RU2806745C2 - Method of production sulfur-containing amino acid or its derivative - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a method of producing a sulfur-containing amino acid or a derivative of a sulfur-containing amino acid. The method includes cultivating a genetically modified microorganism in a culture medium containing thiosulfate, where the microorganism contains a genetic modification to increase the activity of the protein encoded by the ssuABC gene, compared to an unmodified microorganism, where the sulfur-containing amino acid or sulfur-containing amino acid derivative includes at least one (one) selected (o) from the group consisting of methionine, cysteine, cystine, lanthionine, homocysteine, homocystine, homolanthionine and taurine.

EFFECT: invention makes it possible to obtain a sulfur-containing amino acid or a derivative of a sulfur-containing amino acid with a high degree of efficiency.

14 cl, 7 tbl, 8 ex

Description

Field of invention

The present invention relates to a method for producing sulfur-containing amino acids or derivatives of sulfur-containing amino acids.

State of the art

L-amino acids are produced industrially through fermentation methods using microorganisms belonging to the Brevibacterium genus, Corynebacterium genus, Escherichia genus and the like. Such production methods use bacterial strains isolated from a natural source, artificial mutant strains thereof, or strains modified to provide increased activity of an enzyme involved in L-amino acid biosynthesis through recombinant DNA technology.

In this regard, sulfur-containing amino acids are used as ingredients for the synthesis of animal feed, food additives, pharmaceutical injectable liquids and drugs, and research has been conducted on the biological production of sulfur-containing amino acids and their derivatives.

For example, in US patent application no. US 2009-0298135 A1 discloses the production of 0.8 g/L L-methionine by deleting the metJ gene from the Escherichia coli genome and overexpressing the YjeH protein, which is an L-methionine exporter. The BrnF and BrnE polypeptides have also been reported to be L-methionine exporters from Corynebacterium glutamicum (C. Troschel et al., Journal of Bacteriology, pp. 3786-3794, June 2005).

Moreover, during the production of sulfur-containing amino acids, the amount of NADPH consumed by microorganisms may vary depending on the reducing ability of the sulfur source. For example, while sulfides that do not require NADPH have the highest theoretical yields, sulfates that require four molecules of NADPH have low theoretical yields. However, sulfides are disadvantageous in that they are known to cause cell damage and have low stability. Therefore, high production yields of sulfur-containing amino acids can be expected when using thiosulfate, which is a sulfur source with low NADPH requirement and high intracellular stability. However, while the presence of a membrane protein in Escherichia coli capable of using thiosulfate has been confirmed (J Bacteriol. 1995 Jul; 177 14), a membrane protein in microorganisms of the genus Corynebacterium capable of efficiently using thiosulfate has not been disclosed in the prior art.

Technical problem

The inventors of the present invention unexpectedly discovered that the protein encoded by the ssuABC gene is involved in the entry of thiosulfate into a microorganism and confirmed that a microorganism modified to have increased activity of this protein has an increased ability to produce sulfur-containing amino acids using thiosulfate as a sulfur source, thereby thereby carrying out the present invention.

Technical solution

The present invention provides a method for producing a sulfur-containing amino acid and a sulfur-containing amino acid derivative, comprising cultivating a genetically modified microorganism in a culture medium containing thiosulfate, where the microorganism contains a genetic modification to increase the activity of a protein encoded by the ssuABC gene compared to an unmodified microorganism.

The present invention provides a microorganism that produces a sulfur-containing amino acid or a derivative of a sulfur-containing amino acid, containing a genetic modification to increase the activity of the protein encoded by the ssuABC gene compared to an unmodified microorganism.

The present invention provides a composition for producing a sulfur-containing amino acid or a derivative of a sulfur-containing amino acid, comprising a microorganism containing a genetic modification to increase the activity of the protein encoded by the ssuABC gene compared to an unmodified microorganism, or a culture thereof and thiosulfate.

The present invention provides the use of a protein encoded by the ssuABC gene as a thiosulfate transporter.

The present invention provides the use of a microorganism containing a genetic modification to increase the activity of a protein encoded by the ssuABC gene, compared to an unmodified microorganism, to produce a sulfur-containing amino acid or a derivative of a sulfur-containing amino acid.

Beneficial effects

Sulfur-containing amino acids or derivatives thereof can be produced on an industrial scale using a microorganism, a composition and a method for producing a sulfur-containing amino acid or a sulfur-containing amino acid derivative using the same microorganism and composition of the present invention, and thus can be effectively used in the production of useful products, including sulfur-containing amino acids or their derivatives.

Best embodiment of the invention

Each description and embodiment disclosed in the present invention can be applied to various descriptions and embodiments herein. In other words, all combinations of the various components disclosed in the present invention are included within the scope of the present invention. Moreover, the scope of the present invention is not limited to the following descriptions.

One skilled in the art understands or is able to determine, using only routine experimentation, many embodiments equivalent to specific embodiments of the present invention. It is intended that the scope of the present invention cover such equivalent embodiments.

In another aspect of the present invention, there is provided a method for producing a sulfur-containing amino acid and a sulfur-containing amino acid derivative, comprising cultivating a genetically modified microorganism in a culture medium containing thiosulfate.

In one aspect of the present invention, a genetically modified microorganism is provided that produces a sulfur-containing amino acid or a derivative of a sulfur-containing amino acid.

The microorganism may contain a genetic modification to increase the activity of the protein encoded by the ssuABC gene compared to the microorganism before the genetic modification.

The production method may include cultivating a microorganism having increased activity of the protein encoded by the ssuABC gene, compared to natural activity, in a culture medium containing thiosulfate.

In one embodiment of the present invention, the method may be a method of increasing the production of sulfur amino acids or sulfur amino acid derivatives by the ssuABC microorganism compared to natural production.

The production method may include bringing a microorganism having increased activity of a protein encoded by the genomic activity into contact with a thiosulfate.

As used herein, the expression "protein encoded by the ssuABC gene" refers to a protein that encodes the ssuABC gene, or a protein expressed by the ssuABC gene, and may be referred to as "SsuABC protein" (hereinafter referred to as "SsuABC protein") . It is traditionally known that the SsuABC protein is involved in aliphatic sulfonate transport. This protein is one type of ATP-binding cassette transporter (ABC transporter) and is known to be present in microorganisms such as Escherichia coli, Bacillus clausii, Xanthomonas citri and Corynebacterium glutamicum. The SsuABC protein is a complex of the proteins SsuA, SsuB and SsuC, and SsuA is known as a periplasmic binding protein. SsuB is known as a nucleotide binding protein, and SsuC is known as an ABC transporter permease. However, it is not known whether this protein complex is involved in the transport of thiosulfate rather than aliphatic sulfonate.

In the present invention, it was revealed for the first time that the SsuABC protein is involved in thiosulfate transport, and it was confirmed that the production of sulfur-containing amino acid can be increased by increasing the activity of one of the proteins selected from SsuA, SsuB and SsuC, which are components of the SsuABC protein.

The SsuABC protein as defined herein may be derived from, but is not limited to, a microorganism belonging to the genus Corynebacterium.

In particular, the SsuABC protein may originate from Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium crenatum, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutis oli, Corynebacterium imitans, Corynebacterium testudinoris , Corynebacterium pacaense, Corynebacterium suranareeae or Corynebacterium flavescens, more particularly of origin from, but not limited to, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium deserti or Corynebacterium suranareeae. The amino acid sequence obtained for the SsuABC protein belonging to the genus Corynebacterium may be available from a known database such as, but not limited to, GenBank from the US National Center for Biotechnology Information.

The SsuABC protein of the present invention can be considered not only as one or more than one protein and/or protein complex involved in thiosulfate transport, but also as a system including one or more than one protein and/or protein complex as a component, that is , as the thiosulfate transport system itself. That is, in a system in which one or more proteins interact to transport a substrate, the term “transporter” may be understood to include not only each protein, but also two or more proteins or the entire system throughout the present specification.

The SsuA, SsuB and SsuC proteins constituting the SsuABC protein of the present invention may have amino acid sequences having at least 80% identity to the amino acid sequences of SEQ ID NO: 43, 44 and 45, respectively. In particular, the SsuA, SsuB and SsuC proteins may comprise the amino acid sequences of SEQ ID NO: 43, 44 and 45, respectively, or may comprise amino acid sequences having at least 80%, 90%, 95%, 97% or 99% homology or identity to the amino acid sequences of SEQ ID NO: 43, 44 and 45, respectively. It is also apparent that any protein having amino acid sequences including the deletion, modification or addition of multiple amino acids is within the scope of the present invention, provided that these amino acid sequences retain the above-described homology or identity and actions equivalent to those of the polypeptide (i.e., activity , specific for the transport of thiosulfate relative to other sulfur sources).

In addition, any polypeptide having thiosulfate-specific transporter activity and encoded by a polynucleotide that hybridizes to a probe designed using known gene sequences, such as a nucleotide sequence fully or partially complementary to the polynucleotide under stringent conditions, may also be included without limitation.

That is, although the present invention uses the expression "a protein or polypeptide containing the amino acid sequence specified by SEQ ID NO", "a protein or polypeptide consisting of the amino acid sequence specified by SEQ ID NO" or "a protein or polypeptide having the amino acid sequence specified by SEQ ID NO ", it is obvious that any protein including deletion, modification, substitution, conservative substitution or addition of one or more amino acids can be used in the present invention, provided that the protein has an activity identical or equivalent to that of the polypeptide consisting of the amino acid sequence of SEQ ID NO. For example, the addition of a sequence that does not change the function of the protein at the N-terminus and/or C-terminus of an amino acid sequence, a natural mutation, a silent mutation therein, or a conservative substitution therein can be used.

As used herein, the term “conservative substitution” refers to the replacement of one amino acid with another amino acid having similar structural and/or chemical properties. Such amino acid substitution may generally occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residue.

In one embodiment of the present invention, the ssuABC gene may comprise a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the nucleotide sequence of SEQ ID NO: 8 In particular, the ssuABC gene may consist of a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology to the nucleotide sequence of SEQ ID NO: 8 , without limiting this.

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

The polynucleotide may be a polynucleotide (ssuABC gene) encoding the SsuABC protein of the present invention. The polynucleotide of the present invention may include various modifications made to the coding region, provided that the amino acid sequence of the polypeptide expressed from the coding region is not altered due to degeneracy of the genetic code or due to codon preferences of the living organism in which the protein is expressed. The polynucleotide of the present invention may be, for example, a polynucleotide encoding a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity to a protein SsuABC according to the present invention. In particular, for example, polynucleotides encoding proteins containing amino acid sequences having at least 80% homology or identity with the amino acid sequences of SEQ ID NO: 43, 44 and 45, respectively, may be polynucleotides having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity with part of the nucleotide sequence of SEQ ID NO: 8. In particular, polynucleotides encoding proteins containing amino acid sequences having at least 80% identity to the amino acid sequences of SEQ ID NO: 43, 44 and 45, respectively, may represent polynucleotides having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity with at least one sequence selected from the group consisting of a polynucleotide including nucleotides ²⁵³⁰ to ³⁴⁸⁹ ; a polynucleotide including nucleotides ¹⁷⁸⁹ to 2520, and a polynucleotide including nucleotides ¹⁰⁰⁴ to ¹⁷⁷⁴ in the nucleotide sequence of SEQ ID NO: 8, without limitation.

Additionally, it will be appreciated that any polynucleotide that can be translated into a protein comprising an amino acid sequence having at least 80% identity with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 43, 44 may also be included and 45 due to the degeneracy of the genetic code, and a protein having homology or identity with such a protein. Alternatively, any polynucleotide encoding a protein comprising an amino acid sequence having at least 80% identity with at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 43, 44 and 45 and hybridizable to a probe designed using known gene sequences, for example a nucleotide sequence wholly or partially complementary to that polynucleotide sequence, under strict conditions, may be included without limitation. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are disclosed in detail in known documents (for example, 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 hybridization between genes having a high degree of homology or identity, such as 70% or more homology or identity, 80% or more, 85% or more, particularly 90% or more, more particularly 95%, or more, even more specifically 97% or more, or most particularly 99% or more, without hybridization between genes having a lesser degree of homology or identity than the above degree of homology or identity, or a single wash, in particular a two or three wash in traditional washing conditions for Southern hybridization at a salt concentration and temperature of 60°C, 1xSSC and 0.1% SDS, in particular 60°C, 0.1xSSC, 0.1% SDS and more particularly 68°C, 0.1×SSC and 0.1% SDS.

Hybridization requires that the two polynucleotides have complementary sequences, although the bases may not match depending on the stringency of the hybridization. The term "complementary" is used to describe the interaction between nucleotide bases that are capable of hybridizing with each other. For example, in relation to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the present invention may include not only a substantially similar nucleotide sequence, but also a polynucleotide fragment that is separate but complementary to the complete sequence.

In particular, polynucleotides having homology or identity with the polynucleotide of the present invention can be determined using hybridization conditions including a hybridization process performed at a T _m value of 55°C and under the conditions described above. Also, the value of T _m can be 60°C, 63°C or 65°C, but not limited to, and can be suitably specified by one skilled in the art in accordance with the objectives.

The appropriate degree of stringency for polynucleotide hybridization may depend on the lengths and degree of complementarity of the polynucleotides, and its parameters are well known in the art (Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

As used herein, the term “homology” or “identity” refers to the degree of relatedness 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.

The homology or sequence identity of conserved polynucleotides or polypeptides can be determined by a standard alignment algorithm and can use the program's default sequence gap penalties. Substantially homologous or identical sequences may hybridize to each other for at least about 50%, 60%, 70%, 80%, or 90% of the total sequence or full length under moderate to very stringent conditions. Obviously, polynucleotides containing a basic codon or a degenerate codon can also be considered for hybridization.

Homology, similarity or identity between two polynucleotide or polypeptide sequences can be determined using any computer algorithm known in the art, for example, the "FASTA" program, using the default parameters introduced by Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444. Alternatively, homology, similarity or identity can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443^153), which is implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later) (including the GCG software package (Devereux, J. et al., Nucleic Acids Research 12:387 (1984) )), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J MOLEC BIOL 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and CARILLO et al (1988) SIAM J Applied Math 48:1073) For example, homology, similarity or identity can be determined using BLAST from the US National Center for Biotechnology Information or ClustalW database.

Homology, similarity or identity between polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program presented by Needleman et al., (1970), J Mol Biol. 48:443, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines similarity as the number of aligned characters (i.e., nucleotides or amino acids) that are the same, divided by the total number of characters in the shorter of the two sequences. Default parameters for the GAP program may include: (1) a binary comparison matrix (including a value of 1 for matches and 0 for mismatches) and a weighted comparison matrix from Gribskov et al. (1986), Nucl. Acids Res. \A\61A5 as described by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (or EDNAFULL substitution matrix (NCBI NUC4.4 EMBOSS version)); (2) a 3.0 penalty for each skip and an additional 0.10 penalty for each character in each skip (or a 10 penalty for entering a skip and a 0.5 penalty for extending a skip) and (3) no penalty for ending a skip.

Also, homology, similarity or sequence identity between two given polynucleotides or polypeptides can be determined by comparing their sequences by Southern hybridization under certain stringent conditions, and certain stringent hybridization conditions are within the scope of this technology and can be determined in a manner well known to one of ordinary skill in the art. qualifications 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).

As used herein, the term “enhancing” the activity of a polypeptide or protein refers to increasing the activity of the polypeptide compared to its natural activity. The term "amplification" can be used interchangeably with the terms "up-regulation", "overexpression", "amplification" and the like.

In this regard, an increase may include the manifestation of activity that was not present initially, or the manifestation of increased activity compared to natural activity or activity before modification. The term “natural activity” refers to the activity of a particular polypeptide or protein originally possessed by the parent strain or unmodified microorganism prior to transformation, in which the microorganism is transformed by genetic modification caused by a natural or artificial factor. This term can be used interchangeably with the expression "activity before modification". "Enhancing" or "increasing" the activity of a polypeptide or protein relative to natural activity means that the activity of a particular polypeptide or protein is improved compared to the activity originally possessed by the parent strain or unmodified microorganism prior to transformation.

The term "increased activity" can mean an increase achieved by introducing a foreign polypeptide or protein, or increasing the activity of an endogenous polypeptide or protein, particularly achieved by increasing the activity of an endogenous polypeptide or protein. This increase in the activity of the polypeptide or protein can be determined based on the increase in the degree of activity of the polypeptide or protein, its level of expression, or the amount of product it produces.

As used herein, the expression "enhancing or increasing the activity of a protein encoded by the ssuABC gene or a SsuABC protein" may also be referred to as "a genetic modification to increase the activity of a protein encoded by the ssuABC gene", and this means that the activity of at least one protein , selected from the group consisting of the SsuA, SsuB and SsuC proteins that make up the SsuABC protein, is increased compared to natural activity.

Increasing the activity of the SsuABC protein may include both increasing the activity by introducing at least one protein selected from the group consisting of foreign proteins SsuA, SsuB and SsuC and increasing the activity of at least one protein selected from the group consisting of endogenous proteins SsuA, SsuB and SsuC.

As used herein, the term “introducing a protein” refers to providing the activity of a specific protein in a microorganism that does not initially have that protein, or enhancing the activity of the protein compared to the natural activity of the protein or activity before modification. For example, introduction of a protein may refer to the introduction of a particular protein, the introduction of a polynucleotide encoding a particular protein into the chromosome of a microorganism, or the introduction of a vector containing a polynucleotide encoding a particular protein into a microorganism, thereby providing the activity of the protein.

Increasing the activity of a polypeptide or protein can be achieved by using various methods well known in the art, without limitation, provided that the activity of the target polypeptide or protein is enhanced compared to that of the microorganism before modification. In particular, any genetic engineering and/or protein engineering techniques well known in the art as conventional molecular biology techniques may be used (Sitnicka et al. Functional Analysis of Genes. AdvΔNces in Cell Biology. 2010, Vol. 2.1 -16, Sambrook et al. Molecular Cloning 2012 and so on).

Specifically, in the present invention, enhancement of activity can be achieved by:

(1) increasing the number of copies of a gene or polynucleotide encoding a polypeptide or protein in a cell;

(2) replacing the region of regulation of expression of a gene encoding a polypeptide or protein on the chromosome with a sequence with higher activity;

(3) modification of the base sequence encoding the start codon or the 5'-UTR (5'-untranslated region) region of the polypeptide or protein;

(4) modification of the nucleotide sequence on the chromosome to enhance the activity of the polypeptide or protein;

(5) introducing a foreign polynucleotide having the activity of the given polypeptide or protein, or a codon-optimized version of the polynucleotide; or

(6) modifications to enhance activity by any combination of the methods described above, without limitation.

A method of increasing the activity of a polypeptide or protein by protein engineering can be performed by modifying or chemically modifying a surface region selected by analyzing the three-dimensional structure of the polypeptide or protein, but is not limited to this.

Increasing the copy number of a gene or polynucleotide encoding a polypeptide or protein described in (1) above can be accomplished by any method well known in the art, for example, by introducing a vector that replicates and functions independently of the host cell and is operably linked with a gene or polynucleotide encoding a polypeptide or protein into a host cell. Alternatively, copy number expansion may be accomplished by introducing, but not limited to, a vector that is operably linked to a gene and is capable of inserting the gene or polynucleotide into a host cell chromosome into the host cell.

Replacing the expression regulatory region (or expression regulatory sequence) of a gene encoding a polypeptide or protein on a chromosome with a sequence of higher activity described in (2) above can be accomplished by any method known in the art, for example by introducing a mutation into the sequence by deletion, insertion, non-conservative or conservative substitution, or any combination thereof, or by replacing the sequence with a sequence with higher activity to further enhance the activity of the expression regulatory region. The expression control region may comprise a promoter; statement sequence; a sequence encoding a ribosome binding site; and a sequence for regulating transcription and translation termination, but not limited to. For example, the method may be performed by attaching a stronger heterologous promoter in place of the natural promoter, but is not limited to this.

Examples of stronger promoters known in the art may include cj1 - cj7 promoters (US Patent No. US 7,662,943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, lysCP1 promoter (US 2010-0317067 A1), spl1 promoter, spl7 promoter, spl13 promoter (US 10584338 B2), gap A promoter, EF-Tu promoter, groEL promoter, aceA or aceB promoter, 02 promoter (US Patent No. US 10273491 B2), the tkt promoter and the uscA promoter, but are not limited to them.

Modification of the base sequence encoding the initiation codon or the 5'-UTR region of a polypeptide or protein described in (3) above can be accomplished by any method known in the art, for example, by replacing the natural initiation codon with another initiation codon with a higher the expression level of the polypeptide or protein, without limitation.

Modification of a nucleotide sequence on a chromosome to enhance the activity of a polypeptide or protein described in (4) above can be accomplished by any method known in the art, for example by inducing modification of an expression control sequence by deletion, insertion, non-conservative or conservative substitution, or any combinations thereof to further enhance the activity of the nucleotide sequence or replace the sequence with a nucleotide sequence modified to increase activity. The replacement may be, but is not limited to, the insertion of a gene into a chromosome by homologous recombination. The vector used herein may additionally contain a selectable marker to determine insertion into the chromosome.

Administration of a foreign polynucleotide having the activity of a polypeptide or protein described in (5) above can be accomplished by any method known in the art, for example, by introducing a foreign polynucleotide encoding a polypeptide or protein having an activity identical/similar to that of the polypeptide or protein, or introducing a codon-optimized version of this polynucleotide into a host cell. The origin or sequence of the foreign polynucleotide is not particularly limited as long as the foreign polynucleotide exhibits activity identical/similar to that of the polypeptide or protein. In addition, a foreign polynucleotide that is codon optimized for optimized transcription and translation in the host cell may be introduced into the host cell. Administration can be accomplished by any known transformation method suitably selected by one of ordinary skill in the art. As the administered polynucleotide is expressed in the host cell, a polypeptide or protein is produced, thereby increasing its activity.

Finally, the combination of the above methods described in (6) can be accomplished by using one or more than one method described in (1)-(5)- Enhancing the activity of a polypeptide or protein as described above may be an increase in activity or concentration of the polypeptide or protein compared to the activity or concentration of the polypeptide or protein expressed in wild-type or unmodified microorganism strains, or an increase in the amount of product produced by the polypeptide or protein, without limitation.

When used herein, the term "strain before modification" or "microorganism before modification" does not exclude strains containing naturally occurring mutations in the microorganism and may refer to a wild-type strain or a naturally occurring strain or a strain before transformation by genetic modification due to a natural or man-made factor . The terms "strain before modification" or "microorganism before modification" can be used interchangeably with the terms "unmutated strain", "unmodified strain", "unmutated microorganism", "unmodified microorganism" or "reference microorganism".

As used herein, the term “vector” refers to a DNA construct containing the nucleotide sequence of a polynucleotide encoding a target protein and operably linked to a suitable regulatory sequence so as to be capable of expressing the target protein in a suitable host cell. The regulatory sequence may include a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating transcription and translation termination. When a suitable host cell is transformed with a vector, the vector can replicate or function independently of the host genome or can be integrated into the host genome. For example, a polynucleotide encoding a target protein can be inserted into a chromosome using a chromosome insertion vector in cells. Insertion of a polynucleotide into a chromosome can be accomplished by any method known in the art, such as, but not limited to, homologous recombination. The vector may further contain a selectable marker to determine insertion into the chromosome. A selectable marker is used to select cells that are transformed with the vector, that is, to confirm insertion of the desired nucleic acid molecules, and examples of selectable markers may include markers that provide selectable phenotypes such as drug tolerance, nutrient requirements, resistance to cytotoxic agents or surface polypeptide expression. Only cells expressing the selectable marker are able to survive or exhibit different phenotypes under environmental conditions treated with the selection agent, and thus transformed cells can be selected.

The vector used in the present invention is not particularly limited, and any vector known in the art can be used. Examples of vectors commonly used in the art may include natural or recombinant plasmid, cosmid, virus and bacteriophage. For example, pWE15, M13, MBL3, MBL4, GHP, AS NI, ARP, t10, til, Charon4A and Charon21A can be used in as a phage vector or cosmid vector. As the plasmid vector, pBR type, pUC type, pBluescriptll type, pGEM type, pTZ type, pCL type and pET type can be used. In particular, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118 and pCC1BAC can be used. However, the present embodiment is not limited to them.

As used herein, the term “transformation” refers to the process of introducing a vector containing a polynucleotide encoding a target protein into a host cell or microorganism such that the polypeptide encoded by the polynucleotide is expressed in the host cell. The transformed polynucleotide may be present either in a form inserted into the chromosome of the host cell or in a form located outside the chromosome, provided that the protein is expressed in the host cell. In addition, the polynucleotide includes DNA and/or RNA encoding the target protein. The polynucleotide can be introduced into a host cell in any form, provided that the polynucleotide is introduced into the host cell and the polypeptide is expressed therein. For example, the polynucleotide can be introduced into a host cell in the form of an expression cassette, which is a genetic construct that includes all the essential elements necessary for self-replication. An expression cassette may typically comprise a promoter operably linked to a polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of a self-replicating expression vector. Also, the polynucleotide may be introduced into a host cell in its original form and is operably linked to, but is not limited to, a sequence required for expression in the host cell.

In addition, as used herein, the term “operably linked” refers to a functional relationship between a promoter sequence that enables the initiation and process of transcription of a polynucleotide encoding a target protein of the present invention and a gene sequence.

The vector transformation methods of the present invention include any method that allows the introduction of a nucleic acid into a host cell, and can be performed by any suitable standard methods well known in the art, selected in accordance with the host cell. For example, electroporation, calcium phosphate precipitation (CaPO ₄ ), calcium chloride precipitation (CaCl ₂ ), microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method can be used, but the present invention is not limited to this.

The microorganism of the present invention may include both wild-type microorganisms and microorganisms containing natural or artificial genetic modifications, and any microorganism into which a thiosulfate transporter has been introduced or containing a thiosulfate transporter of the present invention may be included herein without limitation.

The microorganism of the present invention may contain at least one of the thiosulfate transporter of the present invention; a polynucleotide encoding such a thiosulfate transporter, and a vector containing such a polynucleotide.

The microorganism may be a microorganism that produces L-amino acids and/or derivatives thereof.

As used herein, the term "microorganism producing L-amino acids and/or derivatives thereof" includes both a microorganism naturally having the ability to produce L-amino acids/derivatives thereof and a microorganism obtained by imparting the ability to produce L-amino acids/derivatives thereof. parental strain unable to produce L-amino acids or their derivatives. In particular, any microorganism containing a genetic modification to produce the target L-amino acid or its derivatives, due to the presence of a certain mechanism, weakened or enhanced by the introduction of an exogenous gene or by enhancing or inactivating the activity of an endogenous gene.

For example, the microorganism may be a microorganism in which the L-amino acid biosynthetic pathway is enhanced or the L-amino acid degradation pathway is weakened. For example, an L-amino acid producing microorganism may be a microorganism in which the L-methionine biosynthetic pathway is enhanced.

For example, the microorganism may be a microorganism in which the activity of the methionine cysteine biosynthesis repressor protein (McbR) or MetJ protein is weakened or eliminated, or a microorganism in which the ability to produce methionine is enhanced and/or increased by increasing the activity of methionine synthase (MetH) or sulfite reductase (CysI). Alternatively, the microorganism may be a microorganism in which the expression of a gene encoding an enzyme involved in the L-amino acid biosynthetic pathway is enhanced or an enzyme involved in the L-amino acid degradation pathway is weakened/inactivated.

In particular, examples of proteins or genes whose expression can be controlled to enhance the L-amino acid biosynthesis pathway or weaken/inactivate the L-amino acid degradation pathway are as follows. They are presented in the following order: protein; a representative gene encoding a protein and its representative EC number. The first letter of the protein name is capitalized, and the gene name is indicated in italics. For example, a thiosulfate serotransferase such as Rdl2p, GlpE, PspE, YgaP, Thil, YbbB, SseA, YnjE, YceA, YibN, NCgl0671, Ncgl1369, NCgl2616, NCgl0053, NCgl0054, NCG12678 and NCgl289 can be used 0; sulfite reductase, CysI; thiosulfate/sulfate transport system, cysPUWA (EC 3.6.3.25); 3'-phosphoadenosine 5'-phosphosulfate reductase, cysH (EC 1.8.4.8); sulfite reductase, cysJI (EC 1.8.1.2); cysteine synthase A, cysK (EC 2.5.1.47); cysteine synthase B, cysM (EC 2.5.1.47); serine acetyltransferase, cysE (EC 2.3.1.30); glycine cleavage system, gcvTHP-lpd (EC 2.1.2.10, EC 1.4.4.2, EC 1.8.1.4); lipoyl synthase, lipA (EC 2.8.1.8); lipoyl protein ligase, lipB (EC 2.3.1.181); phosphoglycerate dehydrogenase, serA (EC 1.1.1.95); 3-phosphoserine phosphatase, serB (EC 3.1.3.3); 3-phosphoserine/phosphohydroxythreonine aminotransferase, serC (EC 2.6.1.52); serine hydroxymethyltransferase, glyA (EC 2.1.2.1); aspartokinase I (EC 2.7.2.4); homoserine dehydrogenase I, thrA (EC 1.1.1.3); aspartate kinase, lysC (EC 2.7.2.4); homoserine dehydrogenase, hom (EC 1.1.1.3); homoserine-(9-acetyltransferase, metX (EC 2.3.1.31); homoserine-O-succinyltransferase, metA (EC 2.3.1.46); cystathionine hashish synthase, metB (EC 2.5.1.48); p-C-S-lyase, aecD (EC 4.4.1.8, beta-lyase); cystathionine beta-lyase, metC (EC 4.4.1.8); B12-independent homocysteine S-methyltransferase, metE (EC 2.1.1.14); methionine synthase, metH (EC 2.1.1.13 ); methylenetetrahydrofolate reductase, metF (EC 1.5.1.20); L-methionine exporter BrnFE; valine exporter YgaZH (B2682, B2683), ygaZH (B2682, B2683); exporter YjeH, b414P, pyridine nucleotide transhydrogenase PntAB, pntAB (EC 1.6.1.2 ); (9-succinyl-homoserine sulfhydrylase, MetZ (EC 2.5.1.48) and phosphoenolpyruvate carboxylase, Rus (EC 4.1.1.31). The biosynthetic pathway of L-amino acids can be enhanced, or the pathway for their cleavage can be weakened by increasing the activity of one or more than one protein described above, or several proteins making up the system, or by overexpression of polynucleotides encoding such proteins Alternatively, at least one protein selected from glucose-6-phosphate isomerase, pgi (EC 5.3.1.9); homoserine kinase, thrB (EC 2.7.1.39); S-adenosine methionine synthase, metK (EC 2.5.1.6); dihydrodipicolinate synthase, dapA (EC 4.2.1.52); phosphoenolpyruvate carboxykinase, rec (EC 4.1.1.49); formyltetrahydrofolate hydrolases, purU (EC 3.5.1.10); pyruvate kinase I, pykF (EC 2.7.1.40); pyruvate kinase II, rukA (EC 2.7.1.40); cystathionine-γ-lyase, cg3086 (EC 4.4.1.1); cystathionine β-synthase, cg2344 (EC 4.2.1.22); regulatory protein Cg3031, cg3031; repressor protein of methionine and cysteine biosynthesis McbR, mcbR; Met transcription repressor protein, metJ; L-methionine transporter MetQNI, metQ, metN, metl; N-acyltransferases, yncA; The sRNA (small RNA) of fnrS and the L-methionine transporter, metP, may be inactivated or attenuated, or the expression of the gene encoding the protein may be suppressed or eliminated.

However, these are only examples, and the microorganism may be a microorganism in which the expression of a gene encoding an enzyme involved in various known L-amino acid biosynthetic pathways is increased, or an enzyme involved in degradation pathways is inactivated/impaired. The increase in protein activity and increase in gene expression are as described above.

As used herein, the term “inactivation” or “attenuation” of a polypeptide or protein is a concept that includes both reduction and elimination of activity compared to natural activity. The terms "inactivation" or "attenuation" can be used interchangeably with the terms "down-regulation", "reduction" and "reduction". Inactivation or attenuation may include the case where the activity of the protein is reduced or eliminated compared to the natural activity of the microorganism by mutation of the gene encoding the protein, modification of the expression control sequence, or complete or partial deletion of the gene; a case where the overall activity of a protein in a cell is lower than that in native strains or unmodified strains due to inhibition of expression or translation of the gene encoding the protein; the case where the gene is not expressed, and the case where there is no activity despite the gene being expressed.

In the present invention, protein inactivation/attenuation can be achieved by various methods well known in the art, without limitation (Nakashima N. et al., Bacterial cellular engineering by genome editing and gene silencing. IntJMol Sci. 2014;15 (2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

Examples of methods include:

(1) complete or partial deletion of a gene encoding a protein,

(2) modification of the expression regulatory region (or expression regulatory sequence) to reduce the expression of the gene encoding the protein,

(3) modification of the sequence of a gene encoding a protein to eliminate or weaken the activity of the protein,

(4) the introduction of an antisense oligonucleotide (for example, the introduction of an antisense RNA) that binds complementarily to the transcript of the gene encoding the protein,

(5) adding a sequence complementary to the Shine-Dalgarno sequence of a protein-coding gene upstream of the Shine-Dalgarno sequence to form a secondary structure that prevents ribosome binding to this sequence,

(6) adding a promoter for reverse transcription at the 3' end of the open reading frame (ORF) of the nucleotide sequence of a gene encoding a protein (Reverse Transcription Technology, RTE), or any combination thereof, but not limited to.

In particular, complete or partial deletion of a protein-coding gene can be accomplished by replacing a polynucleotide encoding a natural target protein on a chromosome with a multi-nucleotide deletion polynucleotide or a marker gene using a vector for insertion into a chromosome in a microorganism. As an example of complete or partial deletion of a polynucleotide, a method for deleting a polynucleotide by homologous recombination can be used, but is not limited to this.

In addition, complete or partial deletion of a gene can be performed by inducing a mutation using radiation such as UV radiation or a chemical and selecting mutant strains with deletion of the target gene. Producing a gene deletion may involve a method using recombinant DNA technology. Recombinant DNA technology can be used by inducing homologous recombination by inserting a nucleotide sequence or vector having homology to a target gene into a microorganism. Additionally, the inserted nucleotide sequence or vector may contain, but is not limited to, a dominant selectable marker.

In addition, modification of the expression control sequence can be accomplished using various methods well known in the art. For example, the modification can be made by inducing a mutation in the expression regulatory region (expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or any combination thereof to further reduce the activity of the expression regulatory region (expression regulatory sequence) or by replacing the sequence with a sequence having weaker activity. The expression control region may comprise, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a transcription and translation termination control sequence.

Also, modification of a gene sequence can be accomplished by inducing a mutation in the gene sequence by deletion, insertion, non-conservative or conservative substitution, or any combination thereof to further reduce the activity of the polypeptide, or by replacing that sequence with a gene sequence modified to have weaker activity, or a gene sequence modified to have no activity, but not limited to.

For example, the expression of a gene can be suppressed or attenuated by the formation of a termination codon by introducing a mutation into the gene sequence.

However, the above methods are examples only, and those of ordinary skill in the art can produce a microorganism that produces L-amino acids and/or derivatives thereof using any methods known in the art.

The L-amino acid and/or a derivative thereof may be a sulfur-containing amino acid and/or a derivative of a sulfur-containing amino acid.

As used herein, the term “sulfur-containing amino acid” or “sulfur-containing amino acid derivative” refers to a sulfur-containing amino acid or derivative thereof, particularly those selected from methionine, cysteine, cystine, lanthionine, homocysteine, homocystine, homolanthionine and taurine, but not limited to them; any sulfur-containing amino acid and its derivatives may be included within the scope of the present invention without limitation.

The microorganism of the present invention may be a microorganism belonging to the genus Corynebacterium sp., the genus Escherichia sp., or the genus Lactobacillus sp., but is not limited to them. The microorganism may include any microorganism having an increased ability to produce L-amino acids and/or derivatives thereof by enhancing the activity of endogenous SsuABC protein or introducing foreign SsuABC protein without limitation.

"Microorganism belonging to the genus Corynebacterium" may include any microorganisms belonging to the genus Corynebacterium. In particular, the microorganism may be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium crenatum, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Coryne bacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens, and more particularly Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium ammoniagenes, Corynebacterium callunae or Corynebacterium deserti, even more particularly Corynebacterium glutamicum, but not limited to them.

"Microorganism belonging to the genus Escherichia'" may include any microorganisms belonging to the genus Escherichia. In particular, the microorganism may be, but is not limited to, Escherichia coli.

The microorganism of the present invention may be any microorganism containing the thiosulfate transporter of the present invention and using thiosulfate as a sulfur source.

The production method of the present invention may include cultivating the microorganism of the present invention in a culture medium containing thiosulfate.

As used herein, the term "cultivation" refers to growing a microorganism under suitably selected conditions. The cultivation process of the present invention can be carried out in accordance with suitable media and culture conditions known in the art. The cultivation process can be easily adapted for use by one skilled in the art according to the strain selected. The cultivation of the microorganism can be performed in a batch process, a continuous process, a fed-batch process, and so on, known in the art, but not limited to them.

As used herein, the term “culture medium” refers to a material in which the nutrients necessary for culturing a microorganism are mixed as the main ingredients, as well as nutrient stores and growth factors, such as water, that are necessary for survival and growth. In particular, although the culture medium and other culture conditions for cultivating the microorganism of the present invention are not particularly limited, as long as the culture media are those commonly used in cultivating microorganisms, the microorganism of the present invention can be cultured in a conventional medium containing suitable carbon sources , nitrogen sources, phosphorus sources, inorganic compounds, amino acids and/or vitamins under aerobic conditions while controlling temperature, pH and the like.

In the present invention, the carbon sources may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid and citric acid; and amino acids such as glutamic acid, methionine and lysine. In addition, natural organic nutrients such as starch hydrolysates, molasses, raw molasses, rice bran, cassava, sugar cane cake and corn extract can be used, and in particular carbohydrates such as glucose and sterile pre-processed molasses (i.e. , molasses converted to reduced sugars), and suitable amounts of any other carbon sources may also be used without limitation. These carbon sources can be used individually or in combination of at least two of them, but not limited to.

Nitrogen sources may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate and ammonium nitrate; and organic nitrogen sources such as amino acids, e.g. glutamic acid, methionine and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn extract, casein hydrolysate, fish or its derivatives and defatted soybean meal or products its processing. These nitrogen sources can be used individually or in combination of at least two of them, without limitation.

Sources of phosphorus may include potassium dihydrogen phosphate, potassium hydrogen phosphate, or their corresponding sodium salts. As inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, calcium carbonate and the like can be used. It is also possible to additionally include amino acids, vitamins and/or suitable precursors. These components and precursors can be added to the culture medium in a batch or continuous process, but are not limited to them.

Also, during the microorganism cultivation process, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, orthophosphoric acid and sulfuric acid can be added to the culture medium in a suitable manner to adjust the pH of the culture medium. Also, during culture, an antifoam agent, such as a polyglycol fatty acid ester, can be added to inhibit foam formation. Additionally, oxygen or an oxygen-containing gas may be introduced into the culture medium to maintain the culture medium under aerobic conditions, or nitrogen, hydrogen, or carbon dioxide gas may be introduced into the culture medium to maintain the culture medium under anaerobic and microaerobic conditions without introducing any other gases, but this embodiment is not limited to this.

The temperature of the culture medium can be maintained in the range of 25°C to 40°C, more particularly 30°C to 37°C, but not limited to. Cultivation may be continued until the desired amount of product is obtained, for example, but not limited to 0.5 hours to 60 hours.

The term "sulfur source" of the present invention can be used interchangeably with the term "sulfur supply source" and refers to a sulfur-containing substance available for the production of a sulfur-containing amino acid.

When cultivating a microorganism, the sulfur source may be an important factor in determining the metabolic pathway of the microorganism. However, the factors involved in the transport of various sulfur sources and the factors involved in their breakdown have not been precisely identified. For example, although wild-type Corynebacterium glutamicum is known to utilize a variety of sulfur sources, the SsuABC protein is not known to be involved in sulfate or sulfite transport, but only in aliphatic sulfonate transport (D. J. Koch, C. Ruckert, D. A. Rey, A. Mix, A. Puhler, J. Kalinowski. 2005. Role of the ssu and sen Genes of Corynebacterium glutamicum ATCC 13032 in Utilization of Sulfonates and Sulfonate Esters as Sulfur Sources. AEM. 71.10.6104-6114. 2005). That is, the protein that transports a source of sulfur into the cell has substrate specificity. Additionally, after a sulfur source is transported into the cell, the enzyme that breaks down the sulfur source may vary, and the metabolic pathway using it may also vary depending on the structure and functional group of the sulfur source. For example, it is known that when sulfate is used as a source of sulfur, CysZ transports the sulfate, and CysDN, CysH and CysI are involved until the sulfide is produced (Bolten, Christoph J., Hartwig Schroder, Jeroen Dickschat, and Christoph Wittmann. Towards Methionine Overproduction in Corynebacterium glutamicum Methanethiol and Dimethyldisulfide as Reduced Sulfur Sources J Microbiol Biotechnol (2010), 20(8), 1196-1203. However, when thiosulfate is used as a source of sulfur for the production of sulfur-containing amino acids, the factors used for the transport and breakdown of thiosulfate have not yet been reliably identified.

The sulfur source may be thiosulfate. Particularly in the present invention, the sulfur source may include a thiosulfate, such as ammonium thiosulfate or sodium thiosulfate, or a mixture of thiosulfate and an organic or inorganic sulfur-containing compound such as sulfite, a reduced feedstock, such as H2S, sulfide, a sulfide derivative, methyl mercaptan, thioglycolate, thiocyanate and thiourea. Alternatively, the sulfur source may contain no substance other than thiosulfate. However, the present embodiment is not limited to this.

A method for producing sulfur-containing amino acids or sulfur-containing amino acid derivatives may include isolating the sulfur-containing amino acids or sulfur-containing amino acid derivatives from a microorganism or a culture medium.

The isolation step can be performed by collecting the desired sulfur-containing amino acids or sulfur-containing amino acid derivatives using a suitable method known in the art in accordance with the culture method of the present invention, such as a batch culture method, a continuous culture method, or a fed-batch culture method. For example, centrifugation, filtration, treatment with a protein precipitation agent (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographic techniques such as molecular sieve chromatography (gel permeation), adsorption chromatography, ion exchange chromatography and affinity chromatography can be used, high performance liquid chromatography (HPLC), and any combination thereof, but not limited to.

The isolation step may further include a purification process. The purification process can be performed using a suitable method known in the art.

In another aspect of the present invention, a composition is provided for producing a sulfur amino acid or a sulfur amino acid derivative, wherein the composition comprises a microorganism having increased activity of the protein encoded by the ssuABC gene compared to its natural activity, or a culture thereof, and thiosulfate.

The protein encoded by the ssuABC gene, microorganism, thiosulfate and sulfur-containing amino acid are as described above.

The culture can be obtained by cultivating the microorganism of the present invention in a culture medium.

The composition for producing a sulfur-containing amino acid or a sulfur-containing amino acid derivative of the present invention may further include any component that can promote the production of sulfur-containing amino acids or sulfur-containing amino acid derivatives, and this component may be suitably selected from those known in the art.

Another aspect of the present invention provides the use of a protein encoded by the ssuABC gene as a thiosulfate transporter.

Another aspect of the present invention provides the use of a microorganism containing a genetic modification to increase the activity of a protein encoded by the ssuABC gene, compared to an unmodified microorganism, to produce sulfur-containing amino acids or derivatives of sulfur-containing amino acids.

The protein encoded by the ssuABC gene, microorganism, cultures, thiosulfate and sulfur-containing amino acid are as described above. Examples

Hereinafter, the present invention will be described in more detail with reference to the following examples and experimental examples. However, the following examples and experimental examples are presented solely to illustrate the present description and do not limit the scope of the present invention.

Example 1: Preparation of a recombinant vector for deleting the mcbR gene

First, in order to obtain a strain producing methionine as a representative sulfur-containing amino acid, Corynebacterium glutamicum strain ATCC 13032 was used to obtain a vector for inactivating the known mcbR gene encoding a protein that regulates the transcription of methionine and cysteine (J. Biotechnol. 103:51-65, 2003).

Specifically, in order to delete the mcbR gene from the chromosome of Corynebacterium glutamicum strain ATCC 13032, a recombinant plasmid vector was prepared according to the following method.

Based on the nucleotide sequence deposited in GenBank of the US National Institutes of Health (NIH), the mcbR gene and flanking sequences (SEQ ID NO: 1) of Corynebacterium glutamicum were obtained.

PCR was carried out using chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template and primers SEQ ID NO: 2, 3, 4 and 5. PCR was carried out under the following 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.

Vector pDZ (US Patent No. US 9109242 B2), unable to replicate in Corynebacterium glutamicum, and amplified fragments of the mcbR gene were treated with the restriction enzyme Smal for insertion into the chromosome, followed by cloning by isothermal assembly. Escherichia coli DH5a was transformed with this vector and plated on solid LB medium containing 25 mg/L kanamycin. Colonies transformed with a vector into which a fragment having a deletion of the target gene had been inserted by PCR were selected, and the plasmid was then isolated by plasmid extraction and designated pDZ-AmcbR.

Example 2: Obtaining and cultivating a strain with a deletion of the mcbR gene

Strain ATCC 13032 was transformed by electroporation with the pDZ-AmcbR vector obtained in Example 1 above by homologous chromosomal recombination (Van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Then a second recombination was carried out in a solid medium containing sucrose. After completion of the second recombination, a transformed Corynebacterium glutamicum strain having a deletion of the mcbR gene was identified by PCR using SEQ ID NOs: 6 and 7, and the recombinant strain was named CM02-0618.

Strain CM02-0618 was deposited at the Korean Microbial Culture Center under the Budapest Agreement on January 4, 2019, with accession number KCCM12425P.

To analyze the ability of the resulting strain CM02-0618 to produce L-methionine, this strain and the parent strain, Corynebacterium glutamicum strain ATCC 13032, were cultivated as follows.

Corynebacterium glutamicum ATCC 13032 and Corynebacterium glutamicum CM02-0618 were inoculated into a 250 ml flask with corner baffles containing 25 ml of seed medium and cultured with shaking at 30°C for 20 hours at 200 rpm. Then 1 ml of this culture broth was inoculated into a 250 ml corner flask containing 24 ml of production medium and cultured with shaking at 30°C for 48 hours at 200 rpm. The composition of the seeding medium and production medium was as follows. In the production environment, (NH ₄ ) ₂ S ₂ O ₃ , which is a type of thiosulfate, was used as a sulfur source.

Sowing 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 calcium pantothenate and 2000 mcg nicotinamide (per 1 liter of distilled water).

Production medium (pH 8.0)

50 g glucose, 12 g (NH ₄ ) ₂ S ₂ O ₃ , 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 of calcium pantothenate, 3000 µg of nicotinamide, 30 g of CaCO ₃ and 1 µg of cyanocobalamin (vitamin B12) (per 1 liter of distilled water).

The strains were cultured according to the above-described culture method and the concentrations of L-methionine contained in the culture broth were analyzed and are presented in Table 1 below.

The strains were cultured according to the above-described culture method and the concentrations of L-methionine contained in the culture broth were analyzed and are presented in Table 1 below.

As a result, it was confirmed that the ability of the mcbR deletion strain to produce L-methionine was increased by 0.04 g/L compared to that of the control strain. It was also confirmed that methionine was produced even when thiosulfate was used as the sole sulfur source.

Example 3: Selecting a Gene Involved in Thiosulfate Influx by Transcript Analysis

Thiosulfate-specific proteins of strains of the genus Corynebacterium are not known. However, as confirmed in Example 2, strain CM02-0618 produced methionine when thiosulfate was used as the sole sulfur source, and therefore an experiment was conducted to select the protein involved in thiosulfate supply.

In more detail, after culturing the strain CM02-0618 obtained in Example 2, changing only the sulfur source (ammonium sulfate and ammonium thiosulfate), transcriptomic analysis (RNA-level analysis) was performed. The same cultivation method was used as in Example 2.

Based on the results of this experiment, it was confirmed that the RNA levels of genes encoding SsuABC (Ncgl1174-76), which is known as a sulfonate transporter, were significantly increased.

Thus, it was confirmed that the SsuABC protein does not interact with sulfate, but specifically interacts with thiosulfate, and therefore it can be assumed that this protein is involved in thiosulfate transport.

Example 4: Confirmation of the effect of a strain with a deletion of the gene encoding the SsuABC protein

A vector was obtained to determine the effect of inactivation of the SsuABC protein, selected as the protein that specifically interacts with thiosulfate in Example 3.

Example 4-1: Preparation of a vector for deleting the gene encoding the SsuABC protein

To delete the gene encoding the SsuABC protein (hereinafter referred to as the ssuABC gene) from the chromosome of Corynebacterium strain ATCC 13032, a recombinant plasmid vector was obtained in accordance with the following method.

Based on nucleotide sequences deposited in GenBank of the US National Institutes of Health (NIH), the ssuABC gene and flanking sequences (SEQ ID NO: 8) of Corynebacterium glutamicum were obtained.

To delete the ssuABC gene, PCR was performed using chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template and primers SEQ ID NO: 9, 10, 11 and 12. PCR was carried out under the following 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, unable to replicate in Corynebacterium glutamicum, and amplified fragments of the ssuABC gene were treated with the restriction enzyme Smal for insertion into the chromosome, followed by cloning by isothermal assembly. Escherichia coli DH5a was transformed with this vector and plated on solid LB medium containing 25 mg/L kanamycin. Colonies transformed with a vector into which a fragment having a deletion of the target gene was inserted by PCR were selected, and then the plasmid was obtained by plasmid extraction and designated pDZ-ΔSsuABC.

Example 4-2: Preparation and cultivation of a strain with a deletion of the ssuABC gene Strain 13032/AmcbR was transformed by electroporation with the pDZ-ΔSsuABC vector obtained in Example 4-1 above by homologous chromosomal recombination (Van der Rest et al., Appl Microbiol Biotechnol 52: 541-545, 1999). Then a second recombination was carried out in a solid medium containing sucrose. After completion of the second recombination, a transformed Corynebacterium glutamicum strain having a deletion of the mcbR gene was identified by PCR using SEQ ID NOs: 13 and 14, and the recombinant strain was named Corynebacterium glutamicum CM02-0618/ΔSsuABC.

Example 4-3: Analysis of the ability of a strain with a deletion of the ssuABC gene to produce methionine

To analyze the ability to produce L-methionine of the resulting strain CM02-0618/ΔSsuABC, this strain and the parent strain, Corynebacterium glutamicum strain ATCC 13032, were cultivated as follows.

Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum CM02-0618 obtained in Example 2, and CM02-0618/ΔSsuABC obtained in Example 4-2 were inoculated into a 250 ml corner baffled flask containing 25 ml of seed medium and cultured with shaking at 30°C for 20 hours at 200 rpm. Then 1 ml of this culture broth was inoculated into a 250 ml corner flask containing 24 ml of production medium and cultured with shaking at 30°C for 48 hours at 200 rpm. The composition of the seeding medium and production medium was as follows.

Sowing 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 calcium pantothenate and 2000 mcg nicotinamide (per 1 liter of distilled water).

Production medium (pH 8.0)

50 g glucose, 12 g (NH ₄ ) ₂ S ₂ O ₃ , 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 of calcium pantothenate, 3000 µg of nicotinamide and 30 g of CaCO ₃ (based on 1 liter of distilled water).

The strains were cultured according to the above-described culture method and the concentrations of L-methionine contained in the culture broth were analyzed and are shown in Table 3 below.

As a result, it was confirmed that the ability of the ssuABC gene deletion strain to produce L-methionine was reduced to approximately 25% compared to that of the control strain. Based on this, the SsuABC protein was confirmed to be a protein involved in thiosulfate uptake.

Example 5: Obtaining and cultivating a strain with increased expression of the ssuABC gene

A vector was obtained to enhance the activity of the SsuABC protein, selected as a protein that specifically interacts with thiosulfate in Example 3.

Example 5-1: Preparation of a vector to enhance the expression of the ssuABC gene

To additionally insert the ssuABC gene into the chromosome of Corynebacterium ATCC 13032, a plasmid vector was obtained in accordance with the following method.

First, a deletion vector for Ncgl1464 (Transposase) was generated to insert the ssuABC gene.

Based on nucleotide sequences deposited in GenBank of the US National Institutes of Health (NIH), the Ncgl1464 gene and flanking sequences (SEQ ID NO: 15) of Corynebacterium glutamicum were obtained. To delete the Ncgl1464 gene, PCR was performed using chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template and primers SEQ ID NO: 16, 17, 18 and 19. PCR was carried out under the following 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, respectively.

The pDZ vector, unable to replicate in Corynebacterium glutamicum, and amplified fragments of the Ncgl1464 gene were treated with the restriction enzyme Smal for insertion into the chromosome, followed by cloning by isothermal assembly. Escherichia coli DH5a was transformed with this vector and plated on solid LB medium containing 25 mg/L kanamycin. Colonies transformed with a vector into which a fragment having a deletion of the target gene was inserted by PCR were selected, and then the plasmid was obtained by plasmid extraction and designated pDZ-ΔNcgl1464.

PCR was then performed to obtain fragments of the ssuABC gene using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template and SEQ ID NO: 20 and 21. Additionally, the PgapA promoter was used to enhance the expression of the ssuABC gene. To obtain them, PCR was performed using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template using primers SEQ ID NO: 22 and 23. PCR was carried out under the following 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, fragments of the ssuABC gene and fragments of the gapA promoter were obtained.

The pDZ-ΔNcgl1464 vector, unable to replicate in Corynebacterium glutamicum, was treated with the restriction enzyme Seal, followed by cloning by isothermal assembly along with two amplified DNA fragments. Escherichia coli DH5a was transformed with this vector and plated on solid LB medium containing 25 mg/L kanamycin. Colonies transformed with the vector into which the target gene was inserted by PCR were selected, and then the plasmid was obtained by plasmid extraction and designated pDZ-ΔNcgl1464-PgapΔSsuABC.

Example 5-2: Preparation and cultivation of a strain with increased expression of the ssuABC gene

Strain CM02-0618 was transformed by electroporation with the vectors pDZ-ΔNcgl1464 and pDZ-ΔNcgl1464-PgapΔSsuABC obtained in Example 5-1 above by homologous chromosomal recombination (Van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Then a second recombination was carried out in a solid medium containing sucrose. After completion of the second recombination, a transformed Corynebacterium glutamicum strain with a deletion of the Ncgl1464 gene and a Corynebacterium glutamicum strain having both a deletion of the Ncgl1464 gene and an insertion of the ssuABC gene were identified by PCR using primers SEQ ID NO: 24 and 25. The strain with the deletion of the Ncgl1464 gene was obtained named CM02-0618/ΔNcgl1464, and the strain having both a deletion of the Ncgl1464 gene and an insertion of the ssuABC gene was named CM02-073. Strain CM02-0735 was deposited at the Korean Microbial Culture Center in accordance with the Budapest Agreement on March 21, 2021, with accession number KCCM12466R.

Example 5-3: Analysis of the ability of a strain with increased expression of the ssuABC gene to produce methionine

To analyze the ability of the resulting strains CM02-0618/ΔNcgl1464 and CM02-0735 to produce L-methionine, these strains and the parent strain, strain CM02-0618, were cultivated as follows.

Strains CM02-0618, CM02-0618/ΔNcgl1464, and CM02-0735 were each inoculated into a 250-ml corner baffled flask containing 25 ml of seed medium and cultured with shaking at 30°C for 20 hours at 200 rpm. Then 1 ml of this culture broth was inoculated into a 250 ml corner flask containing 24 ml of production medium and cultured with shaking at 30°C for 48 hours at 200 rpm. The composition of the seeding medium and production medium was as follows.

Sowing 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 calcium pantothenate and 2000 mcg nicotinamide (per 1 liter of distilled water).

Production medium (pH 8.0)

50 g glucose, 12 g (NH ₄ ) ₂ S ₂ O ₃ , 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 of calcium pantothenate, 3000 µg of nicotinamide, 30 g of CaCO ₃ and 1 µg of cyanocobalamin (vitamin B12) (per 1 liter of distilled water).

The strains were cultured according to the above-described culture method and the concentrations of L-methionine contained in the culture broth were analyzed and are shown in Table 4 below.

As a result, it was confirmed that the ability of the strain overexpressing the ssuABC gene to produce L-methionine was increased by 50% or more compared with that of the control strain. Also, the SsuABC protein was confirmed to be involved in thiosulfate uptake, as confirmed in Example 4.

Example 6: Comparative cultivation with thiosulfate and another sulfonate

The SsuABC protein is known as a sulfonate supply protein (Appl. Environ. Microbial. 71 (10:6104-6114, 2005). Sulfonate, having the structure R-SO3, where R represents an organic group, differs from thiosulfate, which has the structure S- SO3.

Therefore, the effect of thiosulfate on methionine production was determined by a comparative experiment using the sulfonate.

Corynebacterium glutamicum strains CM02-0618 and CM02-0735 were inoculated into a 250 ml flask with corner baffles containing 25 ml of seed medium and cultured with shaking at 30°C for 20 hours at 200 rpm. Then 1 ml of this culture broth was inoculated into a 250 ml corner flask containing 24 ml of production medium and cultured with shaking at 30°C for 48 hours at 200 rpm. The composition of the seeding medium and production medium was as follows.

Sowing 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 calcium pantothenate and 2000 mcg nicotinamide (per 1 liter of distilled water).

Production medium (pH 8.0)

50 g glucose, 12 g (NH ₄ ) ₂ S ₂ O ₃ , methanesulfonate or ethanesulfonate (depending on the sulfur source), 5 g yeast extract, 1 g KH ₂ PO ₄ , 1.2 g MgSO ₄ ⋅7H ₂ O, 100 mcg of biotin, 1000 mcg of thiamine-HCL, 2000 mcg of calcium pantothenate, 3000 mcg of nicotinamide, 30 g of CaCO ₃ and 1 mcg of cyanocobalamin (vitamin B12) (per 1 liter of distilled water).

The strains were cultured according to the above-described culture method and the concentrations of L-methionine contained in the culture broth were analyzed and are shown in Table 5 below.

As a result, when thiosulfate was used as a sulfur source for each strain, methionine production increased by up to 700% compared to the case when sulfonate was used as a sulfur source.

Based on this, it was confirmed that methionine production was highest when thiosulfate was used as a sulfur source, and it was also confirmed that increased SsuABC protein activity was involved in this increase in methionine production.

Example 7: Preparation of a Methionine-producing strain with increased expression of the metH and CysI genes without deletion of the mcbR gene

Example 7-1: Preparation of a recombinant vector to enhance both metH and CysI genes

To determine whether the production of sulfur-containing amino acid would be increased by enhancing the activity of the SsuABC protein of the present invention and using thiosulfate as a sulfur source, the above experiment was conducted with another methionine-producing strain. First, a vector was obtained to enhance both the metH gene (Ncgl1450), encoding methionine synthase, and the cys/(Ncgl2718) gene, encoding sulfite reductase, in strain ATCC 13032.

In particular, a recombinant plasmid vector was obtained for additional insertion of the metH and CysI genes into the chromosome of Corynebacterium glutamicum ATCC 13032 in accordance with the following method. Based on nucleotide sequences deposited in the GenBank of the US National Institutes of Health (NIH), the metH gene and flanking sequences (SEQ ID NO: 26) and the CysI gene and flanking sequences (SEQ ID NO: 27) of Corynebacterium glutamicum were obtained.

First, a Ncgl1201 deletion vector (Transposase) was generated to insert these genes. Based on nucleotide sequences deposited in GenBank of the US National Institutes of Health (NIH), the Ncgl1021 gene and flanking sequences (SEQ ID NO: 28) of Corynebacterium glutamicum were obtained. To delete the Ncgl1021 gene, PCR was performed using chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template and primers SEQ ID NO: 29, 30, 31 and 32. PCR was carried out under the following 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, and amplified fragments of the Ncgl1021 gene were treated with the Xbal restriction enzyme for insertion into the chromosome, followed by cloning by isothermal assembly. Escherichia coli DH5a was transformed with this vector and plated on solid LB medium containing 25 mg/L kanamycin. Colonies transformed with a vector into which a fragment having a deletion of the target gene was inserted by PCR were selected, and then the plasmid was obtained by plasmid extraction and designated pDZ-ΔNcgl1021.

Then, to obtain the metH and CysI genes, PCR was performed using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template and primers SEQ ID NO: 33, 34, 35 and 36. Additionally, the Pcj7 promoter was used to enhance the expression of the metH gene and the Pspll promoter was used to enhance CysI gene expression. To obtain them, PCR was carried out using the chromosomal DNA of Corynebacterium ammoniagenes ATCC 6872 as a template and using primers SEQ ID NO: 37 and 38 for the Pcj7 promoter and PCR was carried out using the DNA of the known spll-GFP vector (US 10584338 B2) as a template and using primers SEQ ID NO: 39 and 40 for the Pspll promoter. PCR was carried out under the following 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 and CysI genes, the Pcj7 promoter (US 7662943 B2) and the Pspll promoter (US 10584338 B2) were obtained.

The pDZ-ΔNcgl1021 vector, unable to replicate in Corynebacterium glutamicum, was treated with the restriction enzyme Seal, and the four amplified DNA fragments were treated with the restriction enzyme Seal, followed by cloning by isothermal assembly. Escherichia coli DH5a was transformed with this vector and plated on solid LB medium containing 25 mg/L kanamycin. Colonies transformed with the vector into which the target gene was inserted by PCR were selected, and then the plasmid was obtained by plasmid extraction and designated pDZ-ΔNcgl1021-Pcj7metH-PspllcysI.

Example 7-2: Development of an L-Methionine-producing strain and detection of L-Methionine production using this strain

Strain ATCC 13032 was transformed by electroporation with the vectors pDZ-ΔNcgl1021 HpDZ-ΔNcgl1021-Pcj7metH-PspllcysI obtained in Example 7-1 above by homologous chromosomal recombination (Van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 199 9). Then a second recombination was carried out in a solid medium containing sucrose. After completion of the second recombination, the insertion of the Pcj7-metH-PspllCysI gene in the transformed strain Corynebacterium glutamicum was detected using SEQ ID NO: and 41 and 42. The recombinant strains were named Corynebacterium glutamicum 13032/ΔNcgl1021 (strain transformed pDZ-ΔNcgl1021) and CM02- 0753 ( transformed pDZ-ΔNcgl1021-Pcj7metH-PspllcysIn).

To analyze the ability of the resulting strains 13032/ΔNcgl 1021 and CM02-0753 to produce L-methionine, these strains and the parent strain, Corynebacterium glutamicum strain ATCC 13032, were cultivated as follows.

Corynebacterium glutamicum ATCC 13032 and the strains of the present invention, i.e., Corynebacterium glutamicum 13032/ΔNcgl1021 and CM02-0753 were inoculated into a 250 ml corner baffled flask containing 25 ml of seed medium and cultured with shaking at 30°C for 20 hours. at 200 rpm. Then 1 ml of this culture broth was inoculated into a 250 ml corner flask containing 24 ml of production medium and cultured with shaking at 30°C for 48 hours at 200 rpm. The composition of the seeding medium and production medium was as follows. Sowing 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 calcium pantothenate and 2000 mcg nicotinamide (per 1 liter of distilled water).

Production medium (pH 8.0)

50 g glucose, 12 g (NH ₄ ) ₂ S ₂ O ₃ , 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 of calcium pantothenate, 3000 µg of nicotinamide, 30 g of CaCO ₃ and 1 µg of cyanocobalamin (vitamin B12) (per 1 liter of distilled water).

The strains were cultured according to the above-described culture method and the concentrations of L-methionine contained in the culture broth were analyzed and are shown in Table 6 below.

As a result, it was confirmed that the ability of the strain in which the mcbR gene is present and the metH and CysI genes are overexpressed to produce L-methionine is increased compared with that of the control strain. Based on this, a strain in which the metH and CysI genes were overexpressed without deletion of the mcbR gene was confirmed to have the ability to produce methionine, and this strain was used in the next experiment.

Example 8: Development of an L-Methionine-producing strain with increased SsuABC activity and containing mcbR, and determination of the ability to produce L-Methionine

A strain with increased activity of the SsuABC protein was obtained using the methionine-producing strain obtained in Example 7, and then its ability to produce L-methionine was determined.

Example 8-1: Preparation of a strain having increased SsuABC activity

In particular, strain CM02-0753 from Example 7 was transformed by electroporation with the vector pDZ-ΔNcgl464-PgapΔSsuABC obtained in Example 5 by homologous chromosomal recombination (Van der Rest et at, Appl Microbiol Biotechnol 52:541-545, 1999). Then a second recombination was carried out in a solid medium containing sucrose.

After completion of the second recombination, the insertion of the PgapA-SsuABC gene into the Ncgl1464 site of the transformed Corynebacterium glutamicum was detected using SEQ ID NOs: 24 and 25. The resulting recombinant strain was named Corynebacterium glutamicum CM02-0755.

CM02-0755 was deposited at the Korean Microbial Culture Center in accordance with the Budapest Agreement on March 21, 2019 with accession number KСSM12467Р.

Example 8-2: Determination of the ability of the resulting strain to produce methionine

To analyze the ability of strain CM02-0753 obtained in Example 7 and strain CM02-0755 obtained in Example 8-1 to produce L-methionine, the strains were cultivated as follows.

Corynebacterium glutamicum strains CM02-0753 and CM02-0755 were inoculated into a 250 ml flask with corner baffles containing 25 ml of seed medium and cultured with shaking at 30°C for 20 hours at 200 rpm. Then 1 ml of this culture broth was inoculated into a 250 ml corner flask containing 24 ml of production medium and cultured with shaking at 30°C for 48 hours at 200 rpm. The composition of the seeding medium and production medium was as follows.

Sowing 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 calcium pantothenate and 2000 mcg nicotinamide (per 1 liter of distilled water).

Production medium (pH 8.0)

50 g glucose, 12 g (NH ₄ ) ₂ S ₂ O ₃ , 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 of calcium pantothenate, 3000 µg of nicotinamide, 30 g of CaCO ₃ and 1 µg of cyanocobalamin (vitamin B12) (per 1 liter of distilled water).

The strains were cultured according to the above-described culture method and the concentrations of L-methionine contained in the culture broth were analyzed and are shown in Table 7 below.

The result confirmed that the ability to produce L-methionine was increased by increasing the activity of SsuABC protein in the presence of mcbR and using thiosulfate as a sulfur source.

These results indicate that sulfur-containing amino acids or derivatives of sulfur-containing amino acids can be produced using thiosulfate as a sulfur source by increasing the activity of the SsuABC protein, which is confirmed for the first time in the present invention to be a thiosulfate supply protein.

The above description of the present invention is presented for illustrative purposes, and one skilled in the art will understand that various changes and modifications can be made without changing the technical idea and essential features of the present invention. It is therefore understood that the above-described embodiments are illustrative in all aspects and do not limit the scope of the present invention. Accordingly, the scope of the present invention is defined not by the detailed description, but by the appended claims and their equivalents, and all embodiments within the scope of the appended claims are to be considered as included in the present specification.

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List of sequences

<110>CJ CheilJedang Corporation

<120> METHOD OF PRODUCING SULFUR-CONTAINING AMINO ACID OR DERIVATIVE

THEREOF

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aaaaggagaa ccacatgctt gccctaggtt ggattacatg gatcattatt ggtggtctag 600

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gcagctcggc tcgagcctgg gcgccatccc ggagcggatt ctgccggcac caaccacgat 1140

cttggccgcc agctgggagg tcgccacaaa tggcacgctt ctcgacgccc tcctcgtctc 1200

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gcgccaagca agtgcggccg cctggctctc actgatcgtg gcggaacagg tcaacgcccg 1620

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ggaacgtcac accttccgct accgaaacgc ataagaaagt tgctcgccat gactgccaca 1800

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gtcctccaag gaatcgacct caccatcaac tgcggcgaag taaccgcgct gatcggacgc 1920

tcaggttcag gaaaatccac catcctgcgc gtgttggcgg gcctatctaa agagcattcc 1980

ggctctgtag aaatttccgg aaacccggcc gttgccttcc aagagcctcg cctgttgccg 2040

tggaaaacgg tgctcgataa tgtgaccttt ggcctcaacc gcactgatat ttcctggtca 2100

gaagcacaag aacgcgcctc ggcactgctt gcagaagtca aacttcccga ctccgacgcc 2160

gcctggcccc tcacgctctc cggcggccaa gcccagcgcg tctcccttgc gcgagcgctc 2220

atctccgagc cagagctttt gcttctcgac gaacccttcg gcgccctcga tgctctgaca 2280

agactgacag cccaagacct gctgctcaaa accgtgaaca cccgaaactt gggagttctg 2340

ctggtcaccc atgatgtttc cgaggccatc gccctggccg accacgtcct tcttcttgac 2400

gacggcgcca tcacacacag tttgactgta gatatccccg gcgatcgccg cacccacccc 2460

tcctttgcct cctacaccgc tcaactcctt gagtggctcg aaatcaccac acctgcctag 2520

aaagaaatca tgaaatttaa gaaaatcgcc ctcgttctcg ccttcggtct aggccttgca 2580

tcctgctcat cagcttctgg cgatcccgcc accaacgccg atggatccat cgatctgagc 2640

aaagtaaccc ttaacatcgg tgatcaaatc gccggaacag aacaagtgct ccaagcttca 2700

ggggagctag atgatgtccc ttataaaatc gaatggtcat catttacctc tggaccaccc 2760

caaatcgaag cattaaacgc aggtcaaatt gatttcgcga tcaccggaaa caccccaccg 2820

atcatcggcg gccccaccaa caccaaagtg gtctccgcct acaacaacga tgctttaggt 2880

gatgtcatct tggtcgcccc ggattcttca ataacctcgg tggctgacct tgctggaaag 2940

aaagtggctg tcgcccgcgg atccagcgcc cacggacacc tcatccaaca actagaaaaa 3000

gcaggcgtga gcgttgacga cgtagaaatc aacctcctcc aaccctccga cgccaaagcc 3060

gctttccaaa acggccaggt agatgcgtgg gcagtgtggg atccctacag ctcacaggcg 3120

gaactggaag gagctcaagt tttggtcagg ggagcgggac tggtcagtgg gcatggattt 3180

ggtgtcgcaa gtgatgaagc gctcgatgac cccgcaaagg aagccgcctt ggcagatttc 3240

ctcgatcgcg tggccgactc ttatgaatgg gctgaagaca acaccgatga atgggcgacg 3300

attttcagcc aagaatccgg ctttgatccg gaggcctctc aactgaacac ccgcagcctg 3360

cgccatcagg tgccgctcga cgagtccgtc aacacctatc agaacgcgct tatcgacgct 3420

ttcgtctccg cgggtctcgt tgaggacttt aatttcgagg acaccgtaga cacccgattt 3480

gagggctaag tatgtctgag tatggcaaag gggcgttgag gtgctcagac ggccatacaa 3540

agcatcgtag tttgtgcata tgagggctgc tttttgccgc tgtgagttgg ctgttcagga 3600

atccgtcctc gggttgggga gaggggctga aaccgaacga atattcgcat aaatctttcg 3660

aattggcgcg aatattcgat cccgctggcc tccgaactag ccattttggt agaaaatttg 3720

catccttgag ccggtattgg cccaaggatg cattttcctg tgtcctactt ctgcagtgag 3780

tagaactgag cactcatcgc tgggacatgc agtgagagtg agtaggcgaa attatcgcgt 3840

ggtgtcgctg ctgcttgaac ggaagtagcg atatcgtttt ctgcaccgag gaattcagca 3900

tcatcagtgt tgagaacgag cttccattcg ccacccgctg caacaccgag ctggtactca 3960

ggctgggagg ttccagacag gttgaataca cacagcatct gggagccgtc gctgccgaaa 4020

cgagtgaacg ccaaaatgtt gttggtggcg tcgtcgccct tattccatgt gaagccttct 4080

ccggtgaaat cctgagtgtg cagcgcaggg gagtctgagt agacaccgtt gagggagcgg 4140

gtcagagtgc ggatggcttc gtggtactcg ccttgccagc cgtcgacaat atcccatggc 4200

agtccctggc cttcagccca ctcttcacgc tgaccaaact cctgacccat gaaaagcagc 4260

ttcttgcctg ggtgtgacca catgtacgca aggaaggtgc gaagaccagc ggccttgttc 4320

cacgtatcgc caggcatacg gtcccacagg gaacccttgc cgtggacgac ttcatcgtga 4380

gagatcggaa gtacaaaacg ctcagagaat gcgtacacca aggagaaagt gagctcactg 4440

tggtggaatg cgcggtgcac agggtttttg gagaagtact ctaaggtgtc gtgcatccag 4500

cccatgttcc acttgaggga gaatccca 4528

<210> 9

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 9

tcgagctcgg tacccagcaa gctagccttg cgcat 35

<210> 10

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 10

cagacataca gtactgtcag gttccttaag aaatt 35

<210> 11

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 11

gaacctgaca gtactgtatg tctgagtatg gcaaa 35

<210> 12

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 12

ctctagagga tccccttgtc gacggctggc aaggc 35

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 13

ccaggtgttt ggggtgcagg 20

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 14

agtttggtca gcgtgaagag 20

<210> 15

<211> 3161

<212> DNA

<213> Corynebacterium glutamicum

<400> 15

agacgattct ggaaggccac tttcttttga tcctggcggc gatttttcgg tgcttggtag 60

cggaatgtca atcgagacca agcgtccccc ggcgggtcga ttttttggtc tcgaatgaca 120

gtttcgctct ctggaaactc tcagtgtcag gtcagtggtg aaccaccatc cttagcaagg 180

agttcatcat gtccatcccc ttctcagtcc ttcaggacta cctggatctg atcagtcccg 240

aagccttacc cgatccca cagcccccgg cccctgcccc cacagcaccc cagctaccac 300

cggcgccgga cccacacagc atcgagtggc cgatcttccc accagatcga atctccgcca 360

acgggcgacg ctactacgag ccacaaacac gactcgagtt catgcggatc tacaccaccc 420

tgccgcacgg ctaccgccag cccttcctta aagccaacaa catcggccac tgcaccgttc 480

gaacctggct agcagcaata agcaccttca gccgacttcc ccatgctttt gatgatgccc 540

accgcttcgg gatcgaacgc accaccccag tcgacgatgt caccacacta acggctgatg 600

acaaacgtga cctggtcata ggatacttag ctcaaccaca cggtcagggc cagcaattcc 660

tcacgtttta ccaactccgt aagcacacca tcatggcctg gtgcgccgct atgaccgacg 720

gggacttaga cgctgatatc tcaccccgcc agatcgggtt gatgaccacc cgaaccgtgg 780

tcgaaatcgt tcgactacgc cacatgattg cccaacaact agaaagagcc acgatcatgg 840

aaaacgagta cctcaaagaa atcgcagcgc tgaagaaaga actcgcgcac tacaagcaaa 900

aagaccatca gaatcaaatg gtgatcgata tcttgggaaa agctattggg accaggccca 960

atcctggcga gggcttagac gaggaggacg ccacctaaac gtggatgagc aacgcgcctt 1020

tgatcaagga ctcaaggaag aaaacacctt gatcacagat ctcaccacct gtgccaggct 1080

gagccataac aaggcattac ggctgatcaa gctgtcgaaa tcaacggcgt attaccgcaa 1140

caagccgcgt ccccgtcctg caccgaaacc tgtcctgcag gccgtgccag caccaacagc 1200

acctggtgtg gaacccacac cagagccttg gcaggggaag gagccagcag tgtcgtcggt 1260

gcgtcaagcg ttggcagaac acgaacgcca gttcattgtt gatgcgatca ccgcgtaccc 1320

acaactgagc gttagtgggg tgtttaacat gttgtttaac aaaggcatct accgcgcatc 1380

actacgtaca tggtggcgtg ttgccaagca gcacaagttg ttacacaaag accgagtcag 1440

tgccctgtcc ccggggaaac gatcaccaac gccacgggtt aagccgaggt tggaagcaac 1500

acagcctggt caggtggtgt gttgggatgt gacgttcttg ccgtcgctgg tacgtggtaa 1560

gacctatgcg ttgcatctgg cgattgattt gttttcccgc aagattgttg gggcgaaggt 1620

cgcgccgacg gaaaatacct ccaccgcggt ggagttgtta acgcaggtgt tagcggataa 1680

tccgggtgtg gtgacggtgc attcggataa tgggtcggcg atgacatcga cgaggtgcg 1740

gcggttgtta gcggatcatg gtgtggcgtt gtcgttgatt cggccgcggg tgagtgatga 1800

taatgcgttt gtggagtcgg tgtttcatac gttgaagtat cggccgtttt atccgaaggt 1860

gtttgcatcg atggatcagg cccggggtgtg ggtggagggag tttgtggtgt attacaacac 1920

ggttcatccg cattctggtg tggctgggca tactccgcag tcggtgtttg atggtagttg 1980

gagggcggct cataggttgc gtgtgcaggc gttggatgcc cattaccggc agttcccgca 2040

gcggtatgtg gggcggccgg tggttcagga agttgctggt gtggtgcgtc ttaatggtgc 2100

gcgtgatgat gggtctgtac aggagagggt tggtggtgta gcgtcgctgt taagtgcttg 2160

agttagcatg tgttcttatc gcccccctgg ttcacaaacc cctggcagcg agcggaaaag 2220

tgcattttta ggccaagggc cctcggatct tcgagcgctt tggtctcttt tgcacgtctg 2280

accgaaccag atcacctaga aacgccaaag gccccgcaag tatcaaacct gcggggcctt 2340

tgaggtacct gtttcctatt ttgttgactt aggaagctgc gcacggcgga taaccaaacc 2400

gcacagcaag gcagccactc cccacgcggt gagccagaac tgctccacga cataaacctg 2460

aatagttgga agcaaacgac ttacgatcac cagggctaca gcgatgctca aagaaatgat 2520

ctgtgtcttt gagcttggct cgtacttgct ggtggtagcg aatgcgccga gaatgatcga 2580

ggcaacgagg atcagaagga atcctggagt gatcacaaat gggctcagca tgccggcggt 2640

aaacagctca attgctgcaa acaccaacaa aaccagacct aaggctacga aagctccacc 2700

gatgcggatt gctgccggaa ctttacgcca gctggcacgg ccttcgaggc tggtgagctt 2760

ttttaatgat cttcccgatg ctgaactcat aatgtgacat accctactag ttctcgtacc 2820

atccccacac aattgacctg ccaagagtgt ggaaatacag gttgaagcct agaacagtgg 2880

gggtagcgtc gggggcgatg tcgagttttt ccacatcaag tgcatgaact gcgaagaggt 2940

aacggtgcgg tgcgtggcca gctggaggtt gcgctccgta gaagccacgc ttgccggaat 3000

cacccttgag ggaaactacg ccttcgatgc cgccgagggt ttcatcgcca gcaccggtgg 3060

ggatctccgt gacagttgtg gggatgttaa acactgccca gtgccagaaa ccagcgccgg 3120

ttggggcatc tgggtcgagg caggtgatcg cgagggattt g 3161

<210> 16

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 16

tcgagctcgg tacccttttt tggtctcgaa tgaca 35

<210> 17

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 17

catgctaaca gtactgttta ggtggcgtcc tcctc 35

<210> 18

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 18

cacctaaaca gtactgttag catgtgttct tatcg 35

<210> 19

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 19

ctcagaggat cccccccacc ggtgctggcg atga 34

<210> 20

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 20

cgccacctaa acagtgaagc ctaaaaacga ccgag 35

<210> 21

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 21

caaggttgta gtcatgttgt gtctcctcta aagat 35

<210> 22

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 22

tagagggagac acaacatgac tacaaccttg actcg 35

<210> 23

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 23

acacatgcta acagtcatgt cccagcgatg agtgc 35

<210> 24

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 24

ctggcggcga tttttcggtg 20

<210> 25

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 25

cgcgatcacc tgcctcgacc 20

<210> 26

<211> 5666

<212> DNA

<213> Corynebacterium glutamicum

<400> 26

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 gttctaggtt cggaggcgga 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 gggccgggcc 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 tgctcatggg 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

cccacaccacc 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

gcaatcggca gaaaccatga aaactgcggt ggcctatttg gaaccgttca tggaagagga 3240

agcagaagct accggatctg cgcaggcaga gggcaagggc aaaatcgtcg tggccaccgt 3300

caagggtgac gtgcacgata tcggcaagaa cttggtggac atcattttgt ccaacaacgg 3360

ttacgacgtg gtgaacttgg gcatcaagca gccactgtcc gccatgttgg aagcagcgga 3420

agaacacaaa gcagacgtca tcggcatgtc gggacttctt gtgaagtcca ccgtggtgat 3480

gaaggaaaac cttgaggaga tgaacaacgc cggcgcatcc aattacccag tcattttggg 3540

tggcgctgcg ctgacgcgta cctacgtgga aaacgatctc aacgaggtgt acaccggtga 3600

ggtgtactac gcccgtgatg ctttcgaggg cctgcgcctg atggatgagg tgatggcaga 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 tttgagagg 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

ccagcccccg 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 caggtggggg 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> 27

<211> 3613

<212> DNA

<213> Corynebacterium glutamicum

<400> 27

tcctgtgggg tgaacttgac ctgtgctggg ccacgacgtc cgaaaacgtg cacttcagtg 60

gccttgtttt ctttgaggga gtcgtagacg ttgtcggaaa tttcggtgac tttgagctcg 120

tcgcctgtct tagccaggat gcgggctacg tcgaggccga cgttaccaac gccgataaca 180

gcgacggact gtgcagacag atcccaggag cgctcgaagc gtgggttgcc gtcgtagaag 240

ccaacgaact cgccggcacc gaaggagcct tctgcttcaa ttccggggat gttgaggtcg 300

cggtctgcaa ctgcgccggt ggagaacacg actgcatcgt agtagtcgcg gagttcttcg 360

acggtgatgt ctttgccgat ttcaatgtta ccgagcaggc gcaggcgtgg cttgtccaac 420

acgttgtgca gggacttaac gatgcccttg atgcgtgggt ggtctggagc aacgccgtaa 480

cggatgagtc cgaacggtgc aggcatttgc tcgaaaaggt caacgaacac ttcgcgctct 540

tcattgcgga tgaggaggtc ggatgcgtaa atgccagcag ggccagctcc gatgacggct 600

acgcgcaggg gagttgtcat gtgtttgaag ttgcctttcg tgagcccttt tatggaaaca 660

agggtgtgaa aatcaagtag ttaaaggtgt ttcaagtcca ggctgtttaa cactcctaga 720

ccgcttggtc tgtaaacgta gcagcgaaat gcgacaatgc gaagactttt gcttaattaa 780

attcaaactc catgaaaaaa ctagacagat cggtctatta tattcacggt gaacctaacc 840

taatatcccc aggttaattc atttaaacgg gcattaggtg actccattgc tttcagtctc 900

atgaatctaa tggttggtct agacagagcg gtacgtctaa gtttgcggat agatcaaacc 960

gagtgacatg tacttcacta gctctttaag gattaactcc ccatgacaac aaccaccgga 1020

agtgcccggc cagcacgtgc cgccaggaag cctaagcccg aaggccaatg gaaaatcgac 1080

ggcaccgagc cgcttaacca tgccgaggaa attaagcaag aagaacccgc ttttgctgtc 1140

aagcagcggg tcattgatat ttactccaag cagggttttt cttccattgc accggatgac 1200

attgccccac gctttaagtg gttgggcatt tacacccagc gtaagcagga tctgggcggt 1260

gaactgaccg gtcagcttcc tgatgatgag ctgcaggatg agtacttcat gatgcgtgtg 1320

cgttttgatg gcggactggc ttcccctgag cgcctgcgtg ccgtgggtga aatttctagg 1380

gattatgctc gttccaccgc ggacttcacc gaccgccaga acattcagct gcactggatt 1440

cgtattgaag atgtgcctgc gatctgggag aagctagaaa ccgtcggact gtccaccatg 1500

cttggttgcg gtgacgttcc acgtgttatc ttgggctccc cagtttctgg cgtagctgct 1560

gaagagctga tcgatgccac cccggctatc gatgcgattc gtgagcgcta cctagacaag 1620

gaagagttcc acaaccttcc tcgtaagttt aagactgcta tcactggcaa ccagcgccag 1680

gatgttaccc acgaaatcca ggacgtttcc ttcgttcctt cgattcaccc agaattcggc 1740

ccaggatttg agtgctttgt gggcggtggc ctgtccacca acccaatgct tgctcagcca 1800

cttggttctt ggattccact tgatgaggtt ccagaagtgt gggctggcgt cgccggaatt 1860

ttccgcgact acggcttccg acgcctgcgt aaccgtgctc gcctcaagtt cttggtggca 1920

cagtggggta ttgagaagtt ccgtgaagtt cttgagaccg aatacctcga gcgcaagctg 1980

atcgatggcc cagttgttac caccaaccct ggctaccgtg accacattgg cattcaccca 2040

caaaaggacg gcaagttcta cctcggtgtg aagccaaccg ttggacacac caccggtgag 2100

cagctcattg ccattgctga tgttgcagaa aagcacggca tcaccaggat tcgtaccacg 2160

gcggaaaagg aactgctctt cctcgatatt gagagaaaga accttactac cgttgcacgc 2220

gacctggatg aaatcggact gtactcttca ccttccgagt tccgccgcgg catcatttcc 2280

tgcaccggct tggagttctg caagcttgcg cacgcaacca ccaagtcacg agcaattgag 2340

cttgtcgacg aactggaaga gcgcctcggc gatttggatg ttcccatcaa gattgcactg 2400

aacggttgcc ctaactcttg tgcacgcacc caggtttccg acatcggatt caagggacag 2460

accgtcactg atgctgacgg caaccgcgtt gaaggtttcc aggttcacct gggcggttcc 2520

atgaacttgg atccaaactt cggacgcaag ctcaagggcc acaaggttat tgccgatgaa 2580

gtgggagagt acgtcactcg cgttgttacc cacttcaagg aacagcgcca cgaggacgag 2640

cacttccgcg attgggtcca gcgggccgct gaggaagatt tggtgtgagt cttcggagga 2700

aacccaatcc caaccgcaac caccctctgt actgcccata ctgcgcggga gaagttcttt 2760

tccccgatga gcaaacagaa ttcgcgtggt tgtgtgcgga ttgcaccaga gtttttgaag 2820

tgaaatatca cggccaggac gatccagtgc acaggccagc accagcaaag tccacatcgc 2880

aagcattaaa agaatctctc gaaagacaca aaagaggtga gtcgcaacaa tgagctttca 2940

actagttaac gccctgaaaa atactggttc ggtaaaagat cccgagatct cacccgaagg 3000

acctcgcacg accacaccgt tgtcaccaga ggtagcaaaa cataacgagg aactcgtcga 3060

aaagcatgct gctgcgttgt atgacgccag cgcgcaagag atcctggaat ggacagccga 3120

gcacgcgccg ggcgctattg cagtgacctt gagcatggaa aacaccgtgc tggcggagct 3180

ggctgcgcgg cacctgccgg aagctgattt cctctttttg gacaccggtt accacttcaa 3240

ggagaccctt gaagttgccc gtcaggtaga tgagcgctat tcccagaagc ttgtcaccgc 3300

gctgccgatc ctcaagcgca cggagcagga ttccatttat ggtctcaacc tgtaccgcag 3360

caacccagcg gcgtgctgcc gaatgcgcaa agttgaaccg ctggcggcgt cgttaagccc 3420

atacgctggc tggatcaccg gcctgcgccg cgctgatggc ccaacccgtg ctcaagcccc 3480

tgcgctgagc ttggatgcca ccggcaggct caagatttct ccaattatca cctggtcatt 3540

ggaggaaacc aacgagttca ttgcggacaa caacctcatc gatcacccac ttacccatca 3600

gggttatcca tca 3613

<210> 28

<211> 3311

<212> DNA

<213> Corynebacterium glutamicum

<400> 28

ctcattccag cgtcacgacg ttccgaaggt actggttacc tggcattggg 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 cccagcctg 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 gaccacggta aaaaggtcac 1320

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 agttgtgggc 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

gattgctcta ggtttccgta atttgaacca ctacattctg cggtgcctta tccattcagg 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

tttttcctgg 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

cgctcctttg atagcgaggt tgttggcttc tgatccaccc gacgtaaacg tcacctgtgt 3240

ggggcgtcct ccgataatgc gggccacccg agttcgagca tcctccagcc ccgcagaggc 3300

gagtcttccc a 3311

<210> 29

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 29

acccggggat cctctagaat gtttgtgatg cgcag 35

<210> 30

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 30

gtcagagagt acttacgctg atcgggaggg aaagc 35

<210> 31

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 31

atcagcgtaa gtactctctg actagcgtca ccctc 35

<210> 32

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 32

ctgcaggtcg actctagaaa agggattgga gtgtt 35

<210> 33

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 33

caacgaaagg aaacaatgtc tacttcagtt acttc 35

<210> 34

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 34

tagtcagaga gtgatttaga cgttaaagta ctttg 35

<210> 35

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 35

atcaaaacag atatcatgac aacaaccacc ggaag 35

<210> 36

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 36

cgctagtcag agagttcaca ccaaatcttc ctcag 35

<210> 37

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 37

ccgatcagcg taagtagaaa catcccagcg ctact 35

<210> 38

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 38

aactgaagta gacattgttt cctttcgttg ggtac 35

<210> 39

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 39

tactttaacg tctaaggtac cggcgcttca tgtca 35

<210> 40

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 40

ggtggttgtt gtcatgatat ctgttttgat ctcct 35

<210> 41

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 41

atccccatcg gcatctttat 20

<210> 42

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 42

cgatcacact gggctgatct 20

<210> 43

<211> 319

<212>PRT

<213> Corynebacterium glutamicum

<400> 43

Met Lys Phe Lys Lys Ile Ala Leu Val Leu Ala Phe Gly Leu Gly Leu

1 5 10 15

Ala Ser Cys Ser Ser Ala Ser Gly Asp Pro Ala Thr Asn Ala Asp Gly

20 25 30

Ser Ile Asp Leu Ser Lys Val Thr Leu Asn Ile Gly Asp Gln Ile Ala

35 40 45

Gly Thr Glu Gln Val Leu Gln Ala Ser Gly Glu Leu Asp Asp Val Pro

50 55 60

Tyr Lys Ile Glu Trp Ser Ser Phe Thr Ser Gly Pro Pro Gln Ile Glu

65 70 75 80

Ala Leu Asn Ala Gly Gln Ile Asp Phe Ala Ile Thr Gly Asn Thr Pro

85 90 95

Pro Ile Ile Gly Gly Pro Thr Asn Thr Lys Val Val Ser Ala Tyr Asn

100 105 110

Asn Asp Ala Leu Gly Asp Val Ile Leu Val Ala Pro Asp Ser Ser Ile

115 120 125

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

130 135 140

Ser Ser Ala His Gly His Leu Ile Gln Gln Leu Glu Lys Ala Gly Val

145 150 155 160

Ser Val Asp Asp Val Glu Ile Asn Leu Leu Gln Pro Ser Asp Ala Lys

165 170 175

Ala Ala Phe Gln Asn Gly Gln Val Asp Ala Trp Ala Val Trp Asp Pro

180 185 190

Tyr Ser Ser Gln Ala Glu Leu Glu Gly Ala Gln Val Leu Val Arg Gly

195 200 205

Ala Gly Leu Val Ser Gly His Gly Phe Gly Val Ala Ser Asp Glu Ala

210 215 220

Leu Asp Asp Pro Ala Lys Glu Ala Ala Leu Ala Asp Phe Leu Asp Arg

225 230 235 240

Val Ala Asp Ser Tyr Glu Trp Ala Glu Asp Asn Thr Asp Glu Trp Ala

245 250 255

Thr Ile Phe Ser Gln Glu Ser Gly Phe Asp Pro Glu Ala Ser Gln Leu

260 265 270

Asn Thr Arg Ser Leu Arg His Gln Val Pro Leu Asp Glu Ser Val Asn

275 280 285

Thr Tyr Gln Asn Ala Leu Ile Asp Ala Phe Val Ser Ala Gly Leu Val

290 295 300

Glu Asp Phe Asn Phe Glu Asp Thr Val Asp Thr Arg Phe Glu Gly

305 310 315

<210> 44

<211> 243

<212>PRT

<213> Corynebacterium glutamicum

<400> 44

Met Thr Ala Thr Leu Ser Leu Lys Pro Ala Ala Thr Val Arg Gly Leu

1 5 10 15

Arg Lys Ser Tyr Gly Thr Lys Glu Val Leu Gln Gly Ile Asp Leu Thr

20 25 30

Ile Asn Cys Gly Glu Val Thr Ala Leu Ile Gly Arg Ser Gly Ser Gly

35 40 45

Lys Ser Thr Ile Leu Arg Val Leu Ala Gly Leu Ser Lys Glu His Ser

50 55 60

Gly Ser Val Glu Ile Ser Gly Asn Pro Ala Val Ala Phe Gln Glu Pro

65 70 75 80

Arg Leu Leu Pro Trp Lys Thr Val Leu Asp Asn Val Thr Phe Gly Leu

85 90 95

Asn Arg Thr Asp Ile Ser Trp Ser Glu Ala Gln Glu Arg Ala Ser Ala

100 105 110

Leu Leu Ala Glu Val Lys Leu Pro Asp Ser Asp Ala Ala Trp Pro Leu

115 120 125

Thr Leu Ser Gly Gly Gln Ala Gln Arg Val Ser Leu Ala Arg Ala Leu

130 135 140

Ile Ser Glu Pro Glu Leu Leu Leu Leu Asp Glu Pro Phe Gly Ala Leu

145 150 155 160

Asp Ala Leu Thr Arg Leu Thr Ala Gln Asp Leu Leu Leu Lys Thr Val

165 170 175

Asn Thr Arg Asn Leu Gly Val Leu Leu Val Thr His Asp Val Ser Glu

180 185 190

Ala Ile Ala Leu Ala Asp His Val Leu Leu Leu Asp Asp Gly Ala Ile

195 200 205

Thr His Ser Leu Thr Val Asp Ile Pro Gly Asp Arg Arg Thr His Pro

210 215 220

Ser Phe Ala Ser Tyr Thr Ala Gln Leu Leu Glu Trp Leu Glu Ile Thr

225 230 235 240

Thr Pro Ala

<210> 45

<211> 256

<212>PRT

<213> Corynebacterium glutamicum

<400> 45

Met Thr Thr Thr Leu Thr Arg Pro Lys Ile Ala Leu Pro Ala Arg Ile

1 5 10 15

Tyr Ser Pro Leu Ala Val Leu Val Phe Trp Gln Leu Gly Ser Ser Leu

20 25 30

Gly Ala Ile Pro Glu Arg Ile Leu Pro Ala Pro Thr Thr Ile Leu Ala

35 40 45

Ala Ser Trp Glu Val Ala Thr Asn Gly Thr Leu Leu Asp Ala Leu Leu

50 55 60

Val Ser Ser Gln Arg Val Leu Leu Gly Phe Ala Leu Gly Ala Val Leu

65 70 75 80

Gly Ile Ser Leu Gly Val Leu Thr Gly Met Ser Arg Phe Ala Asp Thr

85 90 95

Ala Val Asp Pro Leu Ile Gln Ala Ala Arg Ala Leu Pro His Leu Gly

100 105 110

Leu Val Pro Leu Phe Ile Ile Trp Phe Gly Ile Gly Glu Leu Pro Lys

115 120 125

Val Leu Ile Ile Ser Leu Gly Val Leu Tyr Pro Leu Tyr Leu Asn Thr

130 135 140

Ala Ser Gly Phe Arg Gln Ile Asp Pro Lys Leu Leu Glu Ala Gly His

145 150 155 160

Val Met Gly Phe Gly Phe Phe Gln Arg Leu Arg Thr Ile Ile Ile Pro

165 170 175

Ser Ala Ala Pro Gln Leu Phe Val Gly Leu Arg Gln Ala Ser Ala Ala

180 185 190

Ala Trp Leu Ser Leu Ile Val Ala Glu Gln Val Asn Ala Arg Glu Gly

195 200 205

Leu Gly Phe Leu Ile Asn Asn Ala Arg Asp Phe Tyr Arg Thr Asp Leu

210 215 220

Val Ile Phe Gly Leu Ile Val Tyr Ala Ser Leu Gly Leu Leu Ser Glu

225 230 235 240

Ala Leu Ile Arg Ala Trp Glu Arg His Thr Phe Arg Tyr Arg Asn Ala

245 250 255

<110>CJ CheilJedang Corporation

<120> Method for producing a sulfur-containing amino acid or a derivative thereof

<130>OPA20064

<150> KR 10-2019-0077998

<151> 2019-06-28

<160> 45

<170> KoPatentIn 3.0

<210> 1

<211> 2642

<212> DNA

<213> Corynebacterium glutamicum

<400> 1

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

tagtcgtcgg tattatcggt ggtttgttag gcggctggct gcttggaatc ttcggagtgg 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 cgcggttttt 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> 2

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 2

tcgagctcgg tacccctgcc tggtttgtct tgta 34

<210> 3

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 3

cggaaaatga agaaagttcg gccacgtcct ttcgg 35

<210> 4

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 4

aggacgtggc cgaactttct tcattttccg aaggg 35

<210> 5

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 5

ctctagagga tccccgtttc gatgcccact gagca 35

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 6

aatctggatt tccgccaggt 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 7

cttcctaact cctgaggaag 20

<210> 8

<211> 4528

<212> DNA

<213> Corynebacterium glutamicum

<400> 8

atgcgtggat tactgacgct tctttgattg aggcgacaaa acgcttgaag ttcctcgttg 60

cgcttcgccc tgggcagatt ggacctacgc tgtctgctca aatggcttct actttccagc 120

gtctgtctgg caaccgtttg ctgatcaatg tggtcaccgg tggggaagat gcggagcagc 180

gtgcgtttgg tgatttcttg aacaaggagg agcgctacgc ccgtaccgga gaattcttgg 240

atatcgtgag ccgcttgtgg cgaggcgaaa ccgtcacgca ccacggtgaa cacctgcagg 300

tggagcaagc tagccttgcg catccgccag agattattcc ggagattctt tttggtggat 360

cgtcgccagc tgcaggtgag gtggctgcac gttatgcgga cacctatctc acgtggggtg 420

aaactcccga tcaggtggcg cagaaaatca actggatcaa cgagctagca gcacagcgcg 480

gccgggaact gcgccatgga atccgcttcc atgtgatcac ccgcgatacg tctgaagaag 540

catgggtggt ggcagagaag ttgattagcg gggtcactcc agaacaggtc gctaaggctc 600

aagccgggtt tgcaacgtct aagtcggagg ggcagcgccg gatggctgag ctgcacagca 660

agggtcgtgc ctttactagt ggctcaactg ctcgtgatct ggaggtgtat cccaatgtgt 720

gggcaggcgt cggtttgctt cgcggaggtg caggaacagc ccttgtgggc tcgcatgaag 780

aggtcgccga tcgcatcgaa gaatacgcag cactcggctt ggatcagttt gtactgtcgg 840

gttatccaaa cttggaggag gccttccact tcggtgaggg tgtgattccg gagctgctgc 900

gccgcggtgt ggatatcaaa aatcaagaat cacgagtttt ggaacctgtt gggtaaacgg 960

gaagaacgag acgtcgataa gcaaatttct taaggaacct gacatgacta caaccttgac 1020

tcgccccaaa atcgcgctgc ccgcgcgcat ctattcaccg cttgcggtgc ttgttttctg 1080

gcagctcggc tcgagcctgg gcgccatccc ggagcggatt ctgccggcac caaccacgat 1140

cttggccgcc agctgggagg tcgccacaaa tggcacgctt ctcgacgccc tcctcgtctc 1200

aagccaacgc gtccttctag gcttcgccct cggtgctgtc ctaggcattt ccctaggtgt 1260

attgacaggc atgtccagat ttgcagacac cgccgttgat ccgctcattc aagctgcccg 1320

cgcgctgcct cacctgggtc ttgtgccgct gtttatcatc tggttcggta tcggtgagct 1380

gccgaaagta ctgattatta gcctcggcgt gctgtatccg ctgtacctca acaccgccag 1440

cgggttcagg caaattgatc caaagcttct ggaagccggc cacgtgatgg gcttcggatt 1500

tttccagagg ttgcggacca tcatcattcc ttctgccgcg ccgcaacttt ttgtcggcct 1560

gcgccaagca agtgcggccg cctggctctc actgatcgtg gcggaacagg tcaacgcccg 1620

cgaaggactc ggcttcctca tcaacaatgc gcgcgatttt taccgcaccg acctcgttat 1680

tttcggcctc attgtctacg ccagcctcgg tctgctgtct gaagcgctga tcagagcttg 1740

ggaacgtcac accttccgct accgaaacgc ataagaaagt tgctcgccat gactgccaca 1800

ttgtcactca aacccgcagc cactgtccgt ggattgcgca aatcatacgg aactaaagaa 1860

gtcctccaag gaatcgacct caccatcaac tgcggcgaag taaccgcgct gatcggacgc 1920

tcaggttcag gaaaatccac catcctgcgc gtgttggcgg gcctatctaa agagcattcc 1980

ggctctgtag aaatttccgg aaacccggcc gttgccttcc aagagcctcg cctgttgccg 2040

tggaaaacgg tgctcgataa tgtgaccttt ggcctcaacc gcactgatat ttcctggtca 2100

gaagcacaag aacgcgcctc ggcactgctt gcagaagtca aacttcccga ctccgacgcc 2160

gcctggcccc tcacgctctc cggcggccaa gcccagcgcg tctcccttgc gcgagcgctc 2220

atctccgagc cagagctttt gcttctcgac gaacccttcg gcgccctcga tgctctgaca 2280

agactgacag cccaagacct gctgctcaaa accgtgaaca cccgaaactt gggagttctg 2340

ctggtcaccc atgatgtttc cgaggccatc gccctggccg accacgtcct tcttcttgac 2400

gacggcgcca tcacacacag tttgactgta gatatccccg gcgatcgccg cacccacccc 2460

tcctttgcct cctacaccgc tcaactcctt gagtggctcg aaatcaccac acctgcctag 2520

aaagaaatca tgaaatttaa gaaaatcgcc ctcgttctcg ccttcggtct aggccttgca 2580

tcctgctcat cagcttctgg cgatcccgcc accaacgccg atggatccat cgatctgagc 2640

aaagtaaccc ttaacatcgg tgatcaaatc gccggaacag aacaagtgct ccaagcttca 2700

ggggagctag atgatgtccc ttataaaatc gaatggtcat catttacctc tggaccaccc 2760

caaatcgaag cattaaacgc aggtcaaatt gatttcgcga tcaccggaaa caccccaccg 2820

atcatcggcg gccccaccaa caccaaagtg gtctccgcct acaacaacga tgctttaggt 2880

gatgtcatct tggtcgcccc ggattcttca ataacctcgg tggctgacct tgctggaaag 2940

aaagtggctg tcgcccgcgg atccagcgcc cacggacacc tcatccaaca actagaaaaa 3000

gcaggcgtga gcgttgacga cgtagaaatc aacctcctcc aaccctccga cgccaaagcc 3060

gctttccaaa acggccaggt agatgcgtgg gcagtgtggg atccctacag ctcacaggcg 3120

gaactggaag gagctcaagt tttggtcagg ggagcgggac tggtcagtgg gcatggattt 3180

ggtgtcgcaa gtgatgaagc gctcgatgac cccgcaaagg aagccgcctt ggcagatttc 3240

ctcgatcgcg tggccgactc ttatgaatgg gctgaagaca acaccgatga atgggcgacg 3300

attttcagcc aagaatccgg ctttgatccg gaggcctctc aactgaacac ccgcagcctg 3360

cgccatcagg tgccgctcga cgagtccgtc aacacctatc agaacgcgct tatcgacgct 3420

ttcgtctccg cgggtctcgt tgaggacttt aatttcgagg acaccgtaga cacccgattt 3480

gagggctaag tatgtctgag tatggcaaag gggcgttgag gtgctcagac ggccatacaa 3540

agcatcgtag tttgtgcata tgagggctgc tttttgccgc tgtgagttgg ctgttcagga 3600

atccgtcctc gggttgggga gaggggctga aaccgaacga atattcgcat aaatctttcg 3660

aattggcgcg aatattcgat cccgctggcc tccgaactag ccattttggt agaaaatttg 3720

catccttgag ccggtattgg cccaaggatg cattttcctg tgtcctactt ctgcagtgag 3780

tagaactgag cactcatcgc tgggacatgc agtgagagtg agtaggcgaa attatcgcgt 3840

ggtgtcgctg ctgcttgaac ggaagtagcg atatcgtttt ctgcaccgag gaattcagca 3900

tcatcagtgt tgagaacgag cttccattcg ccacccgctg caacaccgag ctggtactca 3960

ggctgggagg ttccagacag gttgaataca cacagcatct gggagccgtc gctgccgaaa 4020

cgagtgaacg ccaaaatgtt gttggtggcg tcgtcgccct tattccatgt gaagccttct 4080

ccggtgaaat cctgagtgtg cagcgcaggg gagtctgagt agacaccgtt gagggagcgg 4140

gtcagagtgc ggatggcttc gtggtactcg ccttgccagc cgtcgacaat atcccatggc 4200

agtccctggc cttcagccca ctcttcacgc tgaccaaact cctgacccat gaaaagcagc 4260

ttcttgcctg ggtgtgacca catgtacgca aggaaggtgc gaagaccagc ggccttgttc 4320

cacgtatcgc caggcatacg gtcccacagg gaacccttgc cgtggacgac ttcatcgtga 4380

gagatcggaa gtacaaaacg ctcagagaat gcgtacacca aggagaaagt gagctcactg 4440

tggtggaatg cgcggtgcac agggtttttg gagaagtact ctaaggtgtc gtgcatccag 4500

cccatgttcc acttgaggga gaatccca 4528

<210> 9

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 9

tcgagctcgg tacccagcaa gctagccttg cgcat 35

<210> 10

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 10

cagacataca gtactgtcag gttccttaag aaatt 35

<210> 11

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 11

gaacctgaca gtactgtatg tctgagtatg gcaaa 35

<210> 12

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 12

ctctagagga tccccttgtc gacggctggc aaggc 35

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 13

ccaggtgttt ggggtgcagg 20

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 14

agtttggtca gcgtgaagag 20

<210> 15

<211> 3161

<212> DNA

<213> Corynebacterium glutamicum

<400> 15

agacgattct ggaaggccac tttcttttga tcctggcggc gatttttcgg tgcttggtag 60

cggaatgtca atcgagacca agcgtccccc ggcgggtcga ttttttggtc tcgaatgaca 120

gtttcgctct ctggaaactc tcagtgtcag gtcagtggtg aaccaccatc cttagcaagg 180

agttcatcat gtccatcccc ttctcagtcc ttcaggacta cctggatctg atcagtcccg 240

aagccttacc cgatccca cagcccccgg cccctgcccc cacagcaccc cagctaccac 300

cggcgccgga cccacacagc atcgagtggc cgatcttccc accagatcga atctccgcca 360

acgggcgacg ctactacgag ccacaaacac gactcgagtt catgcggatc tacaccaccc 420

tgccgcacgg ctaccgccag cccttcctta aagccaacaa catcggccac tgcaccgttc 480

gaacctggct agcagcaata agcaccttca gccgacttcc ccatgctttt gatgatgccc 540

accgcttcgg gatcgaacgc accaccccag tcgacgatgt caccacacta acggctgatg 600

acaaacgtga cctggtcata ggatacttag ctcaaccaca cggtcagggc cagcaattcc 660

tcacgtttta ccaactccgt aagcacacca tcatggcctg gtgcgccgct atgaccgacg 720

gggacttaga cgctgatatc tcaccccgcc agatcgggtt gatgaccacc cgaaccgtgg 780

tcgaaatcgt tcgactacgc cacatgattg cccaacaact agaaagagcc acgatcatgg 840

aaaacgagta cctcaaagaa atcgcagcgc tgaagaaaga actcgcgcac tacaagcaaa 900

aagaccatca gaatcaaatg gtgatcgata tcttgggaaa agctattggg accaggccca 960

atcctggcga gggcttagac gaggaggacg ccacctaaac gtggatgagc aacgcgcctt 1020

tgatcaagga ctcaaggaag aaaacacctt gatcacagat ctcaccacct gtgccaggct 1080

gagccataac aaggcattac ggctgatcaa gctgtcgaaa tcaacggcgt attaccgcaa 1140

caagccgcgt ccccgtcctg caccgaaacc tgtcctgcag gccgtgccag caccaacagc 1200

acctggtgtg gaacccacac cagagccttg gcaggggaag gagccagcag tgtcgtcggt 1260

gcgtcaagcg ttggcagaac acgaacgcca gttcattgtt gatgcgatca ccgcgtaccc 1320

acaactgagc gttagtgggg tgtttaacat gttgtttaac aaaggcatct accgcgcatc 1380

actacgtaca tggtggcgtg ttgccaagca gcacaagttg ttacacaaag accgagtcag 1440

tgccctgtcc ccggggaaac gatcaccaac gccacgggtt aagccgaggt tggaagcaac 1500

acagcctggt caggtggtgt gttgggatgt gacgttcttg ccgtcgctgg tacgtggtaa 1560

gacctatgcg ttgcatctgg cgattgattt gttttcccgc aagattgttg gggcgaaggt 1620

cgcgccgacg gaaaatacct ccaccgcggt ggagttgtta acgcaggtgt tagcggataa 1680

tccgggtgtg gtgacggtgc attcggataa tgggtcggcg atgacatcga cgaggtgcg 1740

gcggttgtta gcggatcatg gtgtggcgtt gtcgttgatt cggccgcggg tgagtgatga 1800

taatgcgttt gtggagtcgg tgtttcatac gttgaagtat cggccgtttt atccgaaggt 1860

gtttgcatcg atggatcagg cccggggtgtg ggtggagggag tttgtggtgt attacaacac 1920

ggttcatccg cattctggtg tggctgggca tactccgcag tcggtgtttg atggtagttg 1980

gagggcggct cataggttgc gtgtgcaggc gttggatgcc cattaccggc agttcccgca 2040

gcggtatgtg gggcggccgg tggttcagga agttgctggt gtggtgcgtc ttaatggtgc 2100

gcgtgatgat gggtctgtac aggagagggt tggtggtgta gcgtcgctgt taagtgcttg 2160

agttagcatg tgttcttatc gcccccctgg ttcacaaacc cctggcagcg agcggaaaag 2220

tgcattttta ggccaagggc cctcggatct tcgagcgctt tggtctcttt tgcacgtctg 2280

accgaaccag atcacctaga aacgccaaag gccccgcaag tatcaaacct gcggggcctt 2340

tgaggtacct gtttcctatt ttgttgactt aggaagctgc gcacggcgga taaccaaacc 2400

gcacagcaag gcagccactc cccacgcggt gagccagaac tgctccacga cataaacctg 2460

aatagttgga agcaaacgac ttacgatcac cagggctaca gcgatgctca aagaaatgat 2520

ctgtgtcttt gagcttggct cgtacttgct ggtggtagcg aatgcgccga gaatgatcga 2580

ggcaacgagg atcagaagga atcctggagt gatcacaaat gggctcagca tgccggcggt 2640

aaacagctca attgctgcaa acaccaacaa aaccagacct aaggctacga aagctccacc 2700

gatgcggatt gctgccggaa ctttacgcca gctggcacgg ccttcgaggc tggtgagctt 2760

ttttaatgat cttcccgatg ctgaactcat aatgtgacat accctactag ttctcgtacc 2820

atccccacac aattgacctg ccaagagtgt ggaaatacag gttgaagcct agaacagtgg 2880

gggtagcgtc gggggcgatg tcgagttttt ccacatcaag tgcatgaact gcgaagaggt 2940

aacggtgcgg tgcgtggcca gctggaggtt gcgctccgta gaagccacgc ttgccggaat 3000

cacccttgag ggaaactacg ccttcgatgc cgccgagggt ttcatcgcca gcaccggtgg 3060

ggatctccgt gacagttgtg gggatgttaa acactgccca gtgccagaaa ccagcgccgg 3120

ttggggcatc tgggtcgagg caggtgatcg cgagggattt g 3161

<210> 16

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 16

tcgagctcgg tacccttttt tggtctcgaa tgaca 35

<210> 17

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 17

catgctaaca gtactgttta ggtggcgtcc tcctc 35

<210> 18

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 18

cacctaaaca gtactgttag catgtgttct tatcg 35

<210> 19

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 19

ctcagaggat cccccccacc ggtgctggcg atga 34

<210> 20

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 20

cgccacctaa acagtgaagc ctaaaaacga ccgag 35

<210> 21

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 21

caaggttgta gtcatgttgt gtctcctcta aagat 35

<210> 22

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 22

tagagggagac acaacatgac tacaaccttg actcg 35

<210> 23

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 23

acacatgcta acagtcatgt cccagcgatg agtgc 35

<210> 24

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 24

ctggcggcga tttttcggtg 20

<210> 25

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 25

cgcgatcacc tgcctcgacc 20

<210> 26

<211> 5666

<212> DNA

<213> Corynebacterium glutamicum

<400> 26

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 gttctaggtt cggaggcgga 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 gggccgggcc 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 tgctcatggg 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

cccacaccacc 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

gcaatcggca gaaaccatga aaactgcggt ggcctatttg gaaccgttca tggaagagga 3240

agcagaagct accggatctg cgcaggcaga gggcaagggc aaaatcgtcg tggccaccgt 3300

caagggtgac gtgcacgata tcggcaagaa cttggtggac atcattttgt ccaacaacgg 3360

ttacgacgtg gtgaacttgg gcatcaagca gccactgtcc gccatgttgg aagcagcgga 3420

agaacacaaa gcagacgtca tcggcatgtc gggacttctt gtgaagtcca ccgtggtgat 3480

gaaggaaaac cttgaggaga tgaacaacgc cggcgcatcc aattacccag tcattttggg 3540

tggcgctgcg ctgacgcgta cctacgtgga aaacgatctc aacgaggtgt acaccggtga 3600

ggtgtactac gcccgtgatg ctttcgaggg cctgcgcctg atggatgagg tgatggcaga 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 tttgagagg 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

ccagcccccg 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 caggtggggg 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> 27

<211> 3613

<212> DNA

<213> Corynebacterium glutamicum

<400> 27

tcctgtgggg tgaacttgac ctgtgctggg ccacgacgtc cgaaaacgtg cacttcagtg 60

gccttgtttt ctttgaggga gtcgtagacg ttgtcggaaa tttcggtgac tttgagctcg 120

tcgcctgtct tagccaggat gcgggctacg tcgaggccga cgttaccaac gccgataaca 180

gcgacggact gtgcagacag atcccaggag cgctcgaagc gtgggttgcc gtcgtagaag 240

ccaacgaact cgccggcacc gaaggagcct tctgcttcaa ttccggggat gttgaggtcg 300

cggtctgcaa ctgcgccggt ggagaacacg actgcatcgt agtagtcgcg gagttcttcg 360

acggtgatgt ctttgccgat ttcaatgtta ccgagcaggc gcaggcgtgg cttgtccaac 420

acgttgtgca gggacttaac gatgcccttg atgcgtgggt ggtctggagc aacgccgtaa 480

cggatgagtc cgaacggtgc aggcatttgc tcgaaaaggt caacgaacac ttcgcgctct 540

tcattgcgga tgaggaggtc ggatgcgtaa atgccagcag ggccagctcc gatgacggct 600

acgcgcaggg gagttgtcat gtgtttgaag ttgcctttcg tgagcccttt tatggaaaca 660

agggtgtgaa aatcaagtag ttaaaggtgt ttcaagtcca ggctgtttaa cactcctaga 720

ccgcttggtc tgtaaacgta gcagcgaaat gcgacaatgc gaagactttt gcttaattaa 780

attcaaactc catgaaaaaa ctagacagat cggtctatta tattcacggt gaacctaacc 840

taatatcccc aggttaattc atttaaacgg gcattaggtg actccattgc tttcagtctc 900

atgaatctaa tggttggtct agacagagcg gtacgtctaa gtttgcggat agatcaaacc 960

gagtgacatg tacttcacta gctctttaag gattaactcc ccatgacaac aaccaccgga 1020

agtgcccggc cagcacgtgc cgccaggaag cctaagcccg aaggccaatg gaaaatcgac 1080

ggcaccgagc cgcttaacca tgccgaggaa attaagcaag aagaacccgc ttttgctgtc 1140

aagcagcggg tcattgatat ttactccaag cagggttttt cttccattgc accggatgac 1200

attgccccac gctttaagtg gttgggcatt tacacccagc gtaagcagga tctgggcggt 1260

gaactgaccg gtcagcttcc tgatgatgag ctgcaggatg agtacttcat gatgcgtgtg 1320

cgttttgatg gcggactggc ttcccctgag cgcctgcgtg ccgtgggtga aatttctagg 1380

gattatgctc gttccaccgc ggacttcacc gaccgccaga acattcagct gcactggatt 1440

cgtattgaag atgtgcctgc gatctgggag aagctagaaa ccgtcggact gtccaccatg 1500

cttggttgcg gtgacgttcc acgtgttatc ttgggctccc cagtttctgg cgtagctgct 1560

gaagagctga tcgatgccac cccggctatc gatgcgattc gtgagcgcta cctagacaag 1620

gaagagttcc acaaccttcc tcgtaagttt aagactgcta tcactggcaa ccagcgccag 1680

gatgttaccc acgaaatcca ggacgtttcc ttcgttcctt cgattcaccc agaattcggc 1740

ccaggatttg agtgctttgt gggcggtggc ctgtccacca acccaatgct tgctcagcca 1800

cttggttctt ggattccact tgatgaggtt ccagaagtgt gggctggcgt cgccggaatt 1860

ttccgcgact acggcttccg acgcctgcgt aaccgtgctc gcctcaagtt cttggtggca 1920

cagtggggta ttgagaagtt ccgtgaagtt cttgagaccg aatacctcga gcgcaagctg 1980

atcgatggcc cagttgttac caccaaccct ggctaccgtg accacattgg cattcaccca 2040

caaaaggacg gcaagttcta cctcggtgtg aagccaaccg ttggacacac caccggtgag 2100

cagctcattg ccattgctga tgttgcagaa aagcacggca tcaccaggat tcgtaccacg 2160

gcggaaaagg aactgctctt cctcgatatt gagagaaaga accttactac cgttgcacgc 2220

gacctggatg aaatcggact gtactcttca ccttccgagt tccgccgcgg catcatttcc 2280

tgcaccggct tggagttctg caagcttgcg cacgcaacca ccaagtcacg agcaattgag 2340

cttgtcgacg aactggaaga gcgcctcggc gatttggatg ttcccatcaa gattgcactg 2400

aacggttgcc ctaactcttg tgcacgcacc caggtttccg acatcggatt caagggacag 2460

accgtcactg atgctgacgg caaccgcgtt gaaggtttcc aggttcacct gggcggttcc 2520

atgaacttgg atccaaactt cggacgcaag ctcaagggcc acaaggttat tgccgatgaa 2580

gtgggagagt acgtcactcg cgttgttacc cacttcaagg aacagcgcca cgaggacgag 2640

cacttccgcg attgggtcca gcgggccgct gaggaagatt tggtgtgagt cttcggagga 2700

aacccaatcc caaccgcaac caccctctgt actgcccata ctgcgcggga gaagttcttt 2760

tccccgatga gcaaacagaa ttcgcgtggt tgtgtgcgga ttgcaccaga gtttttgaag 2820

tgaaatatca cggccaggac gatccagtgc acaggccagc accagcaaag tccacatcgc 2880

aagcattaaa agaatctctc gaaagacaca aaagaggtga gtcgcaacaa tgagctttca 2940

actagttaac gccctgaaaa atactggttc ggtaaaagat cccgagatct cacccgaagg 3000

acctcgcacg accacaccgt tgtcaccaga ggtagcaaaa cataacgagg aactcgtcga 3060

aaagcatgct gctgcgttgt atgacgccag cgcgcaagag atcctggaat ggacagccga 3120

gcacgcgccg ggcgctattg cagtgacctt gagcatggaa aacaccgtgc tggcggagct 3180

ggctgcgcgg cacctgccgg aagctgattt cctctttttg gacaccggtt accacttcaa 3240

ggagaccctt gaagttgccc gtcaggtaga tgagcgctat tcccagaagc ttgtcaccgc 3300

gctgccgatc ctcaagcgca cggagcagga ttccatttat ggtctcaacc tgtaccgcag 3360

caacccagcg gcgtgctgcc gaatgcgcaa agttgaaccg ctggcggcgt cgttaagccc 3420

atacgctggc tggatcaccg gcctgcgccg cgctgatggc ccaacccgtg ctcaagcccc 3480

tgcgctgagc ttggatgcca ccggcaggct caagatttct ccaattatca cctggtcatt 3540

ggaggaaacc aacgagttca ttgcggacaa caacctcatc gatcacccac ttacccatca 3600

gggttatcca tca 3613

<210> 28

<211> 3311

<212> DNA

<213> Corynebacterium glutamicum

<400> 28

ctcattccag cgtcacgacg ttccgaaggt actggttacc tggcattggg 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 cccagcctg 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 gaccacggta aaaaggtcac 1320

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 agttgtgggc 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

gattgctcta ggtttccgta atttgaacca ctacattctg cggtgcctta tccattcagg 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

tttttcctgg 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

cgctcctttg atagcgaggt tgttggcttc tgatccaccc gacgtaaacg tcacctgtgt 3240

ggggcgtcct ccgataatgc gggccacccg agttcgagca tcctccagcc ccgcagaggc 3300

gagtcttccc a 3311

<210> 29

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 29

acccggggat cctctagaat gtttgtgatg cgcag 35

<210> 30

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 30

gtcagagagt acttacgctg atcgggaggg aaagc 35

<210> 31

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 31

atcagcgtaa gtactctctg actagcgtca ccctc 35

<210> 32

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 32

ctgcaggtcg actctagaaa agggattgga gtgtt 35

<210> 33

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 33

caacgaaagg aaacaatgtc tacttcagtt acttc 35

<210> 34

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 34

tagtcagaga gtgatttaga cgttaaagta ctttg 35

<210> 35

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 35

atcaaaacag atatcatgac aacaaccacc ggaag 35

<210> 36

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 36

cgctagtcag agagttcaca ccaaatcttc ctcag 35

<210> 37

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 37

ccgatcagcg taagtagaaa catcccagcg ctact 35

<210> 38

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 38

aactgaagta gacattgttt cctttcgttg ggtac 35

<210> 39

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 39

tactttaacg tctaaggtac cggcgcttca tgtca 35

<210> 40

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 40

ggtggttgtt gtcatgatat ctgttttgat ctcct 35

<210> 41

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 41

atccccatcg gcatctttat 20

<210> 42

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 42

cgatcacact gggctgatct 20

<210> 43

<211> 319

<212>PRT

<213> Corynebacterium glutamicum

<400> 43

Met Lys Phe Lys Lys Ile Ala Leu Val Leu Ala Phe Gly Leu Gly Leu

1 5 10 15

Ala Ser Cys Ser Ser Ala Ser Gly Asp Pro Ala Thr Asn Ala Asp Gly

20 25 30

Ser Ile Asp Leu Ser Lys Val Thr Leu Asn Ile Gly Asp Gln Ile Ala

35 40 45

Gly Thr Glu Gln Val Leu Gln Ala Ser Gly Glu Leu Asp Asp Val Pro

50 55 60

Tyr Lys Ile Glu Trp Ser Ser Phe Thr Ser Gly Pro Pro Gln Ile Glu

65 70 75 80

Ala Leu Asn Ala Gly Gln Ile Asp Phe Ala Ile Thr Gly Asn Thr Pro

85 90 95

Pro Ile Ile Gly Gly Pro Thr Asn Thr Lys Val Val Ser Ala Tyr Asn

100 105 110

Asn Asp Ala Leu Gly Asp Val Ile Leu Val Ala Pro Asp Ser Ser Ile

115 120 125

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

130 135 140

Ser Ser Ala His Gly His Leu Ile Gln Gln Leu Glu Lys Ala Gly Val

145 150 155 160

Ser Val Asp Asp Val Glu Ile Asn Leu Leu Gln Pro Ser Asp Ala Lys

165 170 175

Ala Ala Phe Gln Asn Gly Gln Val Asp Ala Trp Ala Val Trp Asp Pro

180 185 190

Tyr Ser Ser Gln Ala Glu Leu Glu Gly Ala Gln Val Leu Val Arg Gly

195 200 205

Ala Gly Leu Val Ser Gly His Gly Phe Gly Val Ala Ser Asp Glu Ala

210 215 220

Leu Asp Asp Pro Ala Lys Glu Ala Ala Leu Ala Asp Phe Leu Asp Arg

225 230 235 240

Val Ala Asp Ser Tyr Glu Trp Ala Glu Asp Asn Thr Asp Glu Trp Ala

245 250 255

Thr Ile Phe Ser Gln Glu Ser Gly Phe Asp Pro Glu Ala Ser Gln Leu

260 265 270

Asn Thr Arg Ser Leu Arg His Gln Val Pro Leu Asp Glu Ser Val Asn

275 280 285

Thr Tyr Gln Asn Ala Leu Ile Asp Ala Phe Val Ser Ala Gly Leu Val

290 295 300

Glu Asp Phe Asn Phe Glu Asp Thr Val Asp Thr Arg Phe Glu Gly

305 310 315

<210> 44

<211> 243

<212>PRT

<213> Corynebacterium glutamicum

<400> 44

Met Thr Ala Thr Leu Ser Leu Lys Pro Ala Ala Thr Val Arg Gly Leu

1 5 10 15

Arg Lys Ser Tyr Gly Thr Lys Glu Val Leu Gln Gly Ile Asp Leu Thr

20 25 30

Ile Asn Cys Gly Glu Val Thr Ala Leu Ile Gly Arg Ser Gly Ser Gly

35 40 45

Lys Ser Thr Ile Leu Arg Val Leu Ala Gly Leu Ser Lys Glu His Ser

50 55 60

Gly Ser Val Glu Ile Ser Gly Asn Pro Ala Val Ala Phe Gln Glu Pro

65 70 75 80

Arg Leu Leu Pro Trp Lys Thr Val Leu Asp Asn Val Thr Phe Gly Leu

85 90 95

Asn Arg Thr Asp Ile Ser Trp Ser Glu Ala Gln Glu Arg Ala Ser Ala

100 105 110

Leu Leu Ala Glu Val Lys Leu Pro Asp Ser Asp Ala Ala Trp Pro Leu

115 120 125

Thr Leu Ser Gly Gly Gln Ala Gln Arg Val Ser Leu Ala Arg Ala Leu

130 135 140

Ile Ser Glu Pro Glu Leu Leu Leu Leu Asp Glu Pro Phe Gly Ala Leu

145 150 155 160

Asp Ala Leu Thr Arg Leu Thr Ala Gln Asp Leu Leu Leu Lys Thr Val

165 170 175

Asn Thr Arg Asn Leu Gly Val Leu Leu Val Thr His Asp Val Ser Glu

180 185 190

Ala Ile Ala Leu Ala Asp His Val Leu Leu Leu Asp Asp Gly Ala Ile

195 200 205

Thr His Ser Leu Thr Val Asp Ile Pro Gly Asp Arg Arg Thr His Pro

210 215 220

Ser Phe Ala Ser Tyr Thr Ala Gln Leu Leu Glu Trp Leu Glu Ile Thr

225 230 235 240

Thr Pro Ala

<210> 45

<211> 256

<212>PRT

<213> Corynebacterium glutamicum

<400> 45

Met Thr Thr Thr Leu Thr Arg Pro Lys Ile Ala Leu Pro Ala Arg Ile

1 5 10 15

Tyr Ser Pro Leu Ala Val Leu Val Phe Trp Gln Leu Gly Ser Ser Leu

20 25 30

Gly Ala Ile Pro Glu Arg Ile Leu Pro Ala Pro Thr Thr Ile Leu Ala

35 40 45

Ala Ser Trp Glu Val Ala Thr Asn Gly Thr Leu Leu Asp Ala Leu Leu

50 55 60

Val Ser Ser Gln Arg Val Leu Leu Gly Phe Ala Leu Gly Ala Val Leu

65 70 75 80

Gly Ile Ser Leu Gly Val Leu Thr Gly Met Ser Arg Phe Ala Asp Thr

85 90 95

Ala Val Asp Pro Leu Ile Gln Ala Ala Arg Ala Leu Pro His Leu Gly

100 105 110

Leu Val Pro Leu Phe Ile Ile Trp Phe Gly Ile Gly Glu Leu Pro Lys

115 120 125

Val Leu Ile Ile Ser Leu Gly Val Leu Tyr Pro Leu Tyr Leu Asn Thr

130 135 140

Ala Ser Gly Phe Arg Gln Ile Asp Pro Lys Leu Leu Glu Ala Gly His

145 150 155 160

Val Met Gly Phe Gly Phe Phe Gln Arg Leu Arg Thr Ile Ile Ile Pro

165 170 175

Ser Ala Ala Pro Gln Leu Phe Val Gly Leu Arg Gln Ala Ser Ala Ala

180 185 190

Ala Trp Leu Ser Leu Ile Val Ala Glu Gln Val Asn Ala Arg Glu Gly

195 200 205

Leu Gly Phe Leu Ile Asn Asn Ala Arg Asp Phe Tyr Arg Thr Asp Leu

210 215 220

Val Ile Phe Gly Leu Ile Val Tyr Ala Ser Leu Gly Leu Leu Ser Glu

225 230 235 240

Ala Leu Ile Arg Ala Trp Glu Arg His Thr Phe Arg Tyr Arg Asn Ala

245 250 255

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Claims (14)

1. A method for producing a sulfur-containing amino acid or a sulfur-containing amino acid derivative, comprising cultivating a genetically modified microorganism in a culture medium containing thiosulfate, where the microorganism contains a genetic modification to increase the activity of the protein encoded by the ssuABC gene, compared to an unmodified microorganism, where the sulfur-containing amino acid or a sulfur-containing amino acid derivative includes at least one selected from the group consisting of methionine, cysteine, cystine, lanthionine, homocysteine, homocystine, homolanthionine and taurine. 2. The method according to claim 1, wherein the protein encoded by the ssuABC gene has thiosulfate transporter activity. 3. The method according to claim 1, where the protein encoded by the ssuABC gene is a complex of proteins SsuA, SsuB and SsuC, and the microorganism has increased activity of at least one protein selected from the group consisting of proteins SsuA, SsuB and SsuC, according to compared to an unmodified microorganism. 4. The method of claim 3, wherein the SsuA protein contains an amino acid sequence having at least 80% homology to the amino acid sequence of SEQ ID NO: 43. 5. The method of claim 3, wherein the SsuB protein contains an amino acid sequence having at least 80% homology to the amino acid sequence of SEQ ID NO: 44. 6. The method of claim 3, wherein the SsuC protein contains an amino acid sequence having at least 80% homology to the amino acid sequence of SEQ ID NO: 45. 7. The method according to claim 1, where the microorganism is a microorganism belonging to the genus Corynebacterium sp. or the genus Escherichia sp. 8. The method according to claim 1, further comprising isolating the sulfur-containing amino acid and the derivative of the sulfur-containing amino acid from the microorganism or culture medium. 9. The method according to claim 1, where genetic modification to increase protein activity is achieved by: 1) increasing the number of copies of the polynucleotide encoding the protein in the cell; 2) replacing the region of regulation of expression of the polynucleotide encoding the protein with a sequence with higher activity; 3) modification of the initiation codon or 5′-UTR (5′-untranslated region) of the polynucleotide encoding the protein; 4) modification of the nucleotide sequence on the chromosome to increase protein activity; 5) introduction of a foreign polynucleotide that ensures the manifestation of protein activity, or a codon-optimized version of a polynucleotide encoding a protein, or 6) a combination thereof. 10. A microorganism that produces a sulfur-containing amino acid and a derivative of a sulfur-containing amino acid and contains a genetic modification to increase the activity of a protein encoded by the ssuABC gene compared to an unmodified microorganism, where the sulfur-containing amino acid or a derivative of a sulfur-containing amino acid includes at least one selected from the group consisting of methionine, cysteine, cystine, lanthionine, homocysteine, homocystine, homolanthionine and taurine. 11. The microorganism according to claim 10, wherein the microorganism produces a sulfur-containing amino acid or a derivative of a sulfur-containing amino acid using thiosulfate as a sulfur source. 12. The microorganism according to claim 10, where genetic modification to increase protein activity is achieved by: 1) increasing the number of copies of the polynucleotide encoding the protein in the cell; 2) replacing the region of regulation of expression of the polynucleotide encoding the protein with a sequence with higher activity; 3) modification of the start codon or 5′-UTR of the polynucleotide encoding the protein; 4) modification of the nucleotide sequence on the chromosome to increase protein activity; 5) introduction of a foreign polynucleotide that ensures the manifestation of protein activity, or a codon-optimized version of a polynucleotide encoding this protein, or 6) a combination thereof. 13. A composition for producing a sulfur-containing amino acid or a derivative of a sulfur-containing amino acid, containing a microorganism containing a genetic modification to increase the activity of the protein encoded by the ssuABC gene compared to an unmodified microorganism, or a culture thereof; and thiosulfate, wherein the sulfur amino acid or sulfur amino acid derivative includes at least one selected from the group consisting of methionine, cysteine, cystine, lanthionine, homocysteine, homocystine, homolanthionine and taurine. 14. The use of a microorganism containing a genetic modification to increase the activity of a protein encoded by the ssuABC gene, compared to an unmodified microorganism, for the production of a sulfur-containing amino acid or a derivative of a sulfur-containing amino acid, where the sulfur-containing amino acid or a derivative of a sulfur-containing amino acid includes at least one selected( oe) from the group consisting of methionine, cysteine, cystine, lanthionine, homocysteine, homocystine, homolanthionine and taurine.