatct aggaggccta 1500

aaaacgaccg agcctattgg gattaccatt gaagccagtg tgagttgcat cacattggct 1560

tcaaatctga gactttaatt tgtggattca cgggggtgta atgtagttca taattaaccc 1620

cattcgggggg agcagatcgt agtgcgaacg atttcaggtt cgttccctgc aaaaactatt 1680

tagcgcaagt gttggaaatg cccccgtttg gggtcaatgt ccatttttga atgtgtctgt 1740

atgattttgc atctgctgcg aaatctttgt ttccccgcta aagttgagga caggttgaca 1800

1860

cgccatactc gcccttgggt tctgtcagct caagaattct tgagtgaccg atgctctgat 1920

tgacctaact gcttgacaca ttgcatttcc tacaatcttt agaggagaca caacatgacc 1980

aaaactgtcg cccttctcga ctacggatct ggaaaccttc gttctgctca acgcgcacta 2040

gagcgtgccg gtgcagaagt tatcgtgagc tccgatccag aagtttgcac caacgctgat 2100

ggcctcctag ttcctggagt gggcgcattt gatgcctgca tgaagggtttt gaaaaacgtc 2160

ttcggacatc gcattatcgg acagcgtctt gctggtggac gtccagtgat gggtatttgt 2220

gtgggcatgc agatcctgtt cgatgaaggc gatgagcacg gcattaagtc agctggttgc 2280

ggcgagtggc ctggcaaagt ggaacgcctc caagcggaga tcctgcctca catggggtgg 2340

aacacacttg aaatgcctac caactcacca atgtttgagg gaatttcacc tgatgagcgt 2400

ttctacttcg tgcactccta tggtgtgcgc aagtggacgt tggaaaccga cgatctgacc 2460

acgcctccag aggttgtgtg ggcgaagcac gaaaatgatc gttttgtggc agctgtggaa 2520

aacggcacgc tgtgggctac tcaattccac ccagaaaaat caggtgacgc aggcgcacag 2580

ctactgcgaa actggatcaa ctacatctaa cagataggat caatattcat gaccttcact 2640

attcttcctg cagtcgatgt agttaacgga caagcagttc gcctagatca gggcgaggcc 2700

ggcactgaaa agtcttatgg cacccctttg gaatccgcac tgaagtggca ggagcagggt 2760

gcaaagtggt tgcactttgt ggacctggac gcagcgttca accgtggttc caaccatgag 2820

atgatggcgg aaattgtcgg caagctcgat gttgatgtgg agctcactgg cggtatccgt 2880

gatgatgagt ctctggagcg cgcgctggca accggtgcac gtcgtgtaaa cattggtacc 2940

gctgctctgg agaagccaga gtggattgct tctgcgattc aacgctatgg cgagaagatt 3000

3060

gtctccgatg gtggcgatct gtgggaagtt ctcgagcgtt tggattccca aggttgtgca 3120

3180

ctgcgtgagg ttgctgcagc tacagacgca cctatcgtgg catctggtgg aatttctgtt 3240

ttggaagatg ttttggaact agccaagtac caggatgagg gcattgattc cgtcatcatt 3300

3360

ctcggttaat acatggatgc tcgtgggatg ttggccattg cggaggccgt tgtagatgat 3420

gccgaagccc tcttcatgca gggcttcgga gctgcacctg cccatatgaa atccccgggg 3480

gattttgcca cggaagtgga tatggccatc gaatcccata tgcgttcgat gctgaacatg 3540

atgacaggca ttgctgtcat cggtgaagaa ggtggcggtg cgacctccgg cacgcgctgg 3600

gtgattgatc ccatcgacgg caccgccaac ttcgcggcgt ccaacccgat gagcgcgatc 3660

ctggtgtctt tgcttgtcga cgaccagccc gtcctgggta ttacctccat gcccatgctg 3720

ggtaaacgcc tcaccgcttt tgaaggttca ccgctgatga tcaacggtga acctcaggaa 3780

ccattgcaag aacaatccag tttggtatcc cacattggtt ttagttccat ggcctccccg 3840

cgcaatacag cgtttcctgt ggagttgcgt cgggatcttc tgaccgagct cacggaatcg 3900

tatcttcgtc cccgcattac aggttcggtg ggtgttgatc tcgcgttcac tgcgcaggggc 3960

atttttggag catgcgtatc gtttagtcct catgtttgggg acaattccgc aggcgtgatg 4020

ttgatgcgcg ctgctggtgc acaagttact gacaccgaag gccatccgtg ggcaccaggt 4080

aggggagtcg tggccggaac aaaaagggct cacgatgtgc tgttaagtaa gattgaaaaa 4140

gttcggttga tgcatgcaga tgcaggtaat gaccagtcgt taaatgagga gtacaagtaa 4200

aatgggcgtg gcaattcgag ttattccttg cctggacgtg gacaacggcc gggttgttaa 4260

aggcgtgaac tttgaaaacc tccgcgatgc tggcgatcct gtggagttgg caaagcgcta 4320

tgacgaggaa ggggcagatg agctgacctt cctggatgtc accgcctcga agcatggtcg 4380

cggcaccatg ctggatgttg ttcgacgcac cgctgatcag gtgttcatcc ctctgactgt 4440

cggtggcggc gtgcgcagcg aagaagatgt tgatcaattg ctgcgcgctg gcgccgacaa 4500

ggtttcggtg aacacgtctg cgattgcccg tccagaactg ctgtcagagc tgtccaagcg 4560

ttttggtgct cagtgcatcg tgttgtctgt ggatgccagg cgcgttcctg aaggtggaac 4620

tcctcagcca tctggttttg aagtcaccac ccacggcggt tccaagtccg cagaacttga 4680

tgcaatcgag tgggcaaagc gcggcgaaga gctgggcgtt ggcgaaattc tgctcaactc 4740

catggacggc gacggcacca aaaacggctt tgacctagag ctgctggaaa aagttcgcgc 4800

agccgtatcc attcctgtaa tcgcctccgg cggcgctggc aaggcggagc atttcccacc 4860

agctgttgca gctggcgcca acgcagtgct tgccgcgacc attttccact tccgcgaagt 4920

aaccatcgcc gaagtaaagg gagccattaa agatgcagga tttgaggtgc ggaaatgagt 4980

gacaatccac aagagtatga gctggattgg gacgtcgaaa agcgattaaa gcttaacgac 5040

gccggcctgg tgccggcaat cgtccaggcc gacgggacca acgaggtcct catgatggcc 5100

tggatggata cccacgcgct agcctatact ttggcgaccc gccgtggaac ctatttttct 5160

aggtcccgca acgagtactg gatcaagggc ctgacctctg gaaacgtcca agaagtcacc 5220

ggacttgccc tcgactgcga cggcgacacc gtccttctga ccgtgaaaca aaccggcggt 5280

gcgtgccaca ctggtgccca cacatgtttc gacaatgacg ttttgctgta agcggtaccg 5340

gcgcttcatg tcaacaatct ttaacgtttt caagttcaca agtcgtgttc aaatggtgac 5400

aagattggac actgtgctga attggcacca agccctcata aatgatagat ctaaatcgaa 5460

tatcaatata tggtctgttt attggaacgc gtcccagtgg ctgagacgca tccgctaaag 5520

ccccaggaac cctgtgcaga aagaacaaat aatcgtgaat tttggcagca acagcggggc 5580

ctggtataat tgaaaacgtg caaaagcata gattattggga ggagatcaaa acaatgttga 5640

atgtcactga cctgcgaggt caaacaccat ccaagagcga catccgacgt gctttgccac 5700

gtggtggcac tgacgtgtgg tctgtgcttc ccatagtgca gcctgttgta gaagatgtcc 5760

aaaaccgcgg cgctgaagct gctttggatt acggcgagaa gttcgaccat attcgccccg 5820

cctcggtgcg ggtgccagct gaggtttattg ctgcagcaga aaacacctta gatccgttgg 5880

tgcgtgaatc gattgaagag tcgattcgtc gcgtccgcaa ggttcacgct gagcaaaagc 5940

catccgagca caccactgaa ctttcaccag gtggcaccgt cactgagcgt ttcatgccga 6000

ttgatcgcgt gggactgtac gttccaggcg gcaatgcggt gtacccatca agcgtgatta 6060

tgaatactgt cccagctcaa gaggctggtg tgaactccct tgtggttgcg tcgcctcctc 6120

aggctgagca cggtggctgg cctcacccca ccattttggc ggcgtgttcc atcttgggtg 6180

ttgatgaggt gtgggctgtc ggcggcggtc aggccgtggc gttgctggct tatggtgatg 6240

6300

ctgcgaagcg cctggtcagg ggagtggtag gtactgattc tgaggctggc cctacagaaa 6360

tcgctgtgct tgctgatgcc tctgccaacg ccgtcaacgt tgcctacgat ctgatcagcc 6420

aagcagaaca cgatgtcatg gctgcgtccg tgctcatcac tgactccgag cagcttgcca 6480

aggacgtaaa cagggaaatc gaggcgcgtt actcaatcac gcgcaacgcc gagcgcgtcg 6540

cagaagcttt gcgcggggcc cagagtggca tcgtgcttgt cgacgacatt tccgtgggta 6600

6660

ccgtagcaga gcagatcacc aacgcgggtg cgatcttcgt gggcgatttc tcaccagtac 6720

cactgggtga ttactccgca ggatccaacc acgtgctgcc aacctctgga tccgctcgtt 6780

tctccgcagg tctatccacg cacacgttcc ttcgcccagt caacctcatt gaatacgatg 6840

aggctgctct gaaggacgtc tcgcaggttg tcatcaactt tgccaacgcc gaagatcttc 6900

cagcgcacgg cgaagcaatc cgtgcacgct ttgaaaacct ccccaccacc gacgaggcct 6960

aagaaaaatg accaaaatta ctttgagcga tttgccattg cgtgaagaac tgcgcggtga 7020

gcacgcttac ggcgcacccc agctcaacgt tgatattcgc ctcaacacca acgaaaaccc 7080

ttacccaccg tcagaggcat tggtcgctga cttggttgcc accgtggata agatcgccac 7140

cgagctgaac cgctacccag agcgcgatgc tgtggaactg cgtgatgagt tggctgcgta 7200

catcaccaag caaaccggcg tggctgtcac cagggataac ctgtgggctg ccaatggttc 7260

caatgaaatt ctgcagcagc tgctgcaggc ttttggtgga cctggacgca ccgcgttggg 7320

attccaaccc agctattcca tgcacccaat tttggctaaa ggcacccaca ctgaattcat 7380

tgcggtgtcc cgaggtgctg atttccgcat cgatatggat gtggcgctgg aagaaattcg 7440

tgcaaagcag cctgacattg tttttgtcac caccccgaac aacccgaccg gtgatgtgac 7500

ctcgctggac gatgttgagc gcatcatcaa cgttgcccca ggcatcgtga tcgtggatga 7560

agcttatgcg gaattctccc catcaccttc agcaaccact cttctggaga agtacccaac 7620

caagctggtg gtgtcccgca ccatgagtaa ggcttttgat ttcgcaggtg gacgcctcgg 7680

ctacttcgtg gccaacccag cgtttatcga cgccgtgatg ctagtccgcc ttccgtatca 7740

tctttcagcg ctgagccaag cagccgcaat cgtagcgctg cgtcactccg ctgacacgct 7800

gggaaccgtc gaaaagctct ctgtagagcg tgttcgcgtg gcagcacgct tggaggaact 7860

gggctacgct gtggttccaa gtgagtccaa ctttgtgttc tttggagatt tctccgatca 7920

gcacgcggca tggcaggcat ttttggatag gggagtgctc atccgcgatg tgggaatcgc 7980

tgggcacttg cgcactacca ttggtgtgcc tgaggaaaat gatgcgtttt tggacgcagc 8040

tgcagagatc atcaagctga acctgtaaga gagaagaatt tttcatgact gtcgcaccaa 8100

gaattggtac cgcaacccgc accaccagcg aatccgacat caccgtcgag atcaacctgg 8160

acggcaccgg caaagtagat atcgataccg gcctgccatt tttcgaccac atgctcactg 8220

cattcggcgt gcacggcagt tttgatctga aagtccatgc caagggcgac atcgagatcg 8280

acgcacacca caccgtggaa gataccgcca tcgtgctcgg ccaagcactc cttgacgcta 8340

ttggcgacaa gaaaggcatc cgccgtttcg catcctgcca gctgcccatg gatgaggcat 8400

tagtggagtc cgtggtggat atctccggtc gcccatactt cgtgatctcc ggcgaaccag 8460

accacatgat cacctccgtg atcggtggac actacgcaac cgtgatcaac gagcacttct 8520

ttgaaaccct cgcgctcaac tcccgaatca ccctccacgt gatctgccac tacggccgcg 8580

accctcacca catcaccgaa gcagagtaca aggctgttgc ccgtgcgctg cgcggtgccg 8640

tagagatgga tcctcgtcaa acaggaatcc catccactaa gggagcgctc tagagaaaca 8700

tcccagcgct actaataggg agcgttgacc ttccttccac ggaccggtaa tcggagtgcc 8760

taaaaccgca tgcggcttag gctccaagat aggttctgcg cggccgggta atgcatcttc 8820

tttagcaaca agttgagggg taggtgcaaa taagaacgac atagaaatcg tctcctttct 8880

gtttttaatc aacatacacc accacctaaa aattccccga ccagcaagtt cacagtattc 8940

gggcacaata tcgttgccaa aatattgttt cggaatatca tgggatacgt acccaacgaa 9000

aggaaacact catggaaggc atgactaatc cagagcagac acatcccgct gcaagcctcg 9060

aagacatgat caaaaccatc acaaagacct tcgtgattgc tcacgatcag gattctgatg 9120

agcatcttgc gcaggcactg gtgtacaacg ctggacgttt ggcatggcgc atgcgcgaaa 9180

acggtgtgga tacggattac aagacttctg tgtctgatgt ggtcacggat gccgatcgtg 9240

cggccgaggc cttcgtcgca ggcgttcttg aagcgttgcg gcctgaggac ggcgtgcttg 9300

gcgaggaagg cgcggaccgg gcgtcgaaaa gcggaaaaac ctgggtcatc gacccggttg 9360

atggcaccta caacttcacc cagggctcag attattggtg ctcggcgctc gcgctggtcg 9420

agggcgatcc atccgcgcca tcgcgcgtgc ttttcggcgc cgtacaccgc ccagccatgg 9480

gttatacgtg gttcggtggc ccgggaatcc gcaccacgct cgacggcaag gagctagatt 9540

tgcttgtcga cgcccccctc aatcaaatct ccctggccac ctacatccac ccgtcacgca 9600

tcgcggaacc tgatattcaa aaggcgtgga tgagcgttgc cacccaccct gcaacgctgc 9660

gcatgttcgg cgccggctcc atcgatttgg ccaacatcgc cgacggcagc atcggcgcat 9720

gggtgcagca cagcgtcgca gattgggact ggctacccgg ccgcgcactc atcgaaggcg 9780

tcggcggagc atgcatcaaa gtgaccgccg gcggcgtcga atggtccgtt gcaggaaacg 9840

cggaagcagt tagtgagatc tccgaaactt taagcgcact agactaacaa cacatgagca 9900

aatatgcaga cgatttagcc ttagccctcg aacttgccga acttgccgat tccatcaccc 9960

tcgaccgctt cgaagcctct gacctggaag tatcctccaa gccagacatg actcccgtca 10020

gcgatgccga cctggcgacc gaagaagcac tccgtgagaa aatcgccacc gcccgccccg 10080

ccgactccat cctcggtgaa gaattcggtg gcgacgtaga attcagcggc cgccagtgga 10140

tcatcgaccc catcgacggc accaaaaact acgtccgcgg cgtccccgta tgggcaaccc 10200

tgatcgcgct gctcgacaac ggcaaacccg tcgcaggtgt catctccgca cccgcactgg 10260

ctaggcgttg gtgggcatcc gaaggggccg gcgcatggcg caccttcaac ggcagctccc 10320

cacgcaaact gtccgtgtcc caggtgtcca agcttgacga cgcctccctc tccttctcct 10380

ccctctccgg ctgggccgaa cgagatttgc gcgatcagtt cgtctcccta actgatacca 10440

cctggcgact ccgcggctac ggcgacttct tctcctactg cctcgtcgcc gaaggtgccg 10500

tcgatatcgc cgctgaacca gaagtcagcc tctgggatct tgctcccctg tccatcctgg 10560

tcaccgaagc cggaggaaag ttcacctcac tggctggcgt cgatggacca cacggtggcg 10620

atgcagtagc caccaacggc atcctgcacg atgagacgct ggatcgttta aaatag 10676

<210> 90

<211> 281

<212> PRT

<213> Unknown

<220>

<223> polypeptide

<400> 90

Met Leu Lys Ile Ala Val Pro Asn Lys Gly Ser Leu Ser Glu Arg Ala

1 5 10 15

Met Glu Ile Leu Ala Glu Ala Gly Tyr Ala Gly Arg Gly Asp Ser Lys

20 25 30

Ser Leu Asn Val Phe Asp Glu Ala Asn Asn Val Glu Phe Phe Phe Leu

35 40 45

Arg Pro Lys Asp Ile Ala Ile Tyr Val Ala Gly Gly Gln Leu Asp Leu

50 55 60

Gly Ile Thr Gly Arg Asp Leu Ala Arg Asp Ser Gln Ala Asp Val His

65 70 75 80

Glu Val Leu Ser Leu Gly Phe Gly Ser Ser Thr Phe Arg Tyr Ala Ala

85 90 95

Pro Ala Asp Glu Glu Trp Ser Ile Glu Lys Leu Asp Gly Lys Arg Ile

100 105 110

Ala Thr Ser Tyr Pro Asn Leu Val Arg Asp Asp Leu Ala Ala Arg Gly

115 120 125

Leu Ser Ala Glu Val Leu Arg Leu Asp Gly Ala Val Glu Val Ser Ile

130 135 140

Lys Leu Gly Val Ala Asp Ala Ile Ala Asp Val Val Ser Thr Gly Arg

145 150 155 160

Thr Leu Arg Gln Gln Gly Leu Ala Pro Phe Gly Glu Val Leu Cys Thr

165 170 175

Ser Glu Ala Val Ile Val Gly Arg Lys Asp Glu Lys Val Thr Pro Glu

180 185 190

Gln Gln Ile Leu Leu Arg Arg Ile Gln Gly Ile Leu His Ala Gln Asn

195 200 205

Phe Leu Met Leu Asp Tyr Arg Val Asp Arg Asp Asn Leu Asp Ala Ala

210 215 220

Thr Ala Val Thr Pro Gly Leu Ser His Pro Gln Val Ser Pro Leu Ala

225 230 235 240

Arg Asp Asn Trp Val Ala Val Arg Ala Met Val Pro Arg Arg Ser Ala

245 250 255

Asn Ala Ile Met Asp Lys Leu Ala Gly Leu Gly Ala Glu Ala Ile Leu

260 265 270

Ala Ser Glu Ile Arg Ile Ala Arg Ile

275 280

<210> 91

<211> 2642

<212> DNA

<213> Unknown

<220>

<223> polynucleotide

<400> 91

ctcccgcgca ctgctgcaat ccgcaccgtg cccaatgatg gtggttcgcc cacctgagaa 60

gattaagaag tagtttcttt taagtttcga tgccccggtt tcctgatttt gtgcagggag 120

gccggggcat tggtgtttgc gggttagttc gggccattcg aaagggagaa accaagggca 180

gccagacaga cgtgccaaga atctggattt ccgccaggtt ttggcacgcc cgtctggttt 240

aggcaatgag ataccgaaca cacgtgccaa aagttcggct ttttcgccga tcttgtcacg 300

cctgcctggt ttgtcttgta aagagtgatt tcatggccga gactcctaaa agtttgacct 360

cacaggattg cttctaaggg cctctccaat ctccactgag gtacttaatc cttccgggga 420

attcgggcgc ttaaatcgag aaattaggcc atcacctttt aataacaata caatgaataa 480

ttggaatagg tcgacacctt tggagcggag ccggttaaaa ttggcagcat tcaccgaaag 540

aaaaggagaa ccacatgctt gccctaggtt ggattacatg gatcattatt ggtggtctag 600

ctggttggat tgcctccaag attaaaggca ctgatgctca gcaaggaatt ttgctgaaca 660

720

atgttgccgg tggcggcttg atcttcagct tcatcacatg tctgattggt gctgtcattt 780

tgctgacgat cgtgcagttc ttcactcgga agaagtaatc tgctttaaat ccgtagggcc 840

tgttgatatt tcgatatcaa caggcctttt ggtcattttg gggtggaaaa agcgctagac 900

ttgcctgtgg attaaaacta tacgaaccgg tttgtctata ttggtgttag acagttcgtc 960

gtatcttgaa acagaccaac ccgaaaggac gtggccgaac gtggctgcta gcgcttcagg 1020

caagagtaaa acaagtgccg gggcaaaccg tcgtcgcaat cgaccaagcc cccgacagcg 1080

tctcctcgat agcgcaacca accttttcac cacagaaggt attcgcgtca tcggtattga 1140

tcgtatcctc cgtgaagctg acgtggcgaa ggcgagcctc tattcccttt tcggatcgaa 1200

ggacgccttg gttattgcat acctggagaa cctcgatcag ctgtggcgtg aagcgtggcg 1260

tgagcgcacc gtcggtatga aggatccgga agataaaatc atcgcgttct ttgatcagtg 1320

cattgaggaa gaaccagaaa aagatttccg cggctcgcac tttcagaatg cggctagtga 1380

gtaccctcgc cccgaaactg atagcgaaaa gggcattgtt gcagcagtgt tagagcaccg 1440

cgagtggtgt cataagactc tgactgattt gctcactgag aagaacggct acccaggcac 1500

cacccaggcg aatcagctgt tggtgttcct tgatggtgga cttgctggat ctcgattggt 1560

ccacaacatc agtcctcttg agacggctcg cgatttggct cggcagttgt tgtcggctcc 1620

acctgcggac tactcaattt agtttcttca ttttccgaag gggtatcttc gttgggggag 1680

gcgtcgataa gccccttctt tttagcttta acctcagcgc gacgctgctt taagcgctgc 1740

atggcggcgc ggttcatttc acgttgcgtt tcgcgcctct tgttcgcgat ttctttgcgg 1800

gcctgttttg cttcgttgat ttcggcagta cgggttttgg tgagttccac gtttgttgcg 1860

tgaagcgttg aggcgttcca tggggtgaga atcatcaggg cgcggtttt gcgtcgtgtc 1920

cacaggaaga tgcgcttttc tttttgtttt gcgcggtaga tgtcgcgctg ctctaggtgg 1980

tgcactttga aatcgtcggt aagtgggtat ttgcgttcca aaatgaccat catgatgatt 2040

gtttggagga gcgtccacag gttgttgctg acccaataga gtgcgattgc tgtggggaat 2100

ggtcctgtga ggccaaggga cagtgggaag atcggcgcga ggatcgacat cacgatcatg 2160

aacttcagca tgccgttaga gaatccggat gcgtaatcgt tggtttggaa gctgcggtac 2220

atggacatcg ccatgttgat tgcggtgagg attgcggctg tgatgaacag tggcaaaacg 2280

aaactaagaa cttccgcctg cgtggtgctc aaatatttta gctgctcagt gggcatcgaa 2340

acataagcgg gcagaggcac attgctcacg cgaccagcga ggaaagattc cacttcctca 2400

ggagttagga agccgatcga ctggaagacg ggattttcca aaccaccttc agggcgagcc 2460

atgcggagaa gtgcccagta aagaccaagg acaatcggta tctggatcag cccaggcaca 2520

caacctgcca gcgggttaat gccgtattcc ttattcaaat cattctggcg cttctgcaac 2580

tcccgaatgg acgcttcatc gtactttccc ttgtattctt cccggagcgc agcgcggtga 2640

gg 2642

<210> 92

<211> 5666

<212> DNA

<213> Unknown

<220>

<223> polynucleotide

<400> 92

tgtcatgctt ccggaggtgc gcagggctcg agactccgga aagctatttg ccactccgat 60

gtttgggtca ctcgacgaga tacgtgctga tcacctaatt tggtgcacag ggtttcggcc 120

ggcgattagg ccagttcgtc aacttctcaa acacggacaa ccaaaggttc ctggtcttta 180

tttagtaggc tacggagatt ggacgggacc tgggtctgcg actatcacag gggtcgggct 240

ttatgccaag cgagcagcca aagagattgc cgcgtcagtc ggcaaagtcg ttaaatagtt 300

tgaaggctaa gaacttaatg ttaaagcgaa aattgttttg acacctcaac taatgcagcg 360

atgcgttctt tccagaatgc tttcatgaca gggatgctgt cttgatcagg caggcgtctg 420

tgctggatgc cgaagctgga tttattgtcg cctttggagg tgaagttgac gctcactcga 480

gaatcatcgg ccaaccattt ggcattgaat gttctagggtt cggaggcggga ggttttctca 540

attagtgcgg gatcgagcca ctgcgcccgc aggtcatcgt ctccgaagag cttccacact 600

ttttcgaccg gcaggttaag ggttttggag gcattggccg cgaacccatc gctggtcatc 660

ccgggtttgc gcatgccacg ttcgtattca taaccaatcg cgatgccttg agcccaccag 720

ccactgacat caaagttgtc cacgatgtgc tttgcgatgt gggtgtgagt ccaagaggtg 780

gcttttacgt cgtcaagcaa ttttagccac tcttcccacg gctttccggt gccgttgagg 840

atagcttcag gggacatgcc tggtgttgag ccttgcggag tggagtcagt catgcgaccg 900

agactagtgg cgctttgcct gtgttgctta ggcggcgttg aaaatgaact acgaatgaaa 960

agttcgggaa ttgtctaatc cgtactaagc tgtctacaca atgtctactt cagttacttc 1020

accagcccac aacaacgcac attcctccga atttttggat gcgttggcaa accatgtgtt 1080

gatcggcgac ggcgccatgg gcacccagct ccaaggcttt gacctggacg tggaaaagga 1140

tttccttgat ctggaggggt gtaatgagat tctcaacgac acccgccctg atgtgttgag 1200

gcagattcac cgcgcctact ttgaggcggg agctgacttg gttgagacca atacttttgg 1260

ttgcaacctg ccgaacttgg cggattatga catcgctgat cgttgccgtg agcttgccta 1320

caagggcact gcagtggcta gggaagtggc tgatgagatg gggccggggcc gaaacggcat 1380

gcggcgtttc gtggttggtt ccctgggacc tggaacgaag cttccatcgc tgggccatgc 1440

accgtatgca gatttgcgtg ggcactacaa ggaagcagcg cttggcatca tcgacggtgg 1500

tggcgatgcc tttttgattg agactgctca ggacttgctt caggtcaagg ctgcggttca 1560

cggcgttcaa gatgccatgg ctgaacttga tacattcttg cccattattt gccacgtcac 1620

cgtagagacc accggcacca tgctcatgggg ttctgagatc ggtgccgcgt tgacagcgct 1680

gcagccactg ggtatcgaca tgattggtct gaactgcgcc accggcccag atgagatgag 1740

cgagcacctg cgttacctgt ccaagcacgc cgatattcct gtgtcggtga tgcctaacgc 1800

aggtcttcct gtcctgggta aaaacggtgc agaataccca cttgaggctg aggatttggc 1860

gcaggcgctg gctggattcg tctccgaata tggcctgtcc atggtgggtg gttgttgtgg 1920

caccacacct gagcacatcc gtgcggtccg cgatgcggtg gttggtgttc cagagcagga 1980

aacctccaca ctgaccaaga tccctgcagg ccctgttgag caggcctccc gcgaggtgga 2040

gaaagaggac tccgtcgcgt cgctgtacac ctcggtgcca ttgtcccagg aaaccggcat 2100

ttccatgatc ggtgagcgca ccaactccaa cggttccaag gcattccgtg aggcaatgct 2160

gtctggcgat tgggaaaagt gtgtggatat tgccaagcag caaacccgcg atggtgcaca 2220

catgctggat ctttgtgtgg attacgtggg acgagacggc accgccgata tggcgacctt 2280

ggcagcactt cttgctacca gctccacttt gccaatcatg attgactcca ccgagccaga 2340

ggttattcgc acaggccttg agcacttggg tggacgaagc atcgttaact ccgtcaactt 2400

tgaagacggc gatggccctg agtcccgcta ccagcgcatc atgaaactgg taaagcagca 2460

cggtgcggcc gtggttgcgc tgaccattga tgaggaaggc caggcacgta ccgctgagca 2520

caaggtgcgc attgctaaac gactgattga cgatatcacc ggcagctacg gcctggatat 2580

caaagacatc gttgtggact gcctgacctt cccgatctct actggccagg aagaaaccag 2640

gcgagatggc attgaaacca tcgaagccat ccgcgagctg aagaagctct acccagaaat 2700

ccacaccacc ctgggtctgt ccaatatttc cttcggcctg aaccctgctg cacgccaggt 2760

tcttaactct gtgttcctca atgagtgcat tgaggctggt ctggactctg cgattgcgca 2820

cagctccaag attttgccga tgaaccgcat tgatgatcgc cagcgcgaag tggcgttgga 2880

tatggtctat gatcgccgca ccgaggatta cgatccgctg caggaattca tgcagctgtt 2940

tgagggcgtt tctgctgccg atgccaagga tgctcgcgct gaacagctgg ccgctatgcc 3000

tttgtttgag cgtttggcac agcgcatcat cgacggcgat aagaatggcc ttgaggatga 3060

tctggaagca ggcatgaagg agaagtctcc tattgcgatc atcaacgagg accttctcaa 3120

cggcatgaag accgtgggtg agctgtttgg ttccggacag atgcagctgc cattcgtgct 3180

3240

agcagaagct accggatctg cgcaggcaga gggcaagggc aaaatcgtcg tggccaccgt 3300

3360

ttacgacgtg gtgaacttgg gcatcaagca gccactgtcc gccatgttgg aagcagcgga 3420

agaacacaaa gcagacgtca tcggcatgtc gggacttctt gtgaagtcca ccgtggtgat 3480

3540

tggcgctgcg ctgacgcgta cctacgtgga aaacgatctc aacgaggtgt acaccggtga 3600

3660

aaagcgtggt gaaggacttg atcccaactc accagaagct attgagcagg cgaagaagaa 3720

ggcggaacgt aaggctcgta atgagcgttc ccgcaagatt gccgcggagc gtaaagctaa 3780

tgcggctccc gtgattgttc cggagcgttc tgatgtctcc accgatactc caaccgcggc 3840

accaccgttc tggggaaccc gcattgtcaa gggtctgccc ttggcggagt tcttgggcaa 3900

ccttgatgag cgcgccttgt tcatggggca gtggggtctg aaatccaccc gcggcaacga 3960

gggtccaagc tatgaggatt tggtggaaac tgaaggccga ccacgcctgc gctactggct 4020

ggatcgcctg aagtctgagg gcattttgga ccacgtggcc ttggtgtatg gctacttccc 4080

agcggtcgcg gaaggcgatg acgtggtgat cttggaatcc ccggatccac acgcagccga 4140

acgcatgcgc tttagcttcc cacgccagca gcgcggcagg ttcttgtgca tcgcggattt 4200

cattcgccca cgcgagcaag ctgtcaagga cggccaagtg gacgtcatgc cattccagct 4260

ggtcaccatg ggtaatccta ttgctgattt cgccaacgag ttgttcgcag ccaatgaata 4320

ccgcgagtac ttggaagttc acggcatcgg cgtgcagctc accgaagcat tggccgagta 4380

ctggcactcc cgagtgcgca gcgaactcaa gctgaacgac ggtggatctg tcgctgattt 4440

tgatccagaa gacaagacca agttcttcga cctggattac cgcggcgccc gcttctcctt 4500

tggttacggt tcttgccctg atctggaaga ccgcgcaaag ctggtggaat tgctcgagcc 4560

aggccgtatc ggcgtggagt tgtccgagga actccagctg cacccagagc agtccacaga 4620

cgcgtttgtg ctctaccacc cagaggcaaa gtactttaac gtctaacacc tttgagaggg 4680

aaaactttcc cgcacattgc agatcgtgcc actttaacta aggttgacgg catgattaag 4740

gcgattttct gggacatgga cggcacgatg gtggactctg agccacagtg gggcattgct 4800

acctacgagc tcagcgaagc catgggccgc cgcctcaccc cggagctccg ggaactcacc 4860

gtcggctcga gcctgccgcg caccatgcgc ttatgcgcag agcacgcagg cattacattg 4920

agcgacgcgg actacgagcg ctaccgggct ggcatgttcg cccgggtcca tgagcttttc 4980

gacgaatccc tcgtcccaaa tccaggcgtc accgaactcc tgacagagtt gaaggccctc 5040

gagatcccca tgttggtcac caccaacaca gagcgcgatc tcgcgacccg ttcagtcgca 5100

gccgtgggaa atgagttctt catcggttct atcgctggtg atgaagtccc aacagcaaag 5160

ccagccccg acatgtacct cgaagcagca cgacgtgtgg gctttgaccc atcagagtgc 5220

ctcgtgttcg aagattccta caacggcatg ctgggcgctg ttactgcagg ttgccgcgtc 5280

attggtctgc acccagaaga agtccaagcg ccagaaggtg tagtgccttt gcgttccctc 5340

cacggtaaaa actctttcga aggtgtcacc gctgagatgg tcactgcctg gtaccaccag 5400

atcgagccgg caggtgtcgc aaaataaaac caggtgggggg agtgaaatta ttcgactaat 5460

atcctccccc aaacacacat tgataactgt tgtgtggaag aatgtaccga gtgaagacat 5520

ttgactcgct gtacgaagaa cttcttaacc gtgctcagac ccgccctgaa gggtctggaa 5580

ccgtggccgc cttggataaa ggcatccatc atctaggtaa gaaggtcatc gaagaagccg 5640

gagaggtctg gattgcagcc gagtat 5666

<210> 93

<211> 11332

<212> DNA

<213> Unknown

<220>

<223> polynucleotide

<400> 93

tgtcatgctt ccggaggtgc gcagggctcg agactccgga aagctatttg ccactccgat 60

gtttgggtca ctcgacgaga tacgtgctga tcacctaatt tggtgcacag ggtttcggcc 120

ggcgattagg ccagttcgtc aacttctcaa acacggacaa ccaaaggttc ctggtcttta 180

tttagtaggc tacggagatt ggacgggacc tgggtctgcg actatcacag gggtcgggct 240

ttatgccaag cgagcagcca aagagattgc cgcgtcagtc ggcaaagtcg ttaaatagtt 300

tgaaggctaa gaacttaatg ttaaagcgaa aattgttttg acacctcaac taatgcagcg 360

atgcgttctt tccagaatgc tttcatgaca gggatgctgt cttgatcagg caggcgtctg 420

tgctggatgc cgaagctgga tttattgtcg cctttggagg tgaagttgac gctcactcga 480

gaatcatcgg ccaaccattt ggcattgaat gttctagggtt cggaggcggga ggttttctca 540

attagtgcgg gatcgagcca ctgcgcccgc aggtcatcgt ctccgaagag cttccacact 600

ttttcgaccg gcaggttaag ggttttggag gcattggccg cgaacccatc gctggtcatc 660

ccgggtttgc gcatgccacg ttcgtattca taaccaatcg cgatgccttg agcccaccag 720

ccactgacat caaagttgtc cacgatgtgc tttgcgatgt gggtgtgagt ccaagaggtg 780

gcttttacgt cgtcaagcaa ttttagccac tcttcccacg gctttccggt gccgttgagg 840

atagcttcag gggacatgcc tggtgttgag ccttgcggag tggagtcagt catgcgaccg 900

agactagtgg cgctttgcct gtgttgctta ggcggcgttg aaaatgaact acgaatgaaa 960

agttcgggaa ttgtctaatc cgtactaagc tgtctacaca atgtctactt cagttacttc 1020

accagcccac aacaacgcac attcctccga atttttggat gcgttggcaa accatgtgtt 1080

gatcggcgac ggcgccatgg gcacccagct ccaaggcttt gacctggacg tggaaaagga 1140

tttccttgat ctggaggggt gtaatgagat tctcaacgac acccgccctg atgtgttgag 1200

gcagattcac cgcgcctact ttgaggcggg agctgacttg gttgagacca atacttttgg 1260

ttgcaacctg ccgaacttgg cggattatga catcgctgat cgttgccgtg agcttgccta 1320

caagggcact gcagtggcta gggaagtggc tgatgagatg gggccggggcc gaaacggcat 1380

gcggcgtttc gtggttggtt ccctgggacc tggaacgaag cttccatcgc tgggccatgc 1440

accgtatgca gatttgcgtg ggcactacaa ggaagcagcg cttggcatca tcgacggtgg 1500

tggcgatgcc tttttgattg agactgctca ggacttgctt caggtcaagg ctgcggttca 1560

cggcgttcaa gatgccatgg ctgaacttga tacattcttg cccattattt gccacgtcac 1620

cgtagagacc accggcacca tgctcatgggg ttctgagatc ggtgccgcgt tgacagcgct 1680

gcagccactg ggtatcgaca tgattggtct gaactgcgcc accggcccag atgagatgag 1740

cgagcacctg cgttacctgt ccaagcacgc cgatattcct gtgtcggtga tgcctaacgc 1800

aggtcttcct gtcctgggta aaaacggtgc agaataccca cttgaggctg aggatttggc 1860

gcaggcgctg gctggattcg tctccgaata tggcctgtcc atggtgggtg gttgttgtgg 1920

caccacacct gagcacatcc gtgcggtccg cgatgcggtg gttggtgttc cagagcagga 1980

aacctccaca ctgaccaaga tccctgcagg ccctgttgag caggcctccc gcgaggtgga 2040

gaaagaggac tccgtcgcgt cgctgtacac ctcggtgcca ttgtcccagg aaaccggcat 2100

ttccatgatc ggtgagcgca ccaactccaa cggttccaag gcattccgtg aggcaatgct 2160

gtctggcgat tgggaaaagt gtgtggatat tgccaagcag caaacccgcg atggtgcaca 2220

catgctggat ctttgtgtgg attacgtggg acgagacggc accgccgata tggcgacctt 2280

ggcagcactt cttgctacca gctccacttt gccaatcatg attgactcca ccgagccaga 2340

ggttattcgc acaggccttg agcacttggg tggacgaagc atcgttaact ccgtcaactt 2400

tgaagacggc gatggccctg agtcccgcta ccagcgcatc atgaaactgg taaagcagca 2460

cggtgcggcc gtggttgcgc tgaccattga tgaggaaggc caggcacgta ccgctgagca 2520

caaggtgcgc attgctaaac gactgattga cgatatcacc ggcagctacg gcctggatat 2580

caaagacatc gttgtggact gcctgacctt cccgatctct actggccagg aagaaaccag 2640

gcgagatggc attgaaacca tcgaagccat ccgcgagctg aagaagctct acccagaaat 2700

ccacaccacc ctgggtctgt ccaatatttc cttcggcctg aaccctgctg cacgccaggt 2760

tcttaactct gtgttcctca atgagtgcat tgaggctggt ctggactctg cgattgcgca 2820

cagctccaag attttgccga tgaaccgcat tgatgatcgc cagcgcgaag tggcgttgga 2880

tatggtctat gatcgccgca ccgaggatta cgatccgctg caggaattca tgcagctgtt 2940

tgagggcgtt tctgctgccg atgccaagga tgctcgcgct gaacagctgg ccgctatgcc 3000

tttgtttgag cgtttggcac agcgcatcat cgacggcgat aagaatggcc ttgaggatga 3060

tctggaagca ggcatgaagg agaagtctcc tattgcgatc atcaacgagg accttctcaa 3120

cggcatgaag accgtgggtg agctgtttgg ttccggacag atgcagctgc cattcgtgct 3180

3240

agcagaagct accggatctg cgcaggcaga gggcaagggc aaaatcgtcg tggccaccgt 3300

3360

ttacgacgtg gtgaacttgg gcatcaagca gccactgtcc gccatgttgg aagcagcgga 3420

agaacacaaa gcagacgtca tcggcatgtc gggacttctt gtgaagtcca ccgtggtgat 3480

3540

tggcgctgcg ctgacgcgta cctacgtgga aaacgatctc aacgaggtgt acaccggtga 3600

3660

aaagcgtggt gaaggacttg atcccaactc accagaagct attgagcagg cgaagaagaa 3720

ggcggaacgt aaggctcgta atgagcgttc ccgcaagatt gccgcggagc gtaaagctaa 3780

tgcggctccc gtgattgttc cggagcgttc tgatgtctcc accgatactc caaccgcggc 3840

accaccgttc tggggaaccc gcattgtcaa gggtctgccc ttggcggagt tcttgggcaa 3900

ccttgatgag cgcgccttgt tcatggggca gtggggtctg aaatccaccc gcggcaacga 3960

gggtccaagc tatgaggatt tggtggaaac tgaaggccga ccacgcctgc gctactggct 4020

ggatcgcctg aagtctgagg gcattttgga ccacgtggcc ttggtgtatg gctacttccc 4080

agcggtcgcg gaaggcgatg acgtggtgat cttggaatcc ccggatccac acgcagccga 4140

acgcatgcgc tttagcttcc cacgccagca gcgcggcagg ttcttgtgca tcgcggattt 4200

cattcgccca cgcgagcaag ctgtcaagga cggccaagtg gacgtcatgc cattccagct 4260

ggtcaccatg ggtaatccta ttgctgattt cgccaacgag ttgttcgcag ccaatgaata 4320

ccgcgagtac ttggaagttc acggcatcgg cgtgcagctc accgaagcat tggccgagta 4380

ctggcactcc cgagtgcgca gcgaactcaa gctgaacgac ggtggatctg tcgctgattt 4440

tgatccagaa gacaagacca agttcttcga cctggattac cgcggcgccc gcttctcctt 4500

tggttacggt tcttgccctg atctggaaga ccgcgcaaag ctggtggaat tgctcgagcc 4560

aggccgtatc ggcgtggagt tgtccgagga actccagctg cacccagagc agtccacaga 4620

cgcgtttgtg ctctaccacc cagaggcaaa gtactttaac gtctaacacc tttgagaggg 4680

aaaactttcc cgcacattgc agatcgtgcc actttaacta aggttgacgg catgattaag 4740

gcgattttct gggacatgga cggcacgatg gtggactctg agccacagtg gggcattgct 4800

acctacgagc tcagcgaagc catgggccgc cgcctcaccc cggagctccg ggaactcacc 4860

gtcggctcga gcctgccgcg caccatgcgc ttatgcgcag agcacgcagg cattacattg 4920

agcgacgcgg actacgagcg ctaccgggct ggcatgttcg cccgggtcca tgagcttttc 4980

gacgaatccc tcgtcccaaa tccaggcgtc accgaactcc tgacagagtt gaaggccctc 5040

gagatcccca tgttggtcac caccaacaca gagcgcgatc tcgcgacccg ttcagtcgca 5100

gccgtgggaa atgagttctt catcggttct atcgctggtg atgaagtccc aacagcaaag 5160

ccagccccg acatgtacct cgaagcagca cgacgtgtgg gctttgaccc atcagagtgc 5220

ctcgtgttcg aagattccta caacggcatg ctgggcgctg ttactgcagg ttgccgcgtc 5280

attggtctgc acccagaaga agtccaagcg ccagaaggtg tagtgccttt gcgttccctc 5340

cacggtaaaa actctttcga aggtgtcacc gctgagatgg tcactgcctg gtaccaccag 5400

atcgagccgg caggtgtcgc aaaataaaac caggtgggggg agtgaaatta ttcgactaat 5460

atcctccccc aaacacacat tgataactgt tgtgtggaag aatgtaccga gtgaagacat 5520

ttgactcgct gtacgaagaa cttcttaacc gtgctcagac ccgccctgaa gggtctggaa 5580

ccgtggccgc cttggataaa ggcatccatc atctaggtaa gaaggtcatc gaagaagccg 5640

gagaggtctg gattgcagcc gagtattgtc atgcttccgg aggtgcgcag ggctcgagac 5700

tccggaaagc tatttgccac tccgatgttt gggtcactcg acgagatacg tgctgatcac 5760

ctaatttggt gcacagggtt tcggccggcg attaggccag ttcgtcaact tctcaaacac 5820

ggacaaccaa aggttcctgg tctttattta gtaggctacg gagattggac gggacctggg 5880

tctgcgacta tcacaggggt cgggctttat gccaagcgag cagccaaaga gattgccgcg 5940

tcagtcggca aagtcgttaa atagtttgaa ggctaagaac ttaatgttaa agcgaaaatt 6000

gttttgacac ctcaactaat gcagcgatgc gttctttcca gaatgctttc atgacaggga 6060

tgctgtcttg atcaggcagg cgtctgtgct ggatgccgaa gctggattta ttgtcgcctt 6120

tggaggtgaa gttgacgctc actcgagaat catcggccaa ccatttggca ttgaatgttc 6180

taggttcggga ggcgggaggtt ttctcaatta gtgcgggatc gagccactgc gcccgcaggt 6240

catcgtctcc gaagagcttc cacacttttt cgaccggcag gttaagggtt ttggaggcat 6300

tggccgcgaa cccatcgctg gtcatcccgg gtttgcgcat gccacgttcg tattcataac 6360

caatcgcgat gccttgagcc caccagccac tgacatcaaa gttgtccacg atgtgctttg 6420

cgatgtgggt gtgagtccaa gaggtggctt ttacgtcgtc aagcaatttt agccactctt 6480

cccacggctt tccggtgccg ttgaggatag cttcagggga catgcctggt gttgagcctt 6540

gcggagtgga gtcagtcatg cgaccgagac tagtggcgct ttgcctgtgt tgcttaggcg 6600

gcgttgaaaa tgaactacga atgaaaagtt cgggaattgt ctaatccgta ctaagctgtc 6660

tacacaatgt ctacttcagt tacttcacca gcccacaaca acgcacattc ctccgaattt 6720

ttggatgcgt tggcaaacca tgtgttgatc ggcgacggcg ccatgggcac ccagctccaa 6780

ggctttgacc tggacgtgga aaaggatttc cttgatctgg aggggtgtaa tgagattctc 6840

aacgacaccc gccctgatgt gttgaggcag attcaccgcg cctactttga ggcgggagct 6900

gacttggttg agaccaatac ttttggttgc aacctgccga acttggcggga ttatgacatc 6960

gctgatcgtt gccgtgagct tgcctacaag ggcactgcag tggctaggga agtggctgat 7020

gagatggggc cgggccgaaa cggcatgcgg cgtttcgtgg ttggttccct gggacctgga 7080

acgaagcttc catcgctggg ccatgcaccg tatgcagatt tgcgtggggca ctacaaggaa 7140

gcagcgcttg gcatcatcga cggtggtggc gatgcctttt tgattgagac tgctcaggac 7200

ttgcttcagg tcaaggctgc ggttcacggc gttcaagatg ccatggctga acttgataca 7260

ttcttgccca ttatttgcca cgtcaccgta gagaccaccg gcaccatgct catgggttct 7320

gagatcggtg ccgcgttgac agcgctgcag ccactgggta tcgacatgat tggtctgaac 7380

tgcgccaccg gcccagatga gatgagcgag cacctgcgtt acctgtccaa gcacgccgat 7440

attcctgtgt cggtgatgcc taacgcaggt cttcctgtcc tgggtaaaaa cggtgcagaa 7500

tacccacttg aggctgagga tttggcgcag gcgctggctg gattcgtctc cgaatatggc 7560

ctgtccatgg tgggtggttg ttgtggcacc acacctgagc acatccgtgc ggtccgcgat 7620

gcggtggttg gtgttccaga gcaggaaacc tccacactga ccaagatccc tgcaggccct 7680

gttgagcagg cctcccgcga ggtggagaaa gaggactccg tcgcgtcgct gtacacctcg 7740

gtgccattgt ccggaaac cggcatttcc atgatcggtg agcgcaccaa ctccaacggt 7800

tccaaggcat tccgtgaggc aatgctgtct ggcgattggg aaaagtgtgt ggatattgcc 7860

aagcagcaaa cccgcgatgg tgcacacatg ctggatcttt gtgtggatta cgtgggacga 7920

gacggcaccg ccgatatggc gaccttggca gcacttcttg ctaccagctc cactttgcca 7980

atcatgattg actccaccga gccagaggtt attcgcacag gccttgagca cttgggtgga 8040

cgaagcatcg ttaactccgt caactttgaa gacggcgatg gccctgagtc ccgctaccag 8100

cgcatcatga aactggtaaa gcagcacggt gcggccgtgg ttgcgctgac cattgatgag 8160

gaaggccagg cacgtaccgc tgagcacaag gtgcgcattg ctaaacgact gattgacgat 8220

atcaccggca gctacggcct ggatatcaaa gacatcgttg tggactgcct gaccttcccg 8280

atctctactg gccaggaaga aaccaggcga gatggcattg aaaccatcga agccatccgc 8340

gagctgaaga agctctaccc agaaatccac accaccctgg gtctgtccaa tatttccttc 8400

ggcctgaacc ctgctgcacg ccaggttctt aactctgtgt tcctcaatga gtgcattgag 8460

gctggtctgg actctgcgat tgcgcacagc tccaagattt tgccgatgaa ccgcattgat 8520

gatcgccagc gcgaagtggc gttggatatg gtctatgatc gccgcaccga ggattacgat 8580

ccgctgcagg aattcatgca gctgtttgag ggcgtttctg ctgccgatgc caaggatgct 8640

cgcgctgaac agctggccgc tatgcctttg tttgagcgtt tggcacagcg catcatcgac 8700

ggcgataaga atggccttga ggatgatctg gaagcaggca tgaaggagaa gtctcctatt 8760

gcgatcatca acgaggacct tctcaacggc atgaagaccg tgggtgagct gtttggttcc 8820

ggacagatgc agctgccatt cgtgctgcaa tcggcagaaa ccatgaaaac tgcggtggcc 8880

tatttggaac cgttcatgga agaggaagca gaagctaccg gatctgcgca ggcagaggggc 8940

aagggcaaaa tcgtcgtggc caccgtcaag ggtgacgtgc acgatatcgg caagaacttg 9000

gtggacatca ttttgtccaa caacggttac gacgtggtga acttgggcat caagcagcca 9060

ctgtccgcca tgttggaagc agcggaagaa cacaaagcag acgtcatcgg catgtcggga 9120

cttcttgtga agtccaccgt ggtgatgaag gaaaaccttg aggagatgaa caacgccggc 9180

gcatccaatt acccagtcat tttgggtggc gctgcgctga cgcgtaccta cgtggaaaac 9240

gatctcaacg aggtgtacac cggtgaggtg tactacgccc gtgatgcttt cgagggcctg 9300

cgcctgatgg atgaggtgat ggcagaaaag cgtggtgaag gacttgatcc caactcacca 9360

gaagctattg agcaggcgaa gaagaaggcg gaacgtaagg ctcgtaatga gcgttcccgc 9420

aagattgccg cggagcgtaa agctaatgcg gctcccgtga ttgttccgga gcgttctgat 9480

gtctccaccg atactccaac cgcggcacca ccgttctggg gaacccgcat tgtcaagggt 9540

ctgcccttgg cggagttctt gggcaacctt gatgagcgcg ccttgttcat ggggcagtgg 9600

ggtctgaaat ccacccgcgg caacgagggt ccaagctatg aggatttggt ggaaactgaa 9660

ggccgaccac gcctgcgcta ctggctggat cgcctgaagt ctgagggcat tttggaccac 9720

gtggccttgg tgtatggcta cttcccagcg gtcgcggaag gcgatgacgt ggtgatcttg 9780

gaatccccgg atccacacgc agccgaacgc atgcgcttta gcttcccacg ccagcagcgc 9840

ggcaggttct tgtgcatcgc ggatttcatt cgcccacgcg agcaagctgt caaggacggc 9900

caagtggacg tcatgccatt ccagctggtc accatgggta atcctattgc tgatttcgcc 9960

aacgagttgt tcgcagccaa tgaataccgc gagtacttgg aagttcacgg catcggcgtg 10020

cagctcaccg aagcattggc cgagtactgg cactcccgag tgcgcagcga actcaagctg 10080

aacgacggtg gatctgtcgc tgattttgat ccagaagaca agaccaagtt cttcgacctg 10140

gattaccgcg gcgcccgctt ctcctttggt tacggttctt gccctgatct ggaagaccgc 10200

gcaaagctgg tggaattgct cgagccaggc cgtatcggcg tggagttgtc cgaggaactc 10260

cagctgcacc cagagcagtc cacagacgcg tttgtgctct accacccaga ggcaaagtac 10320

tttaacgtct aacacctttg agagggaaaa ctttcccgca cattgcagat cgtgccactt 10380

taactaaggt tgacggcatg attaaggcga ttttctggga catggacggc acgatggtgg 10440

actctgagcc acagtggggc attgctacct acgagctcag cgaagccatg ggccgccgcc 10500

tcaccccgga gctccgggaa ctcaccgtcg gctcgagcct gccgcgcacc atgcgcttat 10560

gcgcagagca cgcaggcatt acattgagcg acgcggacta cgagcgctac cgggctggca 10620

10680

aactcctgac agagttgaag gccctcgaga tccccatgtt ggtcaccacc aacacagagc 10740

gcgatctcgc gacccgttca gtcgcagccg tgggaaatga gttcttcatc ggttctatcg 10800

ctggtgatga agtcccaaca gcaaagccag cccccgacat gtacctcgaa gcagcacgac 10860

gtgtgggctt tgacccatca gagtgcctcg tgttcgaaga ttcctacaac ggcatgctgg 10920

gcgctgttac tgcaggttgc cgcgtcattg gtctgcaccc agaagaagtc caagcgccag 10980

11040

agatggtcac tgcctggtac caccagatcg agccggcagg tgtcgcaaaa taaaaccagg 11100

tgggggagtg aaattattcg actaatatcc tcccccaaac acacattgat aactgttgtg 11160

tggaagaatg taccgagtga agacatttga ctcgctgtac gaagaacttc ttaaccgtgc 11220

tcagacccgc cctgaagggt ctggaaccgt ggccgccttg gataaaggca tccatcatct 11280

aggtaagaag gtcatcgaag aagccggaga ggtctggatt gcagccgagt at 11332

<210> 94

<211> 3311

<212> DNA

<213> Unknown

<220>

<223> polynucleotide

<400> 94

ctcattccag cgtcacgacg ttccgaaggt actggttacc tggcattgggg cactaccgtt 60

tctgcagcac ttggaccagc cctagcactt tttgtcctag gaacatttga ttacgacatg 120

ctgtttatcg tggtcttggc aacctcggtc atctctttga tcgccgtcgt gttcatgtac 180

tttaagacca gcgaccctga gccttctggg gaaccagcca agttcagctt caaatctatt 240

atgaacccaa agatcatccc catcggcatc tttatcttgc ttatttgctt tgcttactct 300

ggcgtcattg cctacatcaa cgcatttgct gaagaacgcg atctgattac gggtgctgga 360

ttgttcttca ttgcctacgc agtatcaatg tttgtgatgc gcagcttcct tggcaaactg 420

caggaccgtc gcggagacaa cgtcgttatt tactttggat tgttcttctt cgttatttcc 480

ttgacgattt tgtcctttgc cacttccaac tggcacgttg tgttgtccgg agtcattgca 540

ggtctgggat acggcacttt gatgccagca gtgcagtcca tcgctgttgg tgtagtagac 600

aaaaccgaat tcggtacggc cttctccact ttgttcctgt ttgtggactt aggttttggc 660

tttggaccta ttatcctggg agcagtttct gcggcaattg gtttcggacc tatgtatgca 720

gcactggcag gtgtgggtgt gattgccgga atcttctacc tgttcacaca cgctcgcacc 780

gatcgagcta agaatggctt tgttaaacac ccagagcctg tcgctttagt tagctagttc 840

tttcagcttt ccctcccgat cagcgtaaac cggcccttcc ggttttgggg tacatcacag 900

aacctgggct agcggtgtag acccgaaaat aaacgagcct tttgtcaggg ttaaggttta 960

ggtatctaag ctaaccaaac accaacaaaa ggctctaccc atgaagtcta ccggcaacat 1020

catcgctgac accatctgcc gcactgcgga actaggactc accatcaccg gcgcttccga 1080

tgcaggtgat tacaccctga tcgaagcaga cgcactcgac tacacctcca cctgcccaga 1140

atgctcccaa cctggggtgt ttcgtcatca cacccaccgg atgctcattg atttacccat 1200

cgtcgggttt cccaccaaac tgtttatccg tctacctcgc taccgctgca ccaaccccac 1260

atgtaagcaa aagtatttcc aagcagaact aagctgcgct

ccaccgggtc acccgctgga ttttacaacg ccttgctatt gaccggatga gtgttcacgc 1380

aaccgcgaaa gcacttgggc tagggtggga tttaacctgc caactagccc tcgatatgtg 1440

ccgtgagctg gtctataacg atcctcacca tcttgatgga gtgtatgtca ttggggtgga 1500

tgagcataag tggtcacata atagggctaa gcatggtgat gggtttgtca ccgtgattgt 1560

cgatatgacc gggcatcggt atgactcacg gtgtcctgcc cggttattag atgtcgtccc 1620

aggtcgtagt gctgatgctt tacggtcctg gcttggctcc cgcggtgaac agttccgcaa 1680

tcagatacgg atcgtgtcca tggatggatt ccaaggctac gccacagcaa gtaaagaact 1740

cattccttct gctcgtcgcg tgatggatcc attccatgtt gtgcggcttg ctggtgacaa 1800

gctcaccgcc tgccggcaac gcctccagcg ggagaaatac cagcgtcgtg gtttaagcca 1860

ggatccgttg tataaaaacc ggaagacctt gttgaccacg cacaagtggt tgagtcctcg 1920

tcagcaagaa agcttggagc agttgtggggc gtatgacaaa gactacgggg cgttaaagct 1980

tgcgtggctt gcgtatcagg cgattattga ttgttatcag atgggtaata agcgtgaagc 2040

gaagaagaaa atgcggacca ttattgatca gcttcgggtg ttgaaggggc cgaataagga 2100

actcgcgcag ttgggtcgta gtttgtttaa acgacttggt gatgtgttgg cgtatttcga 2160

tgttggtgtc tccaacggtc cggtcgaagc gatcaacgga cggttggagc atttgcgtgg 2220

2280

gcagttggtc cataagatca atgcactcta aaacaggaag agcccgtaaa cctctgacta 2340

gcgtcaccct ctgattaagg cgaccgcgga tttaagagca gaggctgcca cgagcgcatc 2400

ttcacggctg tgtgttgtac taaaagtaca gcgcacagcc gttcgtgctt gatcctcctc 2460

aagccccaac gccagcaaca catgggatac ctctccggaa ccacaggcag aaccagggga 2520

gcacacaatg ccttggcgtt ccaattccag aagaacagtt tcagatccta tgctgtcgaa 2580

gagaaaagat gcgtgtccat caatgcgcat cctaggatgt ccagtcaggt gtgctcccgg 2640

gatagtgaga acttcctcga tgaattcgcc aagatctgga taggattccg ccctggccaa 2700

ttccaaggca gtggcaaagg cgatagcccc cgcaacgttt tccgtgccac tacgccgccc 2760

ttttttcctgg ccgccgccat ggattaccgg ctccagggga agctttgacc ataacactcc 2820

aatcccttta ggcgcaccga atttatgacc cgacaaactt aacgcgtcaa ctcccaagtc 2880

aaaggttaaa tgtgcagctt gcactgcatc ggtgtgaaaa ggcgtactgc ttaccgccgc 2940

caactcagct atcggctgaa tggttcccac ctcattgttg gcataaccaa tgctgatcaa 3000

tgtggtgtcc ggcctgactg ctttgcggag accctccggg gagatcagcc cagtgtgatc 3060

gggggatagg taggtgatct cgaaatcatg aaacctttca agataagcag cagtttctag 3120

gacactgtca tgctcgatcg gggtggtgat gaggtgccgg ccacgaggat tagctaagca 3180

3240

ggggcgtcct ccgataatgc gggccacccg agttcgagca tcctccagcc ccgcagaggc 3300

gagtcttccc a 3311

<210> 95

<211> 1230

<212> DNA

<213> Unknown

<220>

<223> polynucleotide

<400> 95

atgagcaaga cctctctcga caagagcaag atccgcttcc ttctgctgga aggcgtccac 60

cagaccgcac tggatacgct caaggctgcc ggctacacca acatcgagta cctgaccggc 120

tcgctgcccg aggaacagtt gaaagagaag atcgccgacg cccacttcat cggcatccgc 180

tcgcgcaccc aactgaccga ggaggtcttc gaccgcgcga agaagctggt cgcggtcggc 240

tgtttctgca tcggcaccaa ccaggtcgac ctggaggccg ctcgcgagcg cggcatcgcg 300

gtgttcaacg ccccctactc gaacacccgc tcggtggccg agctggtgct cgccgaggcg 360

atcctgctgc tgcgcggcat tcccgagaag aacgcggcca gccaccgcgg cggctggctg 420

aagagcgcca gcaactccta cgagatccgc ggcaagaagc tcggcatcat cggctatggc 480

tcgatcggta cccagctctc ggtgctggcc gaaagcctgg gcatgcaggt gctgttctac 540

gacgtggtga ccaagctgcc gctgggcaac gccgcccagg tgggcaacct ctacgacttg 600

ctcggccagg ccgacatcgt caccctgcac gtgccggaaa ccgccgcgac caagtggatg 660

atcggcgaga aggaaatccg cgccatgaag aaaggcgcca tcctgctcaa cgccgcccgc 720

ggcaccgtgg tggacatcga cgcgctggcc gccgccctcc gcgacaagca cctcaacggc 780

gcggccatcg acgtgttcc ggtggaaccg cgctccaaca acgacgagtt cgtcagcccg 840

ctgcgcgagt tcgacaacgt catcctcacc ccgcacgtcg gcggctcgac catggaggcc 900

caggccaaca tcggttcgga agtggccgag aagctggtca agtacagcga caacggcacc 960

tcggtgtcct cggtcaactt ccgggaagtg gccctgccct cccacccgggg caagcaccgc 1020

ctgctgcaca tccacaagaa catccccggga gtgatgagcg agatcaacaa ggtcttcgcc 1080

gagaacggca tcaacatttc cggccagttc ctgcagacca acgagacggt cggctacgtg 1140

gtgatcgatg tcgatgccga gtactcggaa atggccctgg agaaactgca gcaggtgaac 1200

ggcaccatcc gcagccgcgt gctgttctga 1230

<---

Claims (17)

1. An L-amino acid producing microorganism and its precursor, wherein the microorganism is modified by incorporating a protein containing the amino acid sequence of SEQ ID NO: 1. 2. A microorganism according to claim 1, wherein said protein originates from Azotobacter vinelandii . 3. The microorganism according to claim 1, which further has (1) reduced phosphoserine phosphatase activity compared to endogenous phosphoserine phosphatase activity (2) enhanced 3-phosphoserine aminotransferase activity compared to endogenous phosphoserine phosphatase activity, or (3) as reduced phosphoserine phosphatase activity, according to compared with endogenous activity of phosphoserine phosphatase, and increased activity of 3-phosphoserine aminotransferase, compared with endogenous activity of phosphoserine phosphatase. 4. The microorganism of claim 1, which is further modified by enhancing the trp (tryptophan) operon over the endogenous trp operon, tryptophanase (TnaA) inactivation, Mtr membrane protein (Mtr) inactivation, or any combination thereof. 5. The microorganism according to claim 1, which additionally has an enhanced his (histidine) operon compared to the endogenous his operon. 6. The microorganism according to claim 1, which is further modified by inactivating McbR (transcription regulator; mcbR), enhancing methionine synthase (meth) compared to endogenous methionine synthase activity, enhancing sulfite reductase [NADPH] hemoprotein beta component (cysI), compared to endogenous activity of the sulfite reductase [NADPH] hemoprotein beta component, or any combination thereof. 7. The microorganism according to claim 1, which belongs to the genus Corynebacterium or the genus Escherichia . 8. The microorganism according to claim 7, which is Corynebacterium glutamicum or Escherichia coli . 9. The microorganism according to claim 1, wherein the L-amino acid and its precursor are selected from the group consisting of L-serine, L-tryptophan, L-histidine, L-methionine, L-cysteine, O-succinylhomoserine, O-acetylhomoserine, L -homoserine, acetylserine, L-cystothionine, L-homocysteine and O-phosphoserine. 10. The method of obtaining L-amino acids, including the cultivation of the microorganism according to any one of paragraphs. 1-9 in the environment. 11. The method of claim 10, further comprising isolating the L-amino acid from the cultured microorganism or from the culture medium. 12. The method of claim 10, wherein the L-amino acid is selected from the group consisting of serine, tryptophan, histidine, methionine, or L-cysteine. 13. The use of a microorganism according to any one of paragraphs. 1-9 for L-amino acid production. 14. Composition for obtaining L-amino acids, which contains the microorganism according to any one of paragraphs. 1-9, or a protein containing the amino acid sequence of SEQ ID NO: 1, and a buffer. 15. The method of obtaining a precursor of L-amino acids, including the cultivation of the microorganism according to any one of paragraphs. 1-9 in the environment. 16. The use of a microorganism according to any one of paragraphs. 1-9 to obtain an L-amino acid precursor. 17. Composition for obtaining a precursor of L-amino acids, which contains a microorganism according to any one of paragraphs. 1-9, or a protein containing the amino acid sequence of SEQ ID NO: 1, and a buffer.