Title of Invention: Method for producing sulfur-containing amino acid or derivative thereof
technical field
[One]
The present application relates to a method for preparing a sulfur-containing amino acid or a sulfur-containing amino acid derivative.
[2]
background
[3]
L-amino acids are industrially produced by a fermentation method using microorganisms belonging to the genus Brevibacterium, the genus Corynebacterium, the genus Escherichia, and the like. In this production method, a strain isolated from the natural world or an artificial mutant of the strain, or a microorganism mutated to increase the activity of an enzyme involved in L-amino acid biosynthesis by recombinant DNA technology is used.
[4]
On the other hand, sulfur-containing amino acids are used as animal feeds, food additives, pharmaceutical infusions, and synthetic raw materials for pharmaceuticals, and research has been conducted to biologically produce these sulfur-containing amino acids and derivatives thereof.
[5]
For example, in US Patent Publication No. 2009-0298135 A1, by removing the metJ gene on the genomic gene of Escherichia coli and over-expressing the YjeH protein, which is an L-methionine exporter, 0.8 by over-expression reported to produce g/L of L-methionine. In addition, as L-methionine releasing factors of Corynebacterium glutamicum, BrnF and BrnE polypeptides have been reported (C. Troschel, et al, Journal of Bacteriology, p.3786-3794, June 2005) ).
[6]
On the other hand, in the production of sulfur-containing amino acids, the amount of NADPH consumed in microorganisms varies according to the reducing power of the sulfur source. For example, since sulfide does not require NADPH, theoretically the yield is highest, and sulfate requires four NADPHs, so theoretically the yield is low. However, sulfide is known to cause cell damage, so it has a disadvantage of low stability. Therefore, when thiosulfate is used for the production of sulfur-containing amino acids as a sulfur source with a low NADPH requirement and high intracellular stability, a high production yield can be expected. However, studies that can effectively use it in microorganisms of the genus Corynebacterium are insufficient.
[7]
DETAILED DESCRIPTION OF THE INVENTION
technical challenge
[8]
The present inventors have newly identified that the protein encoded by the ssuD gene is involved in the use of thiosulfate, and completed the present application by confirming that the microorganisms with enhanced activity use thiosulfate as a sulfur source to enhance the ability to produce sulfur-containing amino acids. did
[9]
means of solving the problem
[10]
The present application relates to a sulfur-containing amino acid or sulfur-containing, comprising culturing a genetically modified microorganism having a genetic modification that increases the protein activity encoded by the ssuD gene compared to an unmodified microorganism in a medium containing thiosulfate. A method for preparing an amino acid derivative is provided.
[11]
The present application provides a microorganism producing a sulfur-containing amino acid or a sulfur-containing amino acid derivative having a genetic modification that increases the protein activity encoded by the ssuD gene compared to the unmodified microorganism.
[12]
The present application relates to a microorganism having a genetic modification in which the protein activity encoded by the ssuD gene is increased compared to an unmodified microorganism, or a culture thereof; And it provides a composition for preparing a sulfur-containing amino acid or a sulfur-containing amino acid derivative, comprising thiosulfate.
[13]
The present application provides the use of a protein encoded by the ssuD gene as a thiosulfate reductase.
[14]
The present application relates to a microorganism having a genetic modification in which the activity of the protein encoded by the ssuD gene is increased compared to the unmodified microorganism. Provided is a use for preparing a sulfur-containing amino acid or a derivative of a sulfur-containing amino acid.
[15]
Effects of the Invention
[16]
By using the microorganism, composition and the method for producing sulfur-containing amino acids or sulfur-containing amino acids using the microorganisms, compositions of the present application, sulfur-containing amino acids or derivatives thereof can be mass-produced, so it is useful for production of useful products including sulfur-containing amino acids or derivatives thereof can be used
[17]
Best mode for carrying out the invention
[18]
This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present application fall within the scope of the present application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.
[19]
In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present application described herein. Also, such equivalents are intended to be covered by this application.
[20]
[21]
One aspect of the present application provides a genetically modified microorganism to produce a sulfur-containing amino acid or a sulfur-containing amino acid derivative.
[22]
One aspect of the present application provides a method for producing a sulfur-containing amino acid or a sulfur-containing amino acid derivative, comprising culturing a genetically modified microorganism in a medium containing thiosulfate.
[23]
The microorganism may have a genetic modification in which the activity of the protein encoded by the ssuD gene is increased.
[24]
The present application provides a method for producing a sulfur-containing amino acid or a sulfur-containing amino acid derivative, comprising culturing a genetically modified microorganism in a medium containing thiosulfate, wherein the microorganism has a ssuD gene compared to the microorganism before genetic modification It may include a genetic modification in which the activity of the protein encoded by In one embodiment of the present application, the method may be a method of increasing the production of sulfur-containing amino acids or sulfur-containing amino acid derivatives of microorganisms.
[25]
The preparation method may include contacting a microorganism or a culture thereof in which the protein activity encoded by the ssuD gene is enhanced compared to the intrinsic activity with thiosulfate.
[26]
In the present application, the ' protein encoded by the ssuD gene ' is a protein encoded by the ssuD gene or a protein expressed by the ssuD gene, and may be referred to as an 'SsuD protein' (hereinafter referred to as "SsuD protein"). Conventionally, SsuD protein is known as a protein that degrades sulfonate (sulfonate, R-SO 3 ), specifically, as one of alkanesulfonate monooxygenases, sulfonate monooxygenase However, it is not known at all whether the SsuD protein is involved in the utilization of thiosulfate rather than organic sulfate.
[27]
As of the present application, the fact that the SsuD protein is also involved in the use of thiosulfate and, specifically, functions as a reductase for reducing thiosulfate has been newly identified, and sulfur-containing amino acid production by enhancing the activity of the SsuD protein was confirmed to be able to increase.
[28]
[29]
The SsuD protein of the present application may be a protein encoded by an ssuD gene, which may be referred to as 'SsuD protein' or 'thiosulfate reductase', or as 'alkanesulfonate monooxygenase' known in the prior art. . In one embodiment, the SsuD protein of the present application may be 'sulfonate monooxygenase' or 'thiosulfate reductase' derived from the genus Corynebacterium, and specifically, LLM class flavin derived from the genus Corynebacterium. It may be a protein named -dependent oxidoreductase, specifically, the SsuD protein, Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium crudilactis ( Corynebacterium crudilactis ), Corynebacterium deserti ( Corynebacterium deserti ), Corynebacterium efficiens ( Corynebacterium efficiens ), Corynebacterium callunae ( Corynebacterium callunae ), Corynebacterium stationis , Corynebacterium stationis ), ), Corynebacterium halotolerans , Corynebacterium striatum , Corynebacterium ammoniagenes , Corynebacterium pollutisoli , Corynebacterium pollutisoli Nebacterium imitans ( Corynebacterium imitans ), Corynebacterium testudinoris ( Corynebacterium testudinoris ), Corynebacterium Corynebacterium callunae ( Corynebacterium callunae ), Corynebacterium crenatum ( Corynebacterium crenatum ), Coryne Nebacterium deserti ( Corynebacterium deserti ), Corynebacterium flavescens ( Corynebacterium flavescens ), Corynebacterium pacaense ( Corynebacterium pacaense )), or Corynebacterium suranareeae , may be derived, and more specifically, may be a Corynebacterium glutamicum-derived SsuD protein, but is not limited thereto. The sequence of the SsuD protein can be confirmed in a known database such as NCBI.
[30]
In one embodiment, the SsuD protein comprises the amino acid sequence of SEQ ID NO: 43, or at least 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98 with the amino acid sequence of SEQ ID NO: 43 amino acid sequences having % or 99% homology or identity. In addition, as long as it is an amino acid sequence having the homology or identity and exhibiting efficacy corresponding to the polypeptide, it is apparent that some sequences are included within the scope of the present application even if they have an amino acid sequence that is deleted, modified, substituted or added.
[31]
In addition, a probe that can be prepared from a known gene sequence, for example, a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a sequence complementary to all or part of a nucleotide sequence encoding the polypeptide, Polypeptides having thiosulfate reducing activity while having sulfonate monooxygenase activity may also be included without limitation.
[32]
That is, in the present application, 'a protein or polypeptide comprising the amino acid sequence described in a specific SEQ ID NO:', 'a protein or polypeptide comprising the amino acid sequence described in a specific SEQ ID NO:' or 'a protein having an amino acid sequence described in a specific SEQ ID NO: or Even if it is described as 'polypeptide', if it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of the corresponding SEQ ID NO: Some sequences are deleted, modified, substituted, conservatively substituted, or a protein having an added amino acid sequence It is obvious that it can be used in the present application. For example, the amino acid sequence N-terminus and/or C-terminus is a case of adding a sequence that does not alter the function of the protein, a naturally occurring mutation, a silent mutation or a conservative substitution thereof. .
[33]
The term “conservative substitution” means substituting one amino acid for another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may generally occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues.
[34]
In one embodiment of the present application, the SsuD protein may be encoded by the polynucleotide sequence of SEQ ID NO: 8, but is not limited thereto.
[35]
In the present application, the term "polynucleotide" has a meaning comprehensively encompassing DNA or RNA molecules, and nucleotides, which are basic structural units in polynucleotides, may include not only natural nucleotides, but also analogs in which sugar or base sites are modified ( Scheit, Nucleotide Analogs, John Wiley, New York (1980); see Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).
[36]
The polynucleotide may be a polynucleotide encoding the SsuD protein of the present application ( ssuD gene). In the polynucleotide of the present application, various modifications may be made to the coding region within a range that does not change the amino acid sequence due to codon degeneracy or in consideration of codons preferred in an organism to express a protein. A polynucleotide of the present application may be, for example, a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity to the SsuD protein of the present application. It may be a polynucleotide encoding Specifically, the polynucleotide encoding a protein comprising an amino acid sequence having 80% or more homology or identity to the amino acid sequence of SEQ ID NO: 43 is at least 80%, at least 80%, 90%, 92 with the nucleotide sequence of SEQ ID NO: 8 %, 94%, 95%, 96%, 97%, 98% or 99% homology or identity.
[37]
In addition, it is apparent that a polynucleotide that can be translated into a protein comprising an amino acid sequence having 80% or more homology or identity with SEQ ID NO: 43 due to codon degeneracy may also be included. Or an amino acid having 80% or more identity to the amino acid sequence of SEQ ID NO: 43 by hydridation under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of the nucleotide sequence. Any polynucleotide sequence encoding a protein comprising the sequence may be included without limitation. The "stringent conditions" means conditions that allow specific hybridization between polynucleotides. Such conditions are described in, for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology. , John Wiley & Sons, Inc., New York). For example, between genes with high homology or identity, 70% or more, 80% or more, specifically 85% or more, specifically 90% or more, more specifically 95% or more, even more specifically 97% or more , specifically, a condition in which genes having 99% or more homology or identity hybridize with each other, and genes with lower homology or identity do not hybridize than that;
[38]
Hybridization requires that two polynucleotides have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the present application may also encompass substantially similar polynucleotide sequences as well as isolated polynucleotide fragments complementary to the overall sequence.
[39]
Specifically, polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the above-described conditions. In addition, the Tm value may be 60 °C, 63 °C, or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.
[40]
The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotide and the degree of complementarity, and the parameters are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).
[41]
As used herein, the term “homology” or “identity” refers to the degree to which two given amino acid sequences or base sequences are related, and may be expressed as a percentage. The terms homology and identity can often be used interchangeably.
[42]
Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used. Substantially homologous or identical sequences under moderate or high stringent conditions generally contain at least about 50%, 60%, 70%, 80% of the total or full-length of the sequence. or more than 90% hybrid. It is self-evident that hybridization also includes polynucleotides containing degenerate codons instead of common codons in polynucleotides.
[43]
Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444, using a known computer algorithm such as the “FASTA” program. or, as performed in the Needleman Program (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) of the EMBOSS package, The Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine. (GCG program 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 ETA/.] (1988) including SIAM J Applied Math 48: 1073) For example, BLAST of the National Center for Biotechnology Information Database, or ClustalW, can be used to determine homology, similarity or identity.
[44]
Homology, similarity or identity of polynucleotides or polypeptides is described, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, for example, Needleman et al. (1970), J Mol Biol. 48: 443 by comparing the sequence information using a GAP computer program. In summary, the GAP program is defined as the total number of symbols in the shorter of two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids). Default parameters for the GAP program are: (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps. In addition, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be confirmed by comparing the sequences by Southern hybridization experiments under defined stringent conditions,
[45]
[46]
As used herein, the term “enhancement” of the activity of a polypeptide or protein means that the activity of the polypeptide or protein is increased compared to the intrinsic activity. The reinforcement may be used interchangeably with terms such as up-regulation, overexpression, and increase.
[47]
Here, the increase may include both exhibiting an activity that it did not originally have, or exhibiting an improved activity compared to intrinsic activity or activity before modification. The "intrinsic activity" refers to the activity of a specific polypeptide or protein originally possessed by the parent strain or unmodified microorganism before the transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before modification". "Enhancement" or "increase" in the activity of a polypeptide or protein compared to the intrinsic activity means that it is improved compared to the activity of a specific polypeptide or protein originally possessed by the parent strain or unmodified microorganism before transformation.
[48]
The "increase in activity" may be achieved by introducing an exogenous polypeptide or protein or enhancing the activity of an endogenous polypeptide or protein, but specifically, it may be achieved through enhancing the activity of an endogenous polypeptide or protein. Whether the activity of the polypeptide or protein is enhanced can be confirmed from an increase in the activity level, expression level, or amount of a product excreted from the polypeptide or protein.
[49]
In the present application, " enhancing or increasing the activity of the protein encoded by the ssuD gene or the SsuD protein" may be referred to as "a genetic modification in which the activity of the protein encoded by the ssuD gene is increased", which means that the activity of the protein is It may mean enhanced compared to intrinsic activity.
[50]
The increase in activity may include both introducing an exogenous SsuD protein and enhancing the activity of an endogenous SsuD protein.
[51]
In the present application, "introduction of protein" refers to exhibiting the activity of a specific protein that the microorganism did not originally have, or exhibiting improved activity compared to the intrinsic activity or activity prior to modification of the corresponding protein. For example, a specific protein is introduced, a polynucleotide encoding a specific protein is introduced into a chromosome in a microorganism, or a vector including a polynucleotide encoding a specific protein is introduced into a microorganism to exhibit its activity.
[52]
[53]
The enhancement of the activity of the polypeptide or protein can be applied by various methods well known in the art, and may not be limited as long as it can enhance the activity of the target polypeptide or protein compared to the microorganism before modification. The method is not limited thereto, but may use genetic engineering and/or protein engineering well known to those skilled in the art, which are routine methods of molecular biology (Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology). 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012 et al.).
[54]
Specifically, in the present application, the activity enhancement is,
[55]
1) an increase in the intracellular copy number of a gene or polynucleotide encoding the polypeptide or protein;
[56]
2) a method of replacing the gene expression control region on the chromosome encoding the polypeptide or protein with a sequence with strong activity;
[57]
3) a method of modifying the base sequence of the start codon or 5'-UTR region of the polypeptide or protein,
[58]
4) a method of modifying a polynucleotide sequence on a chromosome to enhance the activity of the polypeptide or protein;
[59]
5) introduction of a foreign polynucleotide exhibiting the activity of the polypeptide or protein or a codon-optimized mutant polynucleotide of the polynucleotide, or
[60]
6) It may be performed by a method of modifying to be strengthened by a combination of the above methods, but is not limited thereto.
[61]
The method for enhancing polypeptide or protein activity using protein engineering may be performed by, for example, analyzing the tertiary structure of the polypeptide or protein and selecting an exposed site to modify or chemically modify it, but is limited thereto. doesn't happen
[62]
[63]
1) The increase in the copy number of the gene or polynucleotide encoding the polypeptide or protein is performed by any method known in the art, for example, regardless of the host, to which the gene or polynucleotide encoding the polypeptide or protein is operably linked. This can be performed by introducing a vector capable of replicating and functioning properly into a host cell. Alternatively, a vector capable of inserting the gene or polynucleotide into a chromosome in the host cell, to which the gene is operably linked, may be introduced into the host cell, but is not limited thereto.
[64]
2) The method of replacing the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide or protein with a sequence with strong activity is any method known in the art, for example, the activity of the expression control sequence It can be carried out by inducing a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof to further enhance the nucleic acid sequence, or by replacing the nucleic acid sequence with a nucleic acid sequence having stronger activity. The expression control sequence is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence for controlling the termination of transcription and translation, and the like. The method may specifically be to link a strong heterologous promoter instead of the original promoter, but is not limited thereto.
[65]
Examples of known strong promoters include cj1 to cj7 promoter (US Patent US 7662943 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), gapA promoter, EF-Tu promoter, groEL promoter, aceA or aceB promoter, O2 promoter (US 10273491 B2), tkt promoter and yccA promoter, etc. However, it is not limited thereto.
[66]
3) The method of modifying the base sequence of the start codon or 5'-UTR region of the polypeptide or protein is any method known in the art, for example, the endogenous start codon of the polypeptide or protein is replaced with the endogenous start codon. It may be substituted with another initiation codon having a higher expression rate of a polypeptide or protein, but is not limited thereto.
[67]
4) The method of modifying a polynucleotide sequence on a chromosome to increase polypeptide or protein activity may include any method known in the art, for example, deleting, inserting, or inserting a nucleic acid sequence to further enhance the activity of the polynucleotide sequence; It can be carried out by inducing a mutation in the expression control sequence by non-conservative or conservative substitution or a combination thereof, or by replacing the polynucleotide sequence with an improved polynucleotide sequence to have stronger activity. The replacement may specifically be to insert the gene into the chromosome by homologous recombination, but is not limited thereto. In this case, the vector used may further include a selection marker for confirming whether or not the chromosome is inserted.
[68]
5) The introduction of a foreign polynucleotide sequence exhibiting the activity of a polypeptide or protein can be performed by any method known in the art, for example, a foreign polynucleotide encoding a polypeptide or protein exhibiting the same/similar activity as the polypeptide or protein. , or a codon-optimized mutant polynucleotide thereof may be introduced into a host cell. The foreign polynucleotide may be used without limitation in origin or sequence as long as it exhibits the same/similar activity as the polypeptide or protein. In addition, the introduced foreign polynucleotide may be introduced into the host cell by optimizing its codon so that the optimized transcription and translation are performed in the host cell. The introduction can be carried out by appropriately selecting a known transformation method by those skilled in the art, and by expressing the introduced polynucleotide in a host cell, a polypeptide or protein is produced and its activity can be increased.
[69]
Finally, 6) the combination of the methods may be performed by applying any one or more methods of 1) to 5) together.
[70]
The enhancement of such polypeptide or protein activity is a function in which the activity or concentration of the corresponding polypeptide or protein is increased relative to the activity or concentration of the polypeptide or protein expressed in the wild-type or pre-modified microbial strain, or is produced from the polypeptide or protein. The amount of the product may be increased, but is not limited thereto.
[71]
In the present application, the term "pre-transformation strain" or "pre-transformation microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, and it is either a wild-type strain or a natural-type strain itself, or caused by natural or artificial factors. It may refer to a strain before the trait is changed due to genetic mutation. The “pre-modified strain” or “pre-modified microorganism” may be used interchangeably with “unmodified strain”, “unmodified strain”, “unmodified microorganism”, “unmodified microorganism” or “reference microorganism”.
[72]
[73]
As used herein, the term "vector" refers to a DNA preparation containing a polynucleotide sequence encoding the target protein operably linked to a suitable expression control region or expression control sequence so that the target protein can be expressed in a suitable host. The regulatory sequences may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself. For example, a polynucleotide encoding a target protein may be inserted into a chromosome through a vector for intracellular chromosome insertion. The insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto. The vector may further include a selection marker for confirming whether the chromosome is inserted. The selection marker is used to select cells transformed with the vector, that is, to determine whether a target nucleic acid molecule is inserted, and to confer a selectable phenotype such as drug resistance, auxotrophicity, resistance to cytotoxic agents, or expression of a surface polypeptide. markers may be used. In an environment treated with a selective agent, only cells expressing a selectable marker survive or exhibit other expression traits, and thus transformed cells can be selected.
[74]
The vector of the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pBR-based, pUC-based, and pBluescriptII-based plasmid vectors may be used. , pGEM-based, pTZ-based, pCL-based and pET-based and the like can be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used. However, it is not limited thereto.
[75]
As used herein, the term “transformation” refers to introducing a vector including a polynucleotide encoding a target protein into a host cell or microorganism so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them regardless of whether they are inserted into the chromosome of the host cell or located outside the chromosome, as long as they can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding a target protein. The polynucleotide may be introduced into a host cell and expressed in any form, as long as it can be expressed. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression. The expression cassette may include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, which are usually operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.
[76]
In addition, the term "operably linked" in the present application means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target protein of the present application.
[77]
The method for transforming the vector of the present application includes any method of introducing a nucleic acid into a cell, and may be performed by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and acetic acid Lithium-DMSO method and the like, but is not limited thereto.
[78]
[79]
The microorganisms of the present application include wild-type microorganisms or microorganisms that have been genetically modified either naturally or artificially, and microorganisms into which the thiosulfate reductase of the present application is introduced or included may be included without limitation.
[80]
The microorganism of the present application is the thiosulfate reductase of the present application; a polynucleotide encoding it; And it may include any one or more of the vector containing the polynucleotide.
[81]
[82]
The microorganism may be a microorganism that produces L-amino acids and/or derivatives thereof.
[83]
As used herein, the term “microorganism producing L-amino acid and/or derivatives thereof” refers to a microorganism having a natural ability to produce L-amino acids/derivatives thereof or L-amino acids/derivatives thereof to a parent strain that does not have the ability to produce L-amino acids/derivatives thereof. Includes all microorganisms endowed with the ability to produce Specifically, as a microorganism in which a specific mechanism is weakened or enhanced due to causes such as insertion of an external gene or intensification or inactivation of the activity of an intrinsic gene, genetic modification is performed to produce the desired L-amino acid or derivative thereof. It may be a microorganism comprising
[84]
For example, the microorganism may be a microorganism in which the L-amino acid biosynthesis pathway is enhanced or the degradation pathway is weakened. For example, the microorganism may be a microorganism in which the L-methionine biosynthesis pathway is enhanced.
[85]
For example, in the microorganism, the activity of methionine and cysteine ​​biosynthesis repressor protein (McbR) or MetJ protein is reduced or inactivated, or the activity of methionine synthetase (MetH) or sulfite reductase (CysI) is enhanced to increase methionine production ability. may be enriched and/or added microorganisms. Alternatively, it may be a microorganism that enhances the expression of genes encoding enzymes of other L-amino acid biosynthetic pathways or weakens/inactivates enzymes of degradation pathways.
[86]
Specifically, examples of proteins or genes capable of regulating expression to enhance L-amino acid biosynthetic pathway or to weaken/inactivate degradation pathways are as follows. Proteins, representative genes encoding proteins, and representative EC numbers were described in order. Proteins were written in capital letters, and genes were written in italics. For example, Rdl2p, GlpE, PspE, YgaP, ThiI, YbbB, SseA, YnjE, YceA, YibN, NCgl0671, NCgl1369, NCgl2616, NCgl0053, NCgl0054, NCGl2678, NCgl2890 thiosulphate sulfur transferase; sulfite reductase, cysI ; thiosulphate/sulphate transport system, cysPUWA (EC 3.6.3.25); 3'-phosphoadenosine 5'-phosphosulphate reductase (3'-phosphoadenosine 5''-phosphosulphate reductase), cysH (EC 1.8.4.8); sulphite 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 O-acetyltransferase, metX (EC 2.3.1.31); homoserine O-succinyltransferase, metA (EC 2.3.1.46); cystathionine gamma-synthase, metB (EC 2.5.1.48); β-CS-lyase (β-CS-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 extransporter BrnFE; valine exotransporters YgaZH (B2682, B2683), ygaZH (b2682. b2683 ); exotransporter YjeH , b4141 ; pyridine nucleotide transhydrogenase PntAB, pntAB (EC 1.6.1.2); O-succinylhomoserine sulfhydrylase, MetZ (EC 2.5.1.48); and phosphoenolpyruvate carboxylase, Pyc(EC 4.1.1.31) may enhance the activity of one or more proteins or some proteins constituting the system or a polynucleotide encoding the same may be overexpressed to enhance the L-amino acid biosynthesis pathway or weaken the degradation pathway. or, glucose 6-phosphate isomerase, pgi (EC 5.3.1.9); homoserine kinase, thrB (EC 2.7.1.39); S-adenosylmethionine synthetase, metK (EC 2.5.1.6); dihydrodipicolinate synthetase, dapA (EC 4.2.1.52); phosphoenolpyruvate carboxykinase, pck (EC 4.1.1.49);, formyltetrahydrofolate hydrolase, purU (EC 3.5.1.10); pyruvate kinase I, pykF (EC 2.7.1.40); pyruvate kinase II, pykA (EC 2.7.1.40); cystathionine γ-lyase, cg3086 (EC 4.4.1.1); Cystathionine β-synthetase, cg2344(EC 4.2.1.22); Regulatory proteins Cg3031, cg3031 ; methionine-cysteine ​​biosynthesis repressor protein McbR, mcbR ; L-methionine synthesis transcriptional regulator (Met transcriptional repressor protein), metJ ; L-methionine transporters MetQNI, metQ, metN, metI ; N-acyltransferase, yncA ; sRNA fnrS ; And L-methionine transporter, the activity of one or more proteins selected from the group consisting of metP may be inactivated or attenuated, or the expression of a gene encoding the protein is suppressed or removed.
[87]
However, this is only one example, and may be a microorganism that enhances the expression of genes encoding enzymes of various known L-amino acid biosynthetic pathways or weakens/inactivates enzymes of degradation pathways, but is not limited thereto. The microorganism producing the above L- amino acid can be prepared by applying a variety of known methods. Enhancement of protein activity and increase in gene expression are the same as described above.
[88]
[89]
As used herein, the term "inactivation" or "attenuation" of a polypeptide or protein is a concept that includes both reduced or no activity compared to intrinsic activity. The inactivation or attenuation may be used interchangeably with terms such as down-regulation, decrease, and reduce. . The inactivation or weakening is when the activity of the protein itself is reduced or eliminated compared to the activity of the protein of the original microorganism due to mutation of the gene encoding the protein, modification of the expression control sequence, deletion of part or all of the gene, etc. , when the overall protein activity level in the cell is lower than that of the native strain or strain before transformation due to inhibition of expression or translation inhibition of the gene encoding it, when expression of the gene is not made at all, and expression Even if this occurs, it may also include a case where there is no activity.
[90]
In the present application, inactivation / attenuation of these proteins, but is not limited thereto, can be achieved by application of various methods well known in the art (Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing) Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012 et al.).
[91]
As an example of the method, 1) a method of deleting all or part of the gene encoding the protein,
[92]
2) modification of the expression control region (or expression control sequence) to reduce the expression of the gene encoding the protein;
[93]
3) modification of the gene sequence encoding the protein so that the activity of the protein is eliminated or weakened;
[94]
4) introduction of an antisense oligonucleotide (eg, antisense RNA) that complementarily binds to the transcript of the gene encoding the protein;
[95]
5) By adding a sequence complementary to the Shine-Dalgarno sequence to the front end of the Shine-Dalgarno sequence of the gene encoding the protein, a secondary structure is formed to make attachment of the ribosome impossible method,
[96]
6) There is a method of adding a promoter transcribed in the opposite direction to the 3' end of the open reading frame (ORF) of the polynucleotide sequence of the gene encoding the protein (Reverse transcription engineering, RTE), etc., and a combination thereof can also be achieved, but is not particularly limited thereto.
[97]
Specifically, in the method of deleting part or all of the gene encoding the protein, a polynucleotide encoding an endogenous target protein in a chromosome through a vector for chromosomal insertion in a microorganism is partially deleted from a polynucleotide or a marker gene. This can be done by replacing As an example of a method of deleting part or all of the polynucleotide, a method of deleting a polynucleotide by homologous recombination may be used, but the present invention is not limited thereto.
[98]
In addition, the method of deleting part or all of the gene may be performed by induced mutation using light or chemicals such as ultraviolet light, and selecting a strain in which the target gene is deleted from the obtained mutant. The gene deletion method includes a method by DNA recombination technology. The DNA recombination technique may be accomplished by, for example, injecting a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene into the microorganism to cause homologous recombination. In addition, the injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.
[99]
In addition, the method of modifying the expression control sequence can be achieved by applying various methods well known in the art. As an example of the method, deletion, insertion, non-conservative or conservative substitution of the polynucleotide sequence to further weaken the activity of the expression control region (or expression control sequence), or a combination thereof, the expression control region (or expression control sequence) It can be carried out by inducing a mutation in the phase, or by replacing it with a polynucleotide sequence having a weaker activity. The expression control region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.
[100]
In addition, the method of modifying the gene sequence is performed by inducing a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution of the gene sequence, or a combination thereof to further weaken the activity of the polypeptide, or to exhibit weaker activity. It can be carried out by replacing it with a gene sequence improved to have or a gene sequence improved to have no activity, but is not limited thereto.
[101]
For example, by introducing a mutation in the gene sequence to form a stop codon, the expression of a gene can be inhibited or attenuated.
[102]
However, the above-described method is an example, and a person skilled in the art can prepare a microorganism producing L-amino acids and/or derivatives thereof using known means known in the art.
[103]
[104]
The L-amino acid and/or its derivative may be a sulfur-containing amino acid and/or a derivative of a sulfur-containing amino acid.
[105]
As used herein, the term “sulphur-containing amino acid” or “sulphur-containing amino acid derivative” refers to an amino acid or a derivative thereof containing elemental sulfur, specifically methionine, cysteine, cystine, and lanthionine. (lanthionine), homocysteine ​​(homocysteine), homocystine (homocystine), homolanthionine (homolanthionine), and may be any one selected from taurine (taurine), if the amino acid containing sulfur and its derivatives, the scope of the present application included without limitation.
[106]
[107]
The microorganism of the present application may be a microorganism of Corynebacterium sp., Escherichia sp., or Lactobacillus sp., but is not limited thereto. The microorganism may be included without limitation as long as the intrinsic activity of the SsuD protein is enhanced or the production ability of L-amino acids and/or derivatives thereof is increased by the introduction of the exogenous SsuD protein.
[108]
The "microorganism of the genus Corynebacterium" of the present application may include all microorganisms of the genus Corynebacterium. Specifically, Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium crudilactis ), Corynebacterium crenatum (Corynebacterium crenatum), Corynebacterium deserti ( Corynebacterium deserti ), Corynebacterium efficiens ( Corynebacterium efficiens ), Corynebacterium callunae ( Corynebacterium callunae ), Corynebacterium station seonis ( Corynebacterium stationis ), Corynebacterium singulare ( Corynebacterium singulare ), Corynebacterium Halo Tolerans (Corynebacterium halotolerans ), Corynebacterium striatum ( Corynebacterium striatum ), Corynebacterium ammoniagenes ( Corynebacterium ammoniagenes )), Corynebacterium pollutisoli ( Corynebacterium pollutisoli ), Corynebacterium imitans ( Corynebacterium imitans ), Corynebacterium testudinoris ( Corynebacterium testudinoris ) or Corynebacterium flavescens yl Can, more specifically, Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium callunae ( Corynebacterium callunae ), Corynebacterium crenatum ( Corynebacterium crenatum ), Corynebacterium stasis ( Corynebacterium ) stationis ), Corynebacterium ammoniagenes , or Corynebacterium deserti , and more specifically, Corynebacterium glutamicum, but is not limited thereto.
[109]
"Microorganisms of the genus Escherichia" of the present application may include all microorganisms of the genus Escherichia. Specifically , it may be Escherichia coli , but is not limited thereto.
[110]
The microorganism of the present application may be one using thiosulfate as a sulfur source, including the thiosulfate reductase of the present application.
[111]
The manufacturing method of the present application may include culturing the microorganism of the present application in a medium containing thiosulfate.
[112]
As used herein, the term “cultivation” means growing the microorganism in an appropriately controlled environmental condition. The culturing process of the present application may be made according to a suitable medium and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art according to the selected strain. The step of culturing the microorganism is not particularly limited thereto, but may be performed by a known batch culture method, a continuous culture method, a fed-batch culture method, and the like.
[113]
As used herein, the term “medium” refers to a material in which nutrients required for culturing the microorganism are mixed as a main component, and supplies nutrients and growth factors, including water, which are essential for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganism of the present application may be used without any particular limitation as long as it is a medium used for culturing conventional microorganisms. It can be cultured while controlling temperature, pH, etc. under aerobic conditions in a conventional medium containing compounds, amino acids and/or vitamins.
[114]
As the carbon source in the present application, carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid and the like; Amino acids such as glutamic acid, methionine, lysine, and the like may be included. In addition, natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice winter, cassava, sugar cane offal and corn steep liquor can be used, specifically glucose and sterilized pre-treated molasses (i.e., converted to reducing sugar). molasses) may be used, and other suitable carbon sources may be variously used without limitation. These carbon sources may be used alone or in combination of two or more, but is not limited thereto.
[115]
Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, defatted soybean cake or its degradation products can be used These nitrogen sources may be used alone or in combination of two or more, but is not limited thereto.
[116]
The phosphorus may include potassium first potassium phosphate, second potassium phosphate, or a sodium-containing salt corresponding thereto. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used, and in addition, amino acids, vitamins and/or suitable precursors may be included. These components or precursors may be added to the medium either batchwise or continuously. However, the present invention is not limited thereto.
[117]
In addition, during the culture of the microorganism, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during culturing, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation. In addition, in order to maintain the aerobic state of the medium, oxygen or oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen or carbon dioxide gas may be injected without or without gas to maintain anaerobic and microaerobic conditions, and the present invention is limited thereto. it is not
[118]
The temperature of the medium may be 25°C to 40°C, specifically 30°C to 37°C, but is not limited thereto. The incubation period may be continued until a desired production amount of the useful substance is obtained, for example, 0.5 hours to 60 hours, but is not limited thereto.
[119]
[120]
As used herein, the term “suphur source” is used interchangeably with “sulphur source” and refers to a material containing elemental sulfur that can be used for the production of sulfur-containing amino acids.
[121]
In culturing microorganisms, the sulfur source may be an important factor determining the metabolic pathway in the microorganism. However, with respect to various sulfur sources, it is not clearly known which factors carry them and which factors decompose them. For example, it is known that wild-type Corynebacterium glutamicum can use various sulfur sources, but among them, SsuABC protein is not involved in sulfate or sulfite transport, but is known to be involved in aliphatic sulfonate transport. there is only (DJ Koch, C. Ruckert, DA Rey, A. Mix, A. Puhler, J. Kalinowski. 2005. Role of the ssu and seu 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 the sulfur source into the cell has substrate specificity. In addition, even after the sulfur source is transported into the cell, the enzymes that decompose it are also different depending on the structure and functional group of the sulfur source, and the metabolic pathway using the sulfur source may also vary. For example, when sulfate is used as a sulfur source, CysZ transports it, and it is known that CysDN, CysH, and CysI are involved until sulfide formation. (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, in the production of sulfur-containing amino acids, when thiosulfate is used as a sulfur source, it has not been clearly clarified as to which factors transport and degrade it.
[122]
The sulfur source may mean thiosulfate. In the present application, the sulfur source may include, specifically, thiosulfate such as ammonium thiosulfate or sodium thiosulfate, or sulfite, a reduced raw material such as H 2 S, sulfide, sulfide derivative, methylmercaptan, thio It may be in the form of a mixture of thiosulfate with organic and inorganic sulfur-containing compounds such as glycolite, thiocyanate and thiourea. Alternatively, a material other than thiosulfate may not be included as a sulfur source. However, it is not limited thereto.
[123]
[124]
The method for producing the sulfur-containing amino acid or the sulfur-containing amino acid derivative may include recovering the sulfur-containing amino acid or the sulfur-containing amino acid derivative from the microorganism or culture medium.
[125]
[126]
The recovering step is a method for culturing the microorganism of the present application, for example, a desired sulfur-containing amino acid or a sulfur-containing amino acid derivative from the medium using a suitable method known in the art according to a batch, continuous or fed-batch culture method. can be recovered For example, centrifugation, filtration, treatment with a crystallized protein precipitating agent (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography It may be used in combination of various chromatography, HPLC, and methods thereof, such as, but is not limited to these examples.
[127]
The recovery step may include an additional purification process. The purification process may use a suitable method known in the art.
[128]
[129]
Another aspect of the present application is a microorganism in which the protein activity encoded by the ssuD gene is enhanced compared to the intrinsic activity, or a culture thereof; And it provides a composition for preparing a sulfur-containing amino acid or a sulfur-containing amino acid derivative, comprising thiosulfate.
[130]
Proteins, microorganisms, thiosulfate and sulfur-containing amino acids encoded by the ssuD gene are as described above.
[131]
The culture may be prepared by culturing the microorganism of the present application in a medium.
[132]
The composition for preparing a sulfur-containing amino acid or a sulfur-containing amino acid derivative of the present application may further include any component capable of assisting in the production of a sulfur-containing amino acid or a sulfur-containing amino acid derivative, and these components are from those known in the art. can be appropriately selected.
[133]
[134]
Another aspect of the present application provides the use of a protein encoded by the ssuD gene as a thiosulfate reductase.
[135]
Another aspect of the present application is a microorganism having a genetic modification in which the protein activity encoded by the ssuD gene is increased compared to the unmodified microorganism. Provided is a use for preparing a sulfur-containing amino acid or a derivative of a sulfur-containing amino acid.
[136]
Proteins, microorganisms, cultures, thiosulfate and sulfur-containing amino acids encoded by the ssuD gene are as described above.
[137]
Modes for carrying out the invention
[138]
Hereinafter, the present application will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes of the present application, and the scope of the present application is not limited to these Examples and Experimental Examples.
[139]
[140]
Example 1: Construction of recombinant vector for mcbR gene deletion
[141]
First, in order to prepare a methionine-producing strain, which is a representative sulfur-containing amino acid, with a Corynebacterium glutamicum ATCC13032 strain, mcbR (J. Biotechnol. 103:51-65 encoding a known methionine cysteine ​​transcriptional regulator protein) , 2003) was prepared for the inactivation of the vector.
[142]
Specifically, in order to delete the mcbR gene on the Corynebacterium ATCC13032 chromosome, a recombinant plasmid vector was prepared by the following method.
[143]
Based on the nucleotide sequence reported to the National Institutes of Health's gene bank (NIH Genbank), the mcbR gene and peripheral sequence (SEQ ID NO: 1) of Corynebacterium glutamicum were obtained.
[144]
PCR was performed using the primers of SEQ ID NO: 2 and SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template. PCR conditions were repeated 30 times of denaturation at 95 ° C. for 5 minutes, then denaturation at 95 ° C. for 30 seconds, annealing at 53 ° C. for 30 seconds, and polymerization at 72 ° C. for 30 seconds, followed by polymerization at 72 ° C. for 7 minutes. As a result, each 700 bp DNA fragment was obtained.
[145]
After treating the pDZ vector (US Pat. No. 9109242 B2) that cannot be replicated in Corynebacterium glutamicum and the amplified mcbR gene fragment with a restriction enzyme SmaI for chromosome introduction, after isothermal assembly cloning reaction, E. coli DH5α Transformed and plated on LB solid medium containing kanamycin (25mg / ℓ). After selecting colonies transformed with the vector into which the defective fragments of the desired genes were inserted through PCR, a plasmid was obtained using the plasmid extraction method, and it was named pDZ-ΔmcbR.
[146]
[147]
Example 2: Construction and culture of strains in which the mcbR gene is deleted
[148]
The pDZ-ΔmcbR vector prepared in Example 1 was transformed into the ATCC13032 strain by homologous recombination on the chromosome by electroporation, respectively (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Thereafter, secondary recombination was performed in a solid medium containing sucrose. A strain lacking the mcbR gene was identified through PCR using SEQ ID NOs: 6 and 7 for the Corynebacterium glutamicum transformant on which secondary recombination was completed, and this recombinant strain was named CM02-0618 did
[149]
The CM02-0618 was deposited with the Korea Microorganism Conservation Center, a trustee institution under the Budapest Treaty as of January 4, 2019, and was given an accession number KCCM12425P.
[150]
In order to analyze the L-methionine production ability of the CM02-0618 strain prepared above, it was cultured with the parent strain, Corynebacterium glutamicum ATCC13032 strain, in the following manner.
[151]
Corynebacterium glutamicum ATCC13032 and Corynebacterium glutamicum CM02-0618 were inoculated into a 250 ml corner-baffle flask containing 25 ml of the following seed medium, and shaken at 30° C. for 20 hours at 200 rpm. cultured. Then, 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 24 ml of the production medium and cultured with shaking at 30° C. for 48 hours at 200 rpm. The composition of the species medium and the production medium is as follows, respectively. As a sulfur source in the production medium, a type of thiosulfate (NH 4 ) 2 S 2 O 3 was used.
[152]
[153]

[154]
Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH 2 PO 4 4 g, K 2 HPO 4 8 g, MgSO 4 7H 2 O 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium -Pantothenic acid 2000 ㎍, nicotinamide 2000 ㎍, (based on 1 liter of distilled water)
[155]

[156]
Glucose 50 g, (NH 4 ) 2 S 2 O 3 12 g, Yeast extract 5 g, KH 2 PO 4 1 g, MgSO 4 7H 2 O 1.2 g, biotin 100 μg, thiamine hydrochloride 1000 μg, calcium-pantothenic acid 2000 μg, nicotinamide 3000 μg, CaCO 3 30 g, cyanocobalamin (Vitamin B12) 1 μg (based on 1 liter of distilled water).
[157]
[158]
The L-methionine concentration in the culture medium was analyzed by culturing in the above culture method, and it is shown in Table 1.
[159]
[160]
[Table 1] Confirmation of L-methionine-producing ability of mcbR wild-type and the removed strain
strain L-Methionine (g/L)
Corynebacterium glutamicum ATCC 13032 (wild type) 0.00
CM02-0618 0.04
[161]
[162]
As a result, it was confirmed that the L-methionine production capacity was improved by 0.04 g/L in the mcbR alone removal strain compared to the control strain. In addition, it was confirmed that methionine was produced even when thiosulfate was used as the sole sulfur source. Through this, it was speculated that the protein involved in the use of thiosulfate would also exist in microorganisms of the genus Corynebacterium.
[163]
[164]
Example 3: Selection of genes involved in the use of thiosulfate through transcriptome analysis
[165]
There is no known protein specific for thiosulfate in Corynebacterium strains. However, as confirmed in Example 2, in the case of the CM02-0618 strain, it was confirmed that methionine was produced when thiosulfate was used as the sole sulfur source. An experiment was performed to select proteins involved in the use of thiosulfate. .
[166]
Specifically, the CM02-0618 strain prepared in Example 2 was cultured with different sulfur sources (ammonium sulfate and ammonium thiosulfate), and then Transcriptome (RNA level analysis) analysis was performed. The culture method is the same as in Example 2.
[167]
[168]
[Table 2] Experimental results of major gene transcripts under ammonium sulfate and ammonium thiosulfate conditions of strain CM02-0618.
AMS (signal) ATS (signal) Log2 ratio (ATS/AMS)
SsuD (Ncgl1173) 3691 55539 2.71
[169]
As a result of the experiment, it was found that the RNA level of the gene encoding SsuD (Ncgl1173), known as the conventional sulfonate monooxygenase, was significantly increased.
[170]
Through this, it was confirmed that the SsuD protein did not react to sulfate but specifically reacted to thiosulfate, and it was confirmed that the protein would be involved in using thiosulfate.
[171]
[172]
Example 4: Confirmation of the effect of the ssuD gene deletion strain
[173]
In order to confirm the inactivation effect of SsuD selected as a protein specifically responding to thiosulfate in Example 3, a vector was constructed to delete the ssuD gene.
[174]
Example 4-1: Construction of a vector for ssuD gene deletion
[175]
In order to delete the ssuD gene on the Corynebacterium ATCC13032 chromosome, a recombinant plasmid vector was prepared by the following method.
[176]
Based on the nucleotide sequence reported to the National Institutes of Health's gene bank (NIH Genbank), the ssuD gene and peripheral sequence (SEQ ID NO: 8) of Corynebacterium glutamicum were obtained.
[177]
For the purpose of deleting the ssuD gene, PCR was performed using the primers of SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template. PCR conditions were repeated 30 times of denaturation at 95 ° C. for 5 minutes, then denaturation at 95 ° C. for 30 seconds, annealing at 53 ° C. for 30 seconds, and polymerization at 72 ° C. for 30 seconds, followed by polymerization at 72 ° C. for 7 minutes. As a result, 700 bp DNA fragments were obtained, respectively.
[178]
After the pDZ vector and the amplified ssuD gene fragments, which cannot be replicated in Corynebacterium glutamicum, were treated with a restriction enzyme for chromosome introduction SmaI, after isothermal assembly cloning reaction, transformed into E. coli DH5α and kanamycin (25 mg/ℓ) ) was plated on LB solid medium containing After selecting colonies transformed with the vector into which the defective fragments of the desired genes were inserted through PCR, a plasmid was obtained using the plasmid extraction method, and it was named pDZ-ΔSsuD.
[179]
[180]
Example 4-2: ssuD gene-deficient strain production and culture
[181]
The pDZ-ΔSsuD vector constructed in Example 4-1 was transformed into 13032/ΔmcbR strains by homologous recombination on the chromosome by electroporation (van der Rest et al., Appl Microbiol Biotechnol 52:541-545; 1999). Thereafter, secondary recombination was performed in a solid medium containing sucrose. The strain in which the mcbR gene was deleted was confirmed through PCR using SEQ ID NOs: 13 and 14 for the Corynebacterium glutamicum transformant on which the secondary recombination was completed, and the recombinant strain was treated with Corynebacterium glue It was named Tamicum CM02-0618/ΔSsuD.
[182]
[183]
Example 4-3: Analysis of methionine-producing ability of strains lacking ssuD gene
[184]
In order to analyze the L-methionine production ability of the CM02-0618/ΔSsuD strain prepared above, it was cultured with the parent strain, Corynebacterium glutamicum ATCC13032 strain, in the following way.
[185]
In a 250 ml corner-baffle flask containing 25 ml of the following seed medium, Corynebacterium glutamicum ATCC13032 and Corynebacterium glutamicum CM02-0618 prepared in Example 2, prepared CM02-0618 / ΔSsuD Inoculated, incubated at 30 ℃ for 20 hours, shaking at 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 24 ml of the production medium and cultured with shaking at 30° C. for 48 hours at 200 rpm. The composition of the species medium and the production medium is as follows, respectively.
[186]
[187]

[188]
Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH 2 PO 4 4 g, K 2 HPO 4 8 g, MgSO 4 7H 2 O 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium -Pantothenic acid 2000 ㎍, nicotinamide 2000 ㎍ (based on 1 liter of distilled water)
[189]

[190]
Glucose 50 g, (NH 4 ) 2 S 2 O 3 12 g, Yeast extract 5 g, KH 2 PO 4 1 g, MgSO 4 7H 2 O 1.2 g, biotin 100 μg, thiamine hydrochloride 1000 μg, calcium-pantothenic acid 2000 μg, nicotinamide 3000 μg, CaCO 3 30 g (based on 1 liter of distilled water).
[191]
The L-methionine concentration in the culture medium was analyzed by culturing in the above culture method, and it is shown in Table 3.
[192]
[193]
[Table 3] Confirmation of L-methionine production ability of the strain from which the ssuD gene has been removed
strain L-Methionine (g/L)
CM02-0618 0.04
CM02-0618/ΔSsuD 0.02
[194]
As a result, as the ssuD gene was removed, it was confirmed that the L-methionine production ability was reduced to about 50% compared to the control strain. Through this, it was confirmed that the SsuD protein is a protein involved in the use of thiosulfate.
[195]
[196]
Example 5: Construction and culture of strains with increased ssuD gene expression
[197]
In Example 3, a vector was constructed to enhance the activity of SsuD, which was selected as a protein reacting specifically to thiosulfate.
[198]
[199]
Example 5-1: Construction of a vector for increasing ssuD gene expression
[200]
In order to further insert the ssuD gene onto the Corynebacterium ATCC13032 chromosome, a recombinant plasmid vector was prepared by the following method.
[201]
First , to insert the ssuD gene, a vector for removing Ncgl1464 (Transposase) was prepared.
[202]
Based on the nucleotide sequence reported to the National Institutes of Health's gene bank (NIH Genbank), Ncgl1464 and surrounding sequences of Corynebacterium glutamicum (SEQ ID NO: 15) were obtained. In order to delete the Ncgl1464 gene, PCR was performed using the primers of SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template. PCR conditions were repeated 30 times of denaturation at 95 ° C. for 5 minutes, then denaturation at 95 ° C. for 30 seconds, annealing at 53 ° C. for 30 seconds, and polymerization at 72 ° C. for 30 seconds, followed by polymerization at 72 ° C. for 7 minutes. As a result, each DNA fragment was obtained.
[203]
After the pDZ vector and the amplified Ncgl1464 gene fragment, which cannot be replicated in Corynebacterium glutamicum, were treated with a restriction enzyme for chromosome introduction SmaI, after isothermal assembly cloning reaction, transformed into E. coli DH5α and transformed into kanamycin (25 mg/ℓ) ) was plated on LB solid medium containing After selecting colonies transformed with the vector into which the defective fragments of the desired genes were inserted through PCR, a plasmid was obtained using the plasmid extraction method, and it was named pDZ-ΔNcgl1464.
[204]
Next, for the purpose of obtaining the ssuD gene fragment, PCR was performed using SEQ ID NO: 20 and SEQ ID NO: 21 using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template. In addition , the PgapA promoter was used to enhance the expression of the ssuD gene , and PCR was performed using SEQ ID NOs: 22 and 23 using Corynebacterium glutamicum ATCC 13032 chromosomal DNA as a template for the purpose of obtaining it. PCR conditions were repeated 30 times of denaturation at 95 ° C. for 5 minutes, then denaturation at 95 ° C. for 30 seconds, annealing at 53 ° C. for 30 seconds, and polymerization at 72 ° C. for 30 seconds, followed by polymerization at 72 ° C. for 7 minutes. As a result , the ssuD gene fragment and the gapA promoter fragment were obtained.
[205]
After treating pDZ-ΔNcgl1464 vector, which cannot be replicated in Corynebacterium glutamicum, with restriction enzyme ScaI, after IST reaction with the two amplified DNA fragments, transformed into E. coli DH5α and transformed into kanamycin (25 mg/L ) was plated on LB solid medium containing After selecting colonies transformed with the vector into which the desired gene was inserted through PCR, a plasmid was obtained using a plasmid extraction method, and it was named pDZ-ΔNcgl1464-PgapASsuD.
[206]
[207]
Example 5-2: Construction and culture of strains with enhanced ssuD gene expression
[208]
The vectors, pDZ-ΔNcgl1464 and pDZ-ΔNcgl1464-PgapASsuD, prepared in Example 5-1 were transformed into the CM02-0618 strain by homologous recombination on the chromosome by electroporation, respectively (van der Rest et al., Appl Microbiol). Biotechnol 52:541-545, 1999). Thereafter, secondary recombination was performed in a solid medium containing sucrose. For the Corynebacterium glutamicum transformant on which secondary recombination was completed, a strain in which Ncgl1464 was deleted and a strain in which Ncgl1164 was deleted and the ssuD gene was inserted through PCR using SEQ ID NOs: 24 and 25 were identified. . The strain in which Ncgl1464 was deleted was named CM02-0618/ΔNcgl1464, and the strain in which the ssuD gene was inserted while Ncgl1164 was deleted was named CM02-0736.
[209]
The above CM02-0736 was deposited with the Korea Microorganism Conservation Center, a trustee institution under the Budapest Treaty on May 2, 2019, and was given an accession number KCCM12512P.
[210]
[211]
Example 5-3: Analysis of methionine-producing ability of strains with enhanced ssuD gene expression
[212]
In order to analyze the L-methionine-producing ability of the prepared CM02-0618/ΔNcgl1464 and CM02-0736 strains, they were cultured together with the parent strain CM02-0618 strain in the following manner.
[213]
CM02-0618, CM02-0618 /ΔNcgl1464, and CM02-0736 were inoculated into a 250 ml corner-baffle flask containing 25 ml of the following seed medium, respectively, and cultured with shaking at 30° C. for 20 hours at 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 24 ml of the production medium and cultured with shaking at 30° C. for 48 hours at 200 rpm. The composition of the species medium and the production medium is as follows, respectively.
[214]
[215]

[216]
Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH 2 PO 4 4 g, K 2 HPO 4 8 g, MgSO 4 7H 2 O 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium -Pantothenic acid 2000 ㎍, nicotinamide 2000 ㎍ (based on 1 liter of distilled water)
[217]

[218]
Glucose 50 g, (NH 4 ) 2 S 2 O 3 12 g, Yeast extract 5 g, KH 2 PO 4 1 g, MgSO 4 7H 2 O 1.2 g, biotin 100 μg, thiamine hydrochloride 1000 μg, calcium-pantothenic acid 2000 μg, nicotinamide 3000 μg, CaCO 3 30 g, cobalamin (Vitamin B12) 1 μg (based on 1 liter of distilled water).
[219]
[220]
The L-methionine concentration in the culture medium was analyzed by culturing in the above culture method, and it is shown in Table 4.
[221]
[222]
[Table 4] Confirmation of L-methionine-producing ability of strains with increased ssuD expression
strain L-Methionine (g/L)
CM02-0618 0.04
CM02-0618/ΔNcgl1464 0.04
CM02-0736 0.06
[223]
As a result, it was confirmed that the L-methionine production capacity increased by about 50% compared to the control strain as the ssuD gene expression was enhanced. As confirmed in Example 4, it was confirmed that the SsuD protein is a protein involved in the use of thiosulfate.
[224]
[225]
Example 6: Comparative culture of thiosulfate with other sulfonates.
[226]
The SsuD protein is originally known as a protein involved in the desulfonation of sulfonates. Sulfonate is composed of R-SO3 and R is an organic group. Thiosulfate is different from sulfonate because it consists of S-SO3.
[227]
Therefore, it was attempted to determine how effective the case of using thiosulfate as a sulfur source for methionine production through a comparative experiment with sulfonate.
[228]
Corynebacterium glutamicum CM02-0618 and CM02-0736 were inoculated into a 250 ml corner-baffle flask containing 25 ml of the following seed medium, and cultured with shaking at 30° C. for 20 hours at 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 24 ml of the production medium and cultured with shaking at 30° C. for 48 hours at 200 rpm. The composition of the species medium and the production medium is as follows, respectively.
[229]
[230]

Claims
[Claim 1]
A method for producing a sulfur-containing amino acid or a sulfur-containing amino acid derivative comprising culturing a genetically modified microorganism in a medium containing thiosulfate, wherein the microorganism has an increased activity of the protein encoded by the ssuD gene compared to the unmodified microorganism. Having a genetic modification, the manufacturing method.
[Claim 2]
The method of claim 1, wherein the protein encoded by the ssuD gene has thiosulfate reductase activity.
[Claim 3]
The method according to claim 1, wherein the protein encoded by the ssuD gene comprises an amino acid sequence having at least 80% homology with SEQ ID NO: 43.
[Claim 4]
The method of claim 1, wherein the microorganism is a Corynebacterium sp. or Escherichia sp. microorganism.
[Claim 5]
According to claim 4, wherein the microorganism is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium callunae ( Corynebacterium callunae ), Corynebacterium deserti ( Corynebacterium deserti ), Corynebacterium crenatum ( Corynebacterium crenatum ) Or Escherichia coli ( Escherichia coli ) Will, the manufacturing method.
[Claim 6]
The method according to claim 1, wherein the method comprises recovering a sulfur-containing amino acid or a sulfur-containing amino acid derivative from the microorganism or culture medium.
[Claim 7]
According to claim 1, wherein the genetic modification to increase the protein activity is i) an increase in the intracellular copy number of the polynucleotide encoding the protein, ii) a sequence with a strong activity in the expression control region of the polynucleotide encoding the protein replaced with, iii) a start codon or 5'-UTR modification of the polynucleotide encoding the protein, iv) a polynucleotide sequence on the chromosome is modified to enhance the activity of the protein, v) a foreign polynucleotide exhibiting the activity of the protein Or introduction of a codon-optimized mutant polynucleotide of the polynucleotide encoding the protein, or vi) a combination thereof.
[Claim 8]
The method of claim 1, wherein the sulfur-containing amino acid or sulfur-containing amino acid derivative is methionine, cysteine, cystine, lanthionine, homocysteine, homocysteine, homocystine, homocysteine. Lanthionine (homolanthionine), taurine (taurine) will any one or more selected from, the manufacturing method.
[Claim 9]
A microorganism producing a sulfur-containing amino acid or a derivative of a sulfur-containing amino acid having a genetic modification that increases the protein activity encoded by the ssuD gene compared to an unmodified microorganism.
[Claim 10]
The microorganism according to claim 9, wherein the microorganism uses thiosulfate as a sulfur source to produce a sulfur-containing amino acid or a sulfur-containing amino acid derivative.
[Claim 11]
10. The method of claim 9, wherein the genetic modification to increase the protein activity is i) an increase in the number of intracellular copies of the polynucleotide encoding the protein, ii) a sequence with strong activity in the expression control region of the polynucleotide encoding the protein replacement with, iii) a start codon or 5'-UTR modification of the polynucleotide encoding the protein, iv) modifying the polynucleotide sequence on the chromosome to enhance the activity of the protein, v) a foreign polynucleotide exhibiting the activity of the protein or introduction of a codon-optimized variant polynucleotide of the polynucleotide encoding the protein, or vi) a combination thereof.
[Claim 12]
a microorganism having a genetic modification in which the protein activity encoded by the ssuD gene is intrinsically increased compared to an unmodified microorganism, or a culture thereof; and thiosulfate, a composition for preparing a sulfur-containing amino acid or a sulfur-containing amino acid derivative.
[Claim 13]
Use of a protein encoded by the ssuD gene as a thiosulfate reductase.
[Claim 14]
Use of a sulfur-containing amino acid or a sulfur-containing amino acid derivative of a microorganism having a genetic modification that increases the protein activity encoded by the ssuD gene compared to an unmodified microorganism.