Title of the invention: microorganism producing mycosporine-like amino acid and method for producing mycosporine-like amino acid
Technical field
[One]
The present application relates to a microorganism producing a mycosporine-like amino acid and a method for producing a mycosporine-like amino acid using the microorganism.
[2]
Background
[3]
Ultraviolet rays emitted from the sun consist of UV A (ultraviolet ray A, in the region of about 320-400 nm), UV B (ultraviolet ray B, in the region of about 290-320 nm) and UV C (ultraviolet ray C, in the region of about 100-280 nm) Has been. Ultraviolet A penetrates into the dermal layer and mainly causes pigmentation and skin aging, and is involved in the occurrence of photosensitive skin diseases, and ultraviolet B penetrates the epidermis and the upper dermis with high energy rays and is said to be involved in sunburn, pigmentation and skin cancer Is known.
[4]
There have been attempts to block UV rays to prevent such side effects. Types of sunscreens include chemical sunscreen agents and physical sunscreen agents. Chemical sunscreen agents mainly absorb ultraviolet rays, and physical sunscreen agents prevent the penetration of ultraviolet rays through reflection and scattering. Block.
[5]
[6]
Ingredients included in chemical sunscreens include PABA, which mainly absorbs ultraviolet B, PABA esters (Amyl dimethyl PABA, octyl dimethyl PABA), cinnamates (Cinoxate), salicylate (homomenthyl salicylate), camphor (Camphor) and the like, benzophenone (Oxybenzone, Dioxybenzone, Suliso benzene), dibenzoyl methane (Dibenzoyl methane), and anthranilate (Anthranilate), which mainly absorb ultraviolet A, are known. These chemical sunscreens can absorb and block UV rays, but some of them can irritate the skin or eyes, and in particular, PABA, PABA ester, benzophenone, cinnamate, etc., can lead to contact dermatitis. It is known that it can. In addition, some other problems have been reported, such as causing a photosensitive reaction of the skin. In some countries, the use of chemical sunscreens or the use thereof is restricted.
[7]
[8]
As ingredients included in the physical sunscreen, titanium dioxide, talc (magnesium silicate), magnesium oxide, zinc oxide, kaolin, and the like are known. These have no side effects such as contact dermatitis and have the advantage of not being easily removed by water, but they are difficult to maintain an effective content while realizing a desired formulation, and have disadvantages such as clouding when applied to the skin.
[9]
[10]
Mycosporine-like amino acids (MAAs) are substances that exist in natural organisms and are known to absorb UVA and UVB effectively. MAAs are known to exist in more than 35 species in nature (Mar. Biol., 1991, 108: 157-166; Planta Med., 2015, 81: 813-820). Recently, it has been reported that various sugar-attached forms of MAAs exist in microalgae and have excellent antioxidant properties (Journal of Photochemistry and Photobiology, 2015, 142: 154-168). In addition, MAAs are known to impart resistance to oxidation, osmosis and heat stress, as well as UV blocking ability (Comp. Biochem. Physiol.C Toxicol. Pharmacol., 2007, 146: 60-78; J. Photochem. Photobiol.B., 2007, 89:29-35).
[11]
However, the amount of MAAs produced in microalgae is very low, at the level of several μg, and conditions for separating, extracting, and purifying MAAs by culturing microalgae are complicated, making it difficult to mass-produce MAAs materials.
[12]
[13]
[Prior technical literature]
[14]
[Non-patent literature]
[15]
(Non-Patent Document 1) Comp. Biochem. Physiol. B 1995, 112: 105-114.
[16]
(Non-Patent Document 2) FEMS Microbiol Lett. 2007, 269: 1-10.
[17]
(Non-Patent Document 3) Ann. Rev. Physiol. 2002, 64: 223-262.
[18]
(Non-Patent Document 4) Mar. Biol. 1991, 108: 157-166.
[19]
(Non-Patent Document 5) Journal of Photochemistry and Photobiology B: Biology. 2015, 142: 154-168
[20]
(Non-Patent Document 6) Biol. Rev. 1999, 74: 311-345.
[21]
(Non-Patent Document 7) Mol. Biol. Evol. 2006, 23: 1437-1443.
[22]
(Non-Patent Document 8) Science, 2010, 329: 1653-1656.
[23]
(Non-Patent Document 9) Genomics 2010, 95: 120-128.
[24]
(Non-Patent Document 10) Geomicrobiol. J. 1997. 14: 231-241.
[25]
(Non-Patent Document 11) Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2007. 146: 60-78.
[26]
(Non-Patent Document 12) Can. J. Bot. 2003. 81: 131-138.
[27]
(Non-Patent Document 13) J. Photochem. Photobiol. B. 2007, 89: 29-35.
[28]
(Non-Patent Document 14) J. Bacteriol. 2011. 193(21): 5923-5928.
[29]
(Non-Patent Document 15) Planta Med. 2015. 81: 813-820
[30]
(Non-Patent Document 16) ACS Appl. Mater. Interfaces. 2015. 7: 16558-16564
[31]
(Non-Patent Document 17) Appl Environ Microbiol. 2016, 82(20): 6167-6173
[32]
(Non-Patent Document 18) ChemBioChem. 2015, 16: 320-327
[33]
(Non-Patent Document 19) Methods Mol Biol. 2013, 1073: 43-7
[34]
(Non-Patent Document 20) Nature Review, 2011, 9: 791-802
[35]
Detailed description of the invention
Technical challenge
[36]
As a result of the present inventors' diligent efforts to increase the production of MAAs in microorganisms, the production of MAAs has been improved through various studies that enhance the activity of 2-dehydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase and transketolase proteins in the microorganisms producing MAAs By confirming the increase, the present invention was completed.
[37]
Means of solving the task
[38]
One object of the present application is 2-dihydro-3-deoxyphosphoheptonate aldolase (2-dehydro-3-deoxyphosphoheptonate aldolase), phosphoenolpyruvate synthetase (phosphoenolpyruvate synthetase) and transketolase ( transketolase) to provide a microorganism that produces a mycosporine-like amino acid whose activity is enhanced at least one protein selected from the group consisting of.
[39]
Another object of the present application is culturing the microorganism in a medium; And recovering mycosporine-like amino acids from the cultured microorganism or medium.
[40]
Another object of the present application is to provide the use of the microorganism for producing mycosporine-like amino acids.
[41]
Effects of the Invention
[42]
The microorganisms of the present application have improved ability to produce mycosporine-like amino acids, and thus can be effectively used to produce mycosporine-like amino acids.
[43]
Best mode for carrying out the invention
[44]
Hereinafter, the present application will be described in more detail.
[45]
Meanwhile, each description and embodiment disclosed herein may be applied to each other description and embodiment. That is, all combinations of various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be said that the scope of the present invention is limited by the specific description described below. In addition, a person of ordinary skill in the art may recognize or ascertain using only routine experimentation a number of equivalents to the specific aspects of the invention described in this application. Also, such equivalents are intended to be included in the present invention.
[46]
[47]
One aspect of the present application for achieving the above object is 2-dihydro-3-deoxyphosphoheptonate aldolase (2-dehydro-3-deoxyphosphoheptonate aldolase), phosphoenolpyruvate synthetase ( Phosphoenolpyruvate synthetase) and transketolase (transketolase).
[48]
As used herein, the term “2-dehydro-3-deoxyphosphoheptonate aldolase” refers to an enzyme that catalyzes the reversible reaction of the following reaction formula, specifically, 3 -Deoxy-arabino-heptulosonate-7-phosphate (3-deoxy-D-arabino-heptulosonate-7-phosphate, DAHP) may mean an enzyme that synthesizes, but is not limited thereto.
[49]
[50]
[Reaction Scheme]
[51]

[52]
[53]
In this application, 2-dihydro-3-deoxyphosphoheptonate aldolase is used interchangeably with 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase.
[54]
As used herein, the term "phosphoenolpyruvate synthetase" refers to an enzyme that catalyzes the reversible reaction of the following reaction formula, and specifically, may mean an enzyme that synthesizes phosphoenolpyruvate. , Is not limited thereto.
[55]
[56]
[Reaction Scheme]
[57]

[58]
[59]
As used herein, the term "transketolase" refers to an enzyme that catalyzes the reversible reaction of the following scheme.
[60]
[61]
[Reaction Scheme]
[62]

[63]
or
[64]

[65]
[66]
Genetic information of the 2-dihydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase, and transketolase can be obtained from a known database, for example, National Biological Information of the United States. Center (National Center for Biotechnology Information; NCBI), such as GenBank, but is not limited thereto.
[67]
The 2-dihydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase, and transketolase have differences in the amino acid sequence of a protein exhibiting activity depending on the species or microorganism of the microorganism. In some cases, it is not limited to its origin or sequence.
[68]
Specifically, the 2-dihydro-3-deoxyphosphoheptonate aldolase may be a protein comprising the amino acid sequence of SEQ ID NO: 2, 37 or 124, and the phosphoenolpyruvate synthetase has a sequence It may be a protein including the amino acid sequence of SEQ ID NO: 19 or 98, and the transketolase may be a protein including the amino acid sequence of SEQ ID NO: 24, 96, or 123, but is not limited thereto. As used herein, "protein comprising an amino acid sequence" may be used interchangeably with the expression "protein having an amino acid sequence" or "a protein consisting of an amino acid sequence".
[69]
In addition, in the present application, the enzymes are 80% or more, 85% or more, 90% or more, 91% or more, 92% or more with the amino acid sequence as long as they have the same or corresponding biological activity as each enzyme. , 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more may include proteins that exhibit homology or identity.
[70]
In addition, if an amino acid sequence having a biological activity substantially identical to or corresponding to the enzyme protein of the sequence number described as a sequence having homology or identity to the above sequence, some sequences may have a deleted, modified, substituted or added amino acid sequence. It is obvious that it is also included in the scope of the present application.
[71]
The term “homology or identity” as used herein refers to the degree to which a given amino acid sequence or nucleotide sequence is matched and may be expressed as a percentage. In the present specification, a homologous sequence thereof having the same or similar activity as a given amino acid sequence or nucleotide sequence is expressed as "% homology" or "% identity". For example, standard software that calculates parameters such as score, identity, and similarity, specifically BLAST 2.0, or hybridization used under defined stringent conditions It can be confirmed by comparing sequences by experimentation, and the appropriate hybridization conditions defined are within the scope of the technology, and methods well known to those skilled in the art (eg, 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). In the above, the term "stringent conditions" means conditions that allow specific hybridization between polynucleotides. For example, these conditions are specifically described in the literature (eg, J. Sambrook et al., homolog).
[72]
As long as the 2-dihydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase, and transketolase of the present application have the same or corresponding biological activity as each of the above enzymes, the described sequence numbers Amino acid sequence of or above 80%, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more , It may comprise a polynucleotide encoding a protein exhibiting 99% or more homology or identity.
[73]
In addition, the polynucleotide encoding the enzymes is within a range that does not change the amino acid sequence of the protein expressed from the coding region in consideration of the preferred codon in the organism to express the protein due to the codon degeneracy. Various modifications can be made to the coding region. Accordingly, the polynucleotide may be included without limitation as long as it is a polynucleotide sequence encoding each enzyme protein.
[74]
In addition, a probe that can be prepared from a known gene sequence, for example, is hybridized with a complementary sequence for all or part of the polynucleotide sequence under stringent conditions, and the 2-dihydro-3-deoxyphosphohepto Nate aldolase, phosphoenolpyruvate synthetase, and transketolase enzymes may be included without limitation as long as they are sequences encoding proteins having activity.
[75]
The “strict conditions” refer to conditions that enable specific hybridization between polynucleotides. These conditions are specifically described in the literature (eg, J. Sambrook et al., homolog). For example, between genes with high homology or identity, 40% or more, specifically 90% or more, more specifically 95% or more, more specifically 97% or more, particularly specifically 99% or more homology or Under conditions that hybridize genes with identity and do not hybridize with genes with lower homology or identity, or wash conditions for general Southern hybridization at 60°C, 1XSSC, 0.1% SDS, specifically 60°C, 0.1 At salt concentration and temperature corresponding to XSSC, 0.1% SDS, more specifically 68° C., 0.1XSSC, and 0.1% SDS, the conditions for washing once, specifically two to three times, can be enumerated.
[76]
Hybridization requires that two polynucleotides have a complementary sequence, although a mismatch between bases is possible depending on the stringency of the 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. Thus, the present application may also include substantially similar polynucleotide sequences as well as isolated polynucleotide fragments that are complementary to the entire sequence.
[77]
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. Further, the Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto and may be appropriately adjusted by a person skilled in the art according to the purpose.
[78]
The appropriate stringency to hybridize a polynucleotide depends on the length and degree of complementarity of the polynucleotide and the parameters are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8). "Enhancement of activity" means that the activity of an enzyme protein is introduced, or that the activity is improved compared to the intrinsic activity of the microorganism or the activity before modification. The "introduction" of the above activity means that the activity of a specific protein that the microorganism does not have naturally or artificially appears. Specifically, a microorganism having enhanced activity of an enzyme protein refers to a microorganism having improved activity of an enzyme protein compared to a natural wild-type microorganism or an unmodified microorganism. The enhancement of the activity is, for example, by introducing foreign 2-dihydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase, and/or transketolase; Or enhancing the activity of intrinsic 2-dihydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase and/or transketolase. Specifically, as a method of enhancing activity herein,
[79]
1) increase the copy number of the polynucleotide encoding the enzymes,
[80]
2) modification of the expression control sequence to increase the expression of the polynucleotide,
[81]
3) the type of polynucleotide sequence on the chromosome to enhance the activity of the enzymes, or
[82]
4) It may be performed by a method of transforming to be strengthened by a combination thereof, but is not limited thereto.
[83]
The 1) increase in the copy number of the polynucleotide may be performed in a form operably linked to a vector, but is not particularly limited thereto, or may be performed by being inserted into a chromosome in a host cell. In addition, as an aspect of increasing the copy number, it may be performed by introducing a foreign polynucleotide exhibiting the activity of an enzyme or a codon-optimized variant polynucleotide of the polynucleotide into a host cell. The foreign polynucleotide may be used without limitation in its origin or sequence as long as it exhibits the same/similar activity as the enzyme. The introduction may be performed by appropriately selecting a known transformation method by a person skilled in the art, and the introduced polynucleotide is expressed in a host cell, thereby generating an enzyme and increasing its activity.
[84]
Next, 2) modification of the expression control sequence to increase expression of the polynucleotide is not particularly limited thereto, but deletion, insertion, non-conservative or conservative substitution of the nucleic acid sequence to further enhance the activity of the expression control sequence, or It may be performed by inducing a mutation in the sequence by a combination of, or by replacing with a nucleic acid sequence having a stronger activity. The expression control sequence, although not particularly limited thereto, may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence controlling termination of transcription and translation, and the like.
[85]
Specifically, a strong heterologous promoter may be linked to the upper part of the polynucleotide expression unit instead of the original promoter. Examples of the strong promoter include CJ7 promoter, lysCP1 promoter, EF-Tu promoter, groEL promoter, aceA or aceB promoter, and the like. More specifically, it works with the lysCP1 promoter (WO2009/096689), CJ7 promoter (WO2006/065095), SPL promoter (KR 10-1783170 B), or o2 promoter (KR 10-1632642 B), which are promoters derived from the genus Corynebacterium. It is possible to be linked to improve the expression rate of the polynucleotide encoding the enzyme, but is not limited thereto.
[86]
In addition, 3) modification of the polynucleotide sequence on the chromosome is not particularly limited thereto, but the expression control sequence by deletion, insertion, non-conservative or conservative substitution of the nucleic acid sequence to further enhance the activity of the polynucleotide sequence, or a combination thereof It can be carried out by inducing a phase mutation, or by replacing with a polynucleotide sequence modified to have a stronger activity.
[87]
Finally, 4) the method of modifying to be enhanced by the combination of 1) to 3) is to increase the copy number of the polynucleotide encoding the enzyme, modify the expression control sequence to increase its expression, and the polynucleotide on the chromosome Modification of the sequence and the modification of foreign polynucleotides or codon-optimized variant polynucleotides exhibiting the activity of the enzyme may be performed by applying one or more methods together.
[88]
The polynucleotide may be described as a gene if it is an aggregate of polynucleotides capable of functioning. Herein, polynucleotide and gene may be mixed, and polynucleotide sequence and nucleotide sequence may be mixed.
[89]
As used herein, the term "vector" refers to a DNA preparation containing a base sequence of a polynucleotide encoding the protein of interest operably linked to a suitable regulatory sequence so that the protein of interest can be expressed in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, any 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. Vectors can be transformed into suitable host cells and then replicated or function independently of the host genome, and can be integrated into the genome itself.
[90]
The vector used herein is not particularly limited as long as it can be replicated in a host cell, and any vector known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as a phage vector or a cosmid vector, and as a plasmid vector, pBR system, pUC system, pBluescriptII system , pGEM system, pTZ system, pCL system, pET system, etc. can be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pSKH, pRS-413, pRS-414, pRS-415 vectors, etc. may be used, but are not limited thereto.
[91]
The vector usable herein is not particularly limited, and a known expression vector may be used. In addition, a polynucleotide encoding a target protein may be inserted into a chromosome through a vector for intracellular chromosome insertion. Insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, by homologous recombination, but is not limited thereto. It may further include a selection marker for confirming whether the chromosome is inserted. Selectable markers are used to select cells transformed with a vector, i.e. to confirm the insertion of the target nucleic acid molecule, and give a selectable phenotype such as drug resistance, nutritional demand, resistance to cytotoxic agents or expression of surface proteins Markers can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, and thus transformed cells can be selected.
[92]
As used herein, the term "transformation" refers to introducing a vector containing a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them, whether inserted into the chromosome of the host cell or located outside the chromosome, as long as it can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding the 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 containing all elements necessary for self-expression. The expression cassette may generally include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replicating. 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. The transformation method includes any method of introducing a nucleic acid into a cell, and may be performed by selecting an appropriate standard technique as known in the art according to the host cell. For example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation,
[93]
In addition, the term "operably linked" in the above means that a promoter sequence for initiating and mediating transcription of a polynucleotide encoding a protein of interest of the present application and the polynucleotide sequence are functionally linked. The operable linkage may be prepared using a gene recombination technique known in the art, and site-specific DNA cleavage and linkage may be prepared using a cleavage and linkage enzyme in the art, but is not limited thereto.
[94]
[95]
The microorganism of the present application may additionally have inactivated 3-dehydroquinate dehydratase protein activity.
[96]
As used herein, the term "3-dehydroquinate dehydratase" refers to an enzyme that catalyzes the reversible reaction of the following reaction formula, specifically, 3-dehydroquinate It can be converted to 3-dehydroshikimate, but is not limited thereto.
[97]
[98]
[Reaction Scheme]
[99]

[100]
[101]
3-dihydroquinate dihydratase is not limited to its origin or sequence, since there may be differences in the amino acid sequence of a protein exhibiting activity depending on the species or microorganism of the microorganism. Specifically, it may be a protein including the amino acid sequence of SEQ ID NO: 90, but is not limited thereto. In addition, the amino acid sequence of SEQ ID NO: 90 or an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity thereto may be included. In addition, if such an amino acid sequence having homology or identity and having the same or corresponding biological activity as that of the protein, it is obvious that some sequences are also included in the scope of the present application if they have an amino acid sequence that has been deleted, modified, substituted or added. .
[102]
[103]
As used herein, the term "inactivation" refers to a case in which the activity is weakened compared to the intrinsic activity or pre-modification activity of the enzyme protein possessed by the original microorganism; The protein is not expressed at all; Or, even if it is expressed, it means that there is no activity. The inactivation is a case in which the activity of the enzyme itself is weakened or eliminated compared to the activity of the enzyme of the original microorganism due to mutation of the polynucleotide encoding the enzyme, etc.; When the overall degree of enzyme activity in the cell is lower than that of the native microorganism due to inhibition of expression or translation of the gene encoding the enzyme or the like; Part or all of the gene encoding the enzyme is deleted; And a combination thereof, and is not limited thereto. That is, a microorganism in which the activity of an enzyme protein is inactivated refers to a microorganism in which the activity of an enzyme protein is low or removed compared to a natural wild-type microorganism or an unmodified microorganism.
[104]
[105]
The inactivation of the enzyme activity can be achieved by application of various methods well known in the art. Examples of the method include 1) a method of deleting all or part of a gene on a chromosome encoding the enzyme; 2) modification of the expression control sequence to reduce the expression of the gene on the chromosome encoding the protein, 3) modification of the gene sequence on the chromosome encoding the protein so that the activity of the protein is removed or attenuated, 4) encoding the protein Introduction of antisense oligonucleotides (eg, antisense RNA) that complementarily bind to transcripts of genes on chromosomes; 5) A secondary structure is formed by adding a sequence complementary to the sine-Dalgarno sequence to the front end of the sine-Dalgarno sequence of the gene on the chromosome encoding the protein, making it impossible to attach a ribosome. how to make; 6) There is a method of adding a promoter transcribed in the opposite direction to the 3'end of the ORF (open reading frame) of the polynucleotide sequence encoding the protein (Reverse transcription engineering, RTE), etc., which can also be achieved by a combination thereof. However, this is not particularly limited.
[106]
The method of deleting part or all of the gene on the chromosome encoding the enzyme is to replace the polynucleotide encoding the intrinsic target protein in the chromosome with a polynucleotide or a marker gene in which a part of the nucleotide sequence has been deleted through a vector for chromosome insertion in a microorganism. It can be done by doing. As an example of a method of deleting some or all of these polynucleotides, a method of deleting a polynucleotide by homologous recombination may be used, but is not limited thereto.
[107]
The method of modifying the expression control sequence is performed by inducing a mutation on the expression control sequence by deletion, insertion, non-conservative or conservative substitution of the nucleic acid sequence, or a combination thereof so as to further weaken the activity of the expression control sequence, or It can be carried out by replacing it with an active nucleic acid sequence. The expression control sequence includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence controlling termination of transcription and translation.
[108]
The method of modifying the gene sequence on the chromosome is performed by inducing a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination of the gene sequence to further weaken the activity of the enzyme, or to have weaker activity. It can be performed by replacing with an improved gene sequence or an improved gene sequence such that there is no activity, but is not limited thereto.
[109]
In the above, "some" may be different depending on the type of polynucleotide, specifically 1 to 300, more specifically 1 to 100, more specifically 1 to 50, but particularly limited thereto no.
[110]
[111]
In the microorganism of the present application, the activity of the 3-dehydroquinate synthase protein may be additionally enhanced compared to the unmodified microorganism.
[112]
The 3-dehydroquinate synthase (3-dehydroquinate synthase) refers to an enzyme that catalyzes the reversible reaction of the following reaction formula, and specifically 3-dehydroquinate (3-DHQ) can be synthesized. However, it is not limited thereto.
[113]
[114]
[Reaction Scheme]
[115]

[116]
[117]
As used herein, the term "mycosporine-like amino acids (MAAs)" refers to a cyclic compound that absorbs ultraviolet rays. The mycosporine-like amino acid herein is not limited as long as it can absorb ultraviolet rays, but specifically, a compound having a central ring of cyclohexenone or cyclohexenimine; Or it may be a compound in which various substances such as amino acids are bound to the central ring, and more specifically, Mycosporine-2-glycine, Palythinol, Palythenic acid , Deoxygadusol, Mycosporine-methylaminethreonine, Mycosporine-glycine-valine, Palythine, Asterina-330 (Asterina -330), Shinorine, Porphyra-334, Euharotes-362, Mycosporine-glycine, Mycosporine-ornithine ), Mycosporine-lysine, Mycosporine-glutamic acid-glycine, Mycosporine-methylamine-serine, Mycosporine-taurine ), Palythene,
[118]
Herein, mycosporine-like amino acids may be used interchangeably with MAA and MAAs.
[119]
[120]
As used herein, the term "a microorganism producing a mycosporin-like amino acid" may refer to a gene of an enzyme involved in the biosynthesis of a mycosporin-like amino acid or a microorganism including a cluster of the genes, or a microorganism in which the cluster is introduced or enhanced Can mean In addition, as used herein, the term "mycosporin-like amino acid biosynthesis gene cluster" may mean a group of genes encoding enzymes involved in mycosporin-like amino acid biosynthesis. Specifically, a mycosporine-like amino acid biosynthesis gene, a gene of an enzyme having an activity of attaching an additional amino acid residue to a mycosporine-like amino acid, or a cluster of the genes may be included. The mycosporine-like amino acid biosynthesis gene includes all foreign and/or endogenous genes of the microorganism as long as the microorganism containing the same can produce mycosporine-like amino acid. The foreign gene may be homologous and/or heterologous.
[121]
The mycosporine-like amino acid biosynthesis gene is not limited to the microbial species derived from the genes as long as the microorganism containing the same can produce an enzyme involved in the mycosporine-like amino acid biosynthesis and consequently produce mycosporine-like amino acids. , the cyanobacteria (cyanobacteria) in ahnaba everywhere Varia Billy's ( Anabaena variabilis ), rowing Stock peonti Fort Tome ( Nostoc punctiforme ), rowing lay Leah's pumi dehydrogenase ( Nodularia spumigena ), cyano test in PCC 7424 ( Cyanothece SP . PCC 7424), via line 8106 in PCC ( Lyngbya sp . PCC 8106), micro-Kai seutiseu ah rugi labor ( Microcystis aeruginosa ), micro-house collection greater buttocks flask test ( Microcoleus chthonoplastes ), cyano test in ATCC 51142 ( Cyanothece sp . ATCC 51142), a croissant COSPA Era watt Sony ( Crocosphaera watsonii ), cyano test in CCY 0110 ( Cyanothece SP. CCY 0110), Shirin de los percha stop stag day in PCC 7417 ( cylindrospermum stagnale SP, PCC 7417), APA-no Tess halo blood Utica ( Aphanothece halophytica ) or tricot death Ugly Erie Tribe to help ( Trichodesmium erythraeum ) or, or fungi (fungi) in Magna Forte climb ah ( Magnaporthe orzyae ), blood Reno Fora tree tee Mr. repen teeth ( Pyrenophora tritici - repentis ), Aspergillus Cloud tooth bar ( Aspergillus clavatus ), neck-triazol hematoxylin coca ( Nectria haematococca ), Aspergillus nidulans , Gibberella zeae , Verticillium albo - atrum , Botryotinia fuckeliana , Paeospaeria surf room ( Phaeosphaeria nodorum ), or is nematic Torr telra bekten sheath ( Nematostella vectensis ), heteroaryl CAP Satri Quebec trad ( Heterocapsa triquetra ), oxy-less Marina ( Oxyrrhis marina ), a knife-di nium US Krumlov ( Karlodinium micrum ), evil Martino new Cinema deferring ( Actinosynnema mirum ), but is not limited thereto.
[122]
According to an embodiment, a microorganism producing a mycosporine-like amino acid of the present application includes a mycosporine-like amino acid biosynthesis gene or a cluster thereof. Specifically, in the microorganism, a mycosporin-like amino acid biosynthesis gene cluster is introduced, or the activity of a protein encoded by the gene may be improved compared to intrinsic activity or pre-modification activity, but is not limited thereto.
[123]
In addition, the mycosporine-like amino acid biosynthesis gene is not limited to the name of the enzyme or the derived microorganism as long as the microorganism can produce mycosporine-like amino acid, but 2-dimethyl 4-deoxygadusol synthase (2-demetyl 4-deoxygadusol synthase) ), O-methyltransferase, and one or more, specifically, one or more, two or more, three or more, or all enzyme proteins selected from the group consisting of CN ligase; Or it may include a gene encoding an enzyme protein having the same and/or similar activity.
[124]
For example, the 2-dimethyl 4-deoxygadusol synthase (2-demetyl 4-deoxygadusol synthase) is sedoheptulose-7-phosphite (sedoheptulose-7-phosphate) 2-dimethyl-4-deoxy It is an enzyme that converts to 2-demethyl-4-deoxygadusol. The O-methyltransferase is an enzyme that converts 2-dimethyl-4-deoxygadusol to 4-deoxygadusol, The glycylation of 4-deoxygadusol is catalyzed by the CN ligase.
[125]
Further, the microorganism producing the mycosporine-like amino acid may include a gene of an enzyme having an activity of attaching an additional amino acid residue to the mycosporine-like amino acid or a cluster of the genes. The gene or cluster of genes is not limited to the name of the enzyme or the derived microorganism as long as the microorganism producing the mycosporin-like amino acid can produce the mycosporine-like amino acid to which two or more amino acid residues are attached, but specifically non-ribosomes Peptide synthetase (nonribosomal peptide synthetase: NRPS), non-ribosomal peptide synthetase-like enzyme (NRPS-like enzyme) and D-alanine D-alanine ligase (D-Ala D-Ala ligase: DDL) one or more, specifically, one or more, two or more, three or more, or all enzyme proteins selected from the group consisting of; Or it may include a gene encoding an enzyme protein having the same and/or similar activity.
[126]
Some mycosporine-like amino acids contain a second amino acid residue in mycosporine-glycine. At least one enzyme selected from the group consisting of the non-ribosome peptide synthetase, the non-ribosomal peptide synthetase-like enzyme, and D-alanine D-alanine ligase may attach a second amino acid residue to mycosporine-glycine.
[127]
According to an embodiment, the microorganism producing mycosporine-like amino acid is second to mycosporine-glycine, such as non-ribosome peptide synthetase, non-ribosomal peptide synthetase-like enzyme, and D-alanine D-alanine ligase. If an enzyme has an activity capable of attaching an amino acid, it may be included without limitation on the name of the enzyme or the microorganism species derived from it.
[128]
For example, the non-ribosome peptide synthetase-like enzyme (Ava_3855) in Anabaena variabilis or D-alanine D-alanine ligase (NpF5597) in Nostoc punctiforme is myco Sinorine can be formed by attaching a serine moiety to sporine-glycine. As another example, mycosporin -2-glycine may be formed by attachment of a second glycine residue by D-alanine D-alanine ligase homolog (Ap_3855) in Aphanothece halophytica . . Similarly, in Actinosynnema mirum , serine or alanine can be attached by D-alanine D-alanine ligase to form sinorine or mycosporine -glycine-alanine. The microorganism according to the exemplary embodiment of the present application may include the above-described enzymes or those suitable for producing a mycosporine-like amino acid from among enzymes having the same and/or similar activity.
[129]
[130]
The 2-dimethyl 4-deoxygadusol synthase, O-methyltransferase, CN ligase, non-ribosome peptide synthetase, non-ribosome peptide synthetase-like enzyme and/or D- which can be used in the present invention The alanine D-alanine ligase is not limited to the derived microbial species, and is not limited as long as it is known as an enzyme that performs the same and/or similar functions and roles, and the range of values ​​of homology or identity between them is also not limited. For example, MylA, MylB, MylD, MylE and MylC of Cylindrospermum Stagnal PCC 7417 ( C. stagnale PCC 7417) are Anabaena variabilis and Nostoc Puntiforme ( Nostoc punctiforme ) Derived from 2-dimethyl 4-deoxygadusol synthase, O-methyltransferase, CN ligase, and D-alanine D-alanine ligase (homologous), and the similarity between them is about 61 to 88 % (Appl Environ Microbiol, 2016, 82(20), 6167-6173; J Bacteriol, 2011, 193(21), 5923-5928). That is, the enzyme that can be used in the present invention is not greatly limited to the derived microorganism species or sequence homology or identity as long as it is known to exhibit the same and/or similar functions and effects. In addition, the non-patent documents described in the prior art documents are incorporated by reference herein as a whole.
[131]
In addition, the mycosporine-like amino acid biosynthesis gene may be a polynucleotide encoding a protein including the amino acid sequence of SEQ ID NOs: 115, 116, 117, 118, 119, 120, 121, or 122, but is not limited thereto.
[132]
In addition, the mycosporine-like amino acid biosynthesis gene is 50%, 60%, or 70% or more, specifically 80% or more, and more with the amino acid sequence of SEQ ID NO: 115, 116, 117, 118, 119, 120, 121, or 122 Specifically, it may contain a nucleotide sequence encoding a protein containing an amino acid sequence having homology or identity of 90% or more, more specifically 95% or more, and more specifically 99% or more, and the microorganism is mycosporine A nucleotide sequence encoding a protein out of the homology or identity may be included without limitation as long as it can produce similar amino acids. Specifically, the mycosporine-like amino acid biosynthesis gene may include the nucleotide sequence of SEQ ID NO: 102, 103, 104, 105, 106, 107, 108, or 109, but is not limited thereto.
[133]
In addition, as a sequence having homology or identity to the sequence, if an amino acid sequence having biological activity substantially identical to or corresponding to the protein of the sequence number described above, some sequences may have a deleted, modified, substituted, or added amino acid sequence. It is obvious that it is also included in the scope of the present application.
[134]
In addition, the nucleotide sequence is in the coding region within a range that does not change the amino acid sequence of the protein expressed from the coding region in consideration of codons preferred in organisms to express the protein due to codon degeneracy. Various modifications can be made. Accordingly, the mycosporin-like amino acid biosynthesis gene may be included herein without limitation, as long as it is a nucleotide sequence encoding a protein involved in the mycosporin-like amino acid biosynthesis.
[135]
Or, if a probe that can be prepared from a known gene sequence, for example, a sequence encoding a protein involved in mycosporine-like amino acid biosynthesis by hybridizing under stringent conditions with a complementary sequence for all or part of the nucleotide sequence It may be included herein without limitation.
[136]
[137]
According to an embodiment, a microorganism that produces a mycosporine-like amino acid may include genes for biosynthetic mycosporin-like amino acids that are different from each other.
[138]
In the present application, the enhancement of the activity of the protein and/or the introduction of the gene may be performed simultaneously, sequentially, or in reverse order regardless of the order.
[139]
[140]
The microorganism producing the mycosporine-like amino acid may produce a mycosporine-like amino acid including a mycosporine-like amino acid biosynthetic gene cluster, and additionally, 2-dihydro-3-deoxyphosphoheptonate aldolase, phospho By enhancing the activity of one or more proteins selected from the group consisting of enolpyruvate synthetase and transketolase, it may be a microorganism having an increased ability to produce mycosporine-like amino acids. In addition, the microorganism of the present application enhances the activity of one or more proteins selected from the group consisting of 2-dihydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase, and transketolase, There is no limitation as long as it is a microorganism that increases the production capacity of mycosporine-like amino acids, but specifically, it may be a microorganism of the genus Corynebacterium, a microorganism of the genus Escherichia, or a yeast.
[141]
The microorganisms of the genus Corynebacterium are specifically, Corynebacterium glutamicum , Corynebacterium ammoniagenes , Brevibacterium lactofermentum , Brevibacterium Solarium Plastic pan ( Brevibacterium flavum ), Corynebacterium thermo amino to Ness ( Corynebacterium thermoaminogenes ), Corynebacterium epi syeonseu ( Corynebacterium efficiens , and the like), more specifically, the stiffness, but can Corynebacterium glutamicum Tommy kumil, It is not limited thereto.
[142]
The microorganisms of the genus Escherichia are specifically, Escherichia albertii , Escherichia coli , Escherichia fergusonii , Escherichia hermannii , Escherichia hermannii , S. It may be Escherichia vulneris and the like, and more specifically, Escherichia coli, but is not limited thereto.
[143]
Specifically, the yeast is a microorganism belonging to the Ascomycota Saccharomycotina, Taphrinomycotina, or Basidiomycota, Agaricomycotina, Pucciniomycotina, etc. More specifically, microorganisms in the genus Saccharomyces, microorganisms in the genus Schizosaccharomyces, microorganisms in the genus Phaffia, microorganisms in the genus Kluyveromyces, microorganisms in the genus Pichia It may be a microorganism of the genus Candida. More specifically, it may be Saccharomyces cerevisiae, but is not limited thereto.
[144]
[145]
Another aspect of the present application is culturing the microorganism of the present application in a medium; And recovering mycosporine-like amino acids from the cultured microorganism or medium.
[146]
The "microorganisms" and "mycosporin-like amino acids" are as described above.
[147]
As used herein, "culture" means growing the microorganism in an appropriately controlled environmental condition. The cultivation process of the present application may be performed according to a suitable medium and culture conditions known in the art. This culture process can be easily adjusted and used by a person skilled in the art according to the selected microorganism. 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, or the like. The medium and other culture conditions used for culturing the microorganisms of the present application may be any medium without particular limitation, as long as it is a medium used for cultivation of ordinary microorganisms. In a conventional medium containing amino acids and/or vitamins, it can be cultured while controlling temperature, pH, etc. under aerobic conditions. Specifically, a basic compound (such as sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound (such as phosphoric acid or sulfuric acid) is used to provide an appropriate pH (such as pH 5 to 9, specifically pH 6 to 8, most specifically PH 6.8) can be adjusted, but is not limited thereto. In addition, in order to maintain the aerobic state of the culture, oxygen or oxygen-containing gas may be injected into the culture, or nitrogen, hydrogen, or carbon dioxide gas may be injected without the injection of gas to maintain the anaerobic and microaerobic state. It is not limited. In addition, the culture temperature can be maintained at 20 to 45 ℃, specifically 25 to 40 ℃, and can be cultured for about 10 to 160 hours, but is not limited thereto. In addition,
[148]
In addition, the culture medium used is a carbon source such as sugars and carbohydrates (eg glucose, sucrose, lactose, fructose, maltose, molase, starch and cellulose), fats and fats (eg, soybean oil, sunflower seeds). Oil, peanut oil and coconut oil), fatty acids (such as palmitic acid, stearic acid, and linoleic acid), alcohols (such as glycerol and ethanol), and organic acids (such as acetic acid) can be used individually or in combination. , Is not limited thereto. Nitrogen sources include nitrogen-containing organic compounds (e.g. peptone, yeast extract, broth, malt extract, corn steep liquor, soybean meal and urea), or inorganic compounds (e.g. ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate) or the like may be used individually or in combination, but is not limited thereto. Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and a sodium-containing salt corresponding thereto may be used individually or in combination as a phosphorus source, but are not limited thereto. In addition, the medium may contain essential growth-promoting substances such as other metal salts (eg, magnesium sulfate or iron sulfate), amino acids and vitamins.
[149]
MAAs produced by the culture may be secreted into the medium or remain in the cells.
[150]
[151]
As used herein, the term "medium" means a culture medium in which the microorganisms of the present application are cultivated and/or a product obtained after culturing. The medium is a concept including both a form containing microorganisms and a form in which microorganisms are removed by centrifugation or filtration from the culture medium containing the microorganisms.
[152]
[153]
In the step of recovering the MAAs produced in the culturing step of the present application, the target MAAs may be collected from the culture medium using a suitable method known in the art according to the culture method. For example, centrifugation, filtration, anion exchange chromatography, crystallization and HPLC, and the like can be used, and desired MAAs can be recovered from the cultured microorganism or medium using a suitable method known in the art. The step of recovering the MAAs may additionally include a separation process and/or a purification step.
[154]
[155]
Another aspect of the present application provides the use of a microorganism herein to produce a mycosporine-like amino acid.
[156]
The "microorganisms" and "mycosporin-like amino acids" are as described above.
[157]
Mode for carrying out the invention
[158]
Hereinafter, the present application will be described in more detail by examples. However, these examples are for illustrative purposes only, and the scope of the present application is not limited by these examples.
[159]
[160]

[161]
[162]
Example 1: 2 - dihydro -3- deoxy-phosphonate hept carbonate aldolase (2- dehydro -3-deoxyphosphoheptonate aldolase) activity enhanced production strain
[163]
In order to increase the ability of microorganisms to produce MAAs, Escherichia coli with enhanced activity of 2-dihydro-3-deoxyphosphoheptonate aldolase was prepared. Specifically, aroG (2-dihydro-3- deoxyphosphoheptonate aldolase; SEQ ID NOs: 1 and 2) genes were additionally introduced using E. coli W3110 as the base strain . The templates and primers used in the construction of the plasmid are shown in Table 1 below.
[164]
[165]
[Table 1]
[166]
[167]
After amplifying the gene fragment through PCR using the template and primer, the amplified fragment was added to a pSKH vector (PCT/KR2007/006933) which cut the fhuA arm 1 and fhuA arm 2 gene fragments with BamH1-Spe1 restriction enzyme. It was ligated using In-Fusion R HD cloning kit (clontech), and the prepared vector was named pSKH-ㅿfhuA. Phage infection of E. coli is suppressed due to the fhuA gene deletion.
[168]
The Pn_ aroG gene fragment was ligated to pSKH-fhuA vector digested with Spe1-EcoRV restriction enzyme using In-Fusion R HD cloning kit (clontech), and the Ptrc and Pcj1 gene fragments known as enhanced promoters were Spe1. -Nde1 restriction enzyme digested, aroG gene fragment was digested with Nde1-EcoRV restriction enzyme, and then Ptrc and aroG gene fragments or Pcj1 (Korea Patent Registration No. It was ligated to the cut pSKH-ㅿfhuA vector using In-Fusion R HD cloning kit (clontech). The prepared vector are each pSKH- DELTA fhuA-Pn- aroG , pSKH- DELTA fhuA-Ptrc- aroG, pSKH- DELTA fhuA-Pcj1- aroG was named.
[169]
After confirming the cloning of the vector and the gene sequence of the vector through sequencing, the vector was transformed into a wild-type E. coli W3110 strain by electroporation. The transformed gene was introduced into the chromosome by primary recombination (crossing), and then excision of the plasmid site from the chromosome occurred through secondary recombination (crossing). The introduction of the aroG gene was confirmed through PCR using primers of SEQ ID NOs: 14 (forward) and 8 (reverse) targeting the E. coli transformed lines for which the secondary recombination was completed .
[170]
[171]
Example 2: Construction of a vector overexpressing a microalgal-derived sinorin biosynthesis gene
[172]
The synorin biosynthesis gene cluster based on A. variabilis is 2-dimethyl 4-deoxygadusol synthase, O-methyltransferase, and CN ligase. , And non-ribosomal peptide synthetase (non-ribosomal peptide synthetase), consisting of four genes (Ava_ABCD) encoding, A. variabilis ATCC29413 genomic DNA was used to identify the synorin biosynthetic gene cluster. Using the pECCG117_Pcj1_GFP_terminator vector, a vector containing a synorin biosynthesis gene derived from A. variabilis ATCC29413 was constructed. The names of the synorin biosynthetic gene expression vector and each template and primer used to construct the vector are shown in Table 2 below.
[173]
[174]
[Table 2]
[175]
[176]
After obtaining the gene fragments using the template and primers, each of the gene fragments was ligated to the pECCG 117_Pcj1_GFP_terminator vector treated with EcoRV-XbaI restriction enzyme using In-Fusion R HD cloning kit (clontech). The prepared vector was named pECCG117_Pcj1_Ava_ABCD, and the cloning status and gene sequence of the vector were confirmed through sequencing. The nucleotide sequence of Ava_ABCD is specified in SEQ ID NO: 17.
[177]
[178]
Example 3: 2 - dihydro -3- deoxy-phosphonate hept carbonate aldolase Shino Lin producing ability evaluation of activity enhanced strain
[179]
The pECCG117_Pcj1_Ava_ABCD plasmid prepared in Example 2 was introduced into the aroG gene-enhancing strain and the wild-type W3110 strain prepared in Example 1 through electroporation, respectively, and then plated on LB solid medium. After culturing the strain overnight in a 37 ℃ incubator, 25 mL titer medium [media composition: glucose 40 g/L, KH 2 PO 4 0.3 g/L, K 2 HPO 4 0.6 g/L, (NH 4 ) 2 SO 4 15 g/L, MgSO 4 7H 2 O 1 g/L, NaCl 2.5 g/L, Sodium citrate 1.2 g/L, Yeast extract 2.5 g/L, Calcium carbonate 40 g/L: pH 7.0] After each inoculation, it was incubated for 48 hours in an incubator at 37° C. and 200 rpm, and the analysis results thereof using HPLC (waters) are shown in [Table 3].　
[180]
[181]
[Table 3]
[182]
[183]
As shown in [Table 3], the concentration of synorin produced in the strain (W3110ㅿfhuA:: Pn-aroG /pECCG117_Pcj1_Ava_ABCD) enriched with the gene aroG increased by about 20% compared to the control group. In particular, to enhance gene promoters aroG the reinforcing strain (W3110 DELTA fhuA :: Ptrc- aroG / pECCG117_Pcj1_Ava_ABCD, W3110 DELTA fhuA :: Pcj1- aroG For / pECCG117_Pcj1_Ava_ABCD) was increased 69%, 94%.
[184]
[185]
Example 4: play pyruvate in phosphorylation bait synthetase ( phosphoenolpyruvate synthetase activity enhanced strain produced in)
[186]
In order to increase the ability of microorganisms to produce MAAs, E. coli with enhanced activity of phosphoenolpyruvate synthetase was prepared. Specifically, the pps (phosphoenolpyruvate synthetase; SEQ ID NOs: 18 and 19) genes were additionally introduced using E. coli W3110 as the base strain . The templates and primers used in the construction of the plasmid are shown in Table 4 below.
[187]
[188]
[Table 4]
[189]
[190]
After amplifying a gene fragment by PCR using the template and primers, Pn_ pps gene fragment at a In-Fusion pSKH- DELTA fhuA vector digested with the EcoRV restriction enzyme Spe1- R binding by the HD cloning kit (clontech) (ligation), the Ptrc and Pcj1 gene fragments prepared in Example 1 were digested with Spe1-Nde1 restriction enzyme, and the pps gene fragment was cut with Nde1-EcoRV restriction enzyme, and then Ptrc and pps gene fragment or Pcj1 and pps gene fragment Was ligated to pSKH-fhuA vector digested with Spe1-EcoRV restriction enzyme, respectively, using In-Fusion R HD cloning kit (clontech). The prepared vectors were respectively named pSKH-ㅿfhuA-Pn- pps, pSKH-ㅿfhuA-Ptrc- pps, and pSKH-ㅿfhuA-Pcj1- pps .
[191]
After confirming the cloning of the vector and the gene sequence of the vector through sequencing, the wild-type E. coli W3110 strain was transformed by electroporation. The transformed gene was introduced into the chromosome by primary recombination (crossover), and then excision of the plasmid site from the chromosome occurred through secondary recombination (crossover). The introduction of the pps gene was confirmed through PCR using primers of SEQ ID NOs: 14 (forward) and 21 (reverse) for the E. coli transformed lines for which the secondary recombination was completed .
[192]
[193]
Example 5: phosphoenolpyruvate synthetase activity enhancing strain of synorin production ability evaluation
[194]
The pECCG117_Pcj1_Ava_ABCD plasmid prepared in Example 2 was introduced into the pps gene-transducing strain prepared in Example 4 and the wild-type W3110 strain through electroporation, respectively, and then plated on LB solid medium. After culturing the strain in an incubator at 37° C. overnight, one platinum was inoculated into the 25 mL titer medium of Example 3, and then cultured for 48 hours in an incubator at 37° C. and 200 rpm, and the results are shown in [Table 5] Shown in.
[195]
[196]
[Table 5]
[197]
[198]
As shown in [Table 5], the concentration of synorin produced in the strain enriched with gene pps increased by 41%, and when its activity was enhanced by substitution with a strong promoter, it increased by up to 60% compared to the control group.
[199]
[200]
Example 6: Preparation of strains for enhancing activity of transketolase I/II
[201]
In order to increase the ability of microorganisms to produce MAAs, E. coli with enhanced transketolase activity was prepared. Specifically, a tktA (transketolase; SEQ ID NOs: 23 and 24) gene was additionally introduced using E. coli W3110 as the base strain . The templates and primers used in the construction of the plasmid are shown in Table 6 below.
[202]
[203]
[Table 6]
[204]
[205]
After amplifying the gene fragment through PCR using the template and primer, the Pn_ tktA gene fragment was bound to the pSKH-ㅿfhuA vector digested with Spe1-EcoRV restriction enzyme using In-Fusion R HD cloning kit (clontech). (ligation), the Ptrc and Pcj1 gene fragments prepared in Example 1 were digested with Spe1 and Nde1 restriction enzymes, and the tktA gene fragment was digested with Nde1-EcoRV restriction enzymes, and then Ptrc and tktA gene fragments or Pcj1 and tktA gene fragments. Was ligated to pSKH-fhuA vector digested with Spe1-EcoRV restriction enzyme, respectively , using an In-Fusion R HD cloning kit (clontech). The prepared vector was named as pSKH-ㅿfhuA-Pn- tktA , pSKH-ㅿfhuA-Ptrc- tktA , pSKH-ㅿfhuA-Pcj1-tktA, respectively .
[206]
After confirming the cloning of the vector and the gene sequence of the vector through sequencing, the vector was transformed into a wild-type E. coli W3110 strain by electroporation. The transformed gene was introduced into the chromosome by primary recombination (crossing), and then excision of the plasmid site from the chromosome occurred through secondary recombination (crossing). The introduction of the tktA gene was confirmed by PCR using primers of SEQ ID NOs: 14 (forward) and 26 (reverse) targeting the E. coli transformed lines for which the secondary recombination was completed .
[207]
[208]
Example 7: Evaluation of synorin production ability of strains enhancing the activity of transketolase
[209]
The pECCG117_Pcj1_Ava_ABCD plasmid prepared in Example 2 was introduced into the tktA gene-transducing strain prepared in Example 6 and the wild-type W3110 strain through electroporation, respectively, and then plated on LB solid medium. After culturing the strain in an incubator at 37° C. overnight, one platinum was inoculated into the 25 mL titer medium of Example 3, and then cultured for 48 hours in an incubator at 37° C. and 200 rpm, and the results are shown in [Table 7] Shown in.　
[210]
[211]
[Table 7]
[212]
[213]
As shown in [Table 7], the concentration of synorin produced in the strain enriched with the gene tktA increased by 4.5%, and when its activity was enhanced by substitution with a strong promoter, it increased by up to 32% compared to the control group.
[214]
[215]
Example 8: 2 - dihydro -3- deoxy-phosphonate hept carbonate aldolase / phosphorylation play pyruvate to enhance the activity of synthetase / trans-ketol cyclase production strain
[216]
To increase the ability of microorganisms to produce MAAs, Escherichia coli with enhanced activity of 2-dihydro-3-deoxyphosphoheptonate aldolase/phosphoenolpyruvate synthetase/transketolase was prepared. Specifically, aroG , pps , and tktA genes were additionally introduced using E. coli W3110 as the base strain . The templates and primers used in the construction of the plasmid are shown in Table 8 below.
[217]
[218]
[Table 8]
[219]
[220]
After amplifying the gene fragment through PCR using the above template and primer, it was ligated to the pSKH-ㅿfhuA vector digested with Spe1-EcoRV restriction enzyme using In-Fusion R HD cloning kit (clontech). The prepared vector was named pSKH-ㅿfhuA-Pcj1- aroG -Pcj1- ppsA -Pcj1- tktA .
[221]
After confirming the cloning of the vector and the gene sequence of the vector through sequencing, the vector was transformed into a wild-type E. coli W3110 strain by electroporation. The transformed gene was introduced into the chromosome by primary recombination (crossover), and then excision of the plasmid site from the chromosome occurred through secondary recombination (crossover). The introduction of aroG , pps , and tktA genes was confirmed through PCR using primers of SEQ ID NOs: 14 (forward) and 26 (reverse) targeting the E. coli transformed lines for which the secondary recombination was completed .
[222]
[223]
Example 9: 2 - dihydro -3- deoxy-phosphonate hept carbonate aldolase / play pyruvate in phosphorylation bait Shino Lin producing ability evaluation of the active strain of the reinforcing synthetase / trans-ketol cyclase
[224]
The pECCG117 Pcj1_Ava_ABCD plasmid prepared in Example 2 was introduced into the aroG , pps , tktA gene transduction strain and wild-type W3110 strain prepared in Example 8 through electroporation, and then plated on LB solid medium. After culturing the strain in a 37°C incubator overnight, one platinum was inoculated into the 25 mL titer medium of Example 3, and then cultured for 48 hours in an incubator at 37°C and 200 rpm, and the results are shown in [Table 9]. Indicated.
[225]
[226]
[Table 9]
[227]
[228]
As shown in [Table 9], the concentration of synorin produced in the strain enhanced by combining three genes ( aroG , pps , tktA ) increased by 267% compared to the control group. This is a result of unexpected improvement over the sum of the maximum increase obtained by replacing the promoter of each gene with a strong promoter. That is, when the three genes aroG , pps , and tktA are combined, it was confirmed that synorin production at a higher concentration is possible.
[229]
[230]
Example 10: 3 - dihydro-quinolyl carbonate di hydratase (3- dehydroquinate produced inert strains dehydratase)
[231]
In order to increase the ability of microorganisms to produce MMAs , a strain in which 3-dihydroquinate dihydratase ( aroD ) is inactivated was prepared.
[232]
Specifically, a chloramphenicol resistance gene of pKD3 was used as a gene insertion marker, and the aroD deletion cassette containing a portion of the aroD gene and the chloramphenicol resistance gene of the pKD3 plasmid was prepared with primers of SEQ ID NOs: 32 (forward) and 33 (reverse). Was produced through PCR. Wild-type E. coli W3110 and the aroG , pps, and tktA gene-transducing strain prepared in Example 8 were transformed with the pKD46 plasmid containing the lambda red recombinase gene, and arabinose was used to induce the expression of the gene Tunt cells were prepared. After the aroD deletion cassette was introduced into the competent cells by electroporation , it was plated on LB solid medium containing 30 mg/L of chloramphenicol. The thus obtained strain was PCR using primers of SEQ ID NOs: 34 (forward) and 35 (reverse), and aroD gene deletion was confirmed through observation of the fragment amplified by 1300 bp .
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Example 11: 3 - Evaluation of the ability to produce sinorine of strains in which dihydroquinate dihydratase is inactivated
[235]
The pECCG117 Pcj1_Ava_ABCD plasmid prepared in Example 2 was introduced into the strain in which the aroD gene was deleted prepared in Example 10 through electroporation, and then plated on LB solid medium. After culturing the strain in an incubator at 37° C. overnight, one platinum was inoculated into the 25 mL titer medium of Example 3, and then cultured for 48 hours in an incubator at 37° C. and 200 rpm, and the results are shown in [Table 10]. Indicated.　
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[Table 10]
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As shown in [Table 10], the aroG , pps , and tktA -enhanced synorin -producing strains, in addition to the aroD -deficient strains, increased the concentration of synorin by 66%. The aroG , PPS , tktA an enhanced strain W3110 DELTA :: Pcj1- fhuA aroG -Pcj1- PPS -Pcj1- tktA / pECCG117_PCJ1_Ava_ABCD strain was named CB06-0020, South Korea preserved microorganisms under the Budapest Treaty on February 14, 2018 It was deposited with the Korean Culture Center of Microorganisms (KCCM) and was given the deposit number KCCM12224P.
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< Corynebacterium glutamicum - based MAAs production recombinant microorganism production and MAAs production using the same>
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Example 12: 2 - dihydro -3- deoxy-phosphonate hept carbonate aldolase activity enhance vector production and Lin Sino-producing ability evaluation
[244]
In order to increase the ability of microorganisms to produce MAAs, a microorganism of the genus Corynebacterium having enhanced activity of 2-dihydro-3-deoxyphosphoheptonate aldolase was prepared. Specifically, aroG (2-dihydro-3- deoxyphosphoheptonate aldolase; SEQ ID NOs: 36 and 37) genes were additionally introduced using Corynebacterium glutamicum ATCC13032 as the based strain . The templates and primers used in the construction of the plasmid are shown in Table 11 below.
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[Table 11]
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After obtaining a gene fragment using the template and primers, each of the gene fragments was treated with EcoRV/XbaI restriction enzyme pECCG 117, pECCG 117_Pcj7_GFP_terminator (Korea Patent Registration No. 10-620092, p117-cj7-gfp) vector. It was ligated using In-Fusion R HD cloning kit (clontech). The prepared vectors were named pECCG117_Pn_cgl aroG and pECCG117_Pcj7_cgl aroG , respectively . The vectors were cloned and the gene sequence of the vector was confirmed through sequencing.
[249]
First, since the microorganisms in the genus Corynebacterium cannot produce synorin, a strain introduced into the synorin biosynthesis pathway was prepared. Specifically, pECCG117_Ptrc_Ava_ABCD was used as a template, and Ava_ABCD was PCR using a primer pair of SEQ ID NOs: 42 (forward) and 43 (reverse). A PCR fragment of about 7 kb was ligated to a pDZTn vector treated with NdeI restriction enzyme (International Publication No. WO 2009-125992A) using an In-Fusion R HD cloning kit (clontech) to prepare pDZTn_Ava_ABCD. Subsequently, the O2 promoter (Korea Patent Registration No. 10-1632642) fragment was PCR with a primer pair of SEQ ID NOs: 44 (forward) and 45 (reverse), and pDZTn_Ava_ABCD was treated with NdeI restriction enzyme In-Fusion R HD cloning kit (clontech) was used to prepare pDZTn_PO2_Ava_ABCD by ligation.
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The recombinant plasmid was transformed into wild Corynebacterium glutamicum ATCC13032 by electroporation (van der Rest et al. 1999), and the plasmid was introduced into the chromosome by primary recombination (crossover), Plasmids were excised from the chromosomes through secondary recombination (crossover).
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The introduction of the Ava_ABCD gene was confirmed by PCR using each gene-specific primer pair, SEQ ID NO: 42 and 43, targeting the Corynebacterium glutamicum transformants having completed the secondary recombination. The prepared strain was named Corynebacterium glutamicum 13032 ΔN1021PO2_Ava_ABCD.
[252]
The Corynebacterium glutamicum 13032 ΔN1021_PO2_Ava_ABCD strain was transformed with pECCG117_Pn_cgl aroG and pECCG117_Pcj7_cgl aroG vectors , respectively , by electroporation.
[253]
The prepared strain and the control Corynebacterium glutamicum ATCC13032 (c.gl 13032) were cultured overnight in BHIS solid medium containing kanamycin, and the strains were cultured in 25 mL titer medium [Medium composition: glucose 40 g /L, KH 2 PO 4 1 g/L, (NH 4 ) 2 SO 4 10 g/L, MgSO 4 7H 2 O 5g/L, NaCl 5 g/L, yeast extract 5 g/L, calcium carbonate 30 g /L: pH7.0] was inoculated with one platinum each, and then cultured for 48 hours in an incubator at 37° C. and 200 rpm, and the results are shown in [Table 12].　
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[Table 12]
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As shown in [Table 12] above, when the aroG expression level was increased in the strain containing the synorin biosynthesis gene, the concentration of synorin was increased by 39%. In particular, it was confirmed that when the promoter was strengthened, the concentration of sinorine could be improved up to 80%.
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[259]
Example 13: the phosphorylation play pyruvate synthetase / trans-ketol cyclase enhance vector production and activity-producing ability evaluation Shino Lin
[260]
In order to increase the ability of microorganisms to produce MAAs, Corynebacterium glutamicum with enhanced tkt or pps activity was prepared. Specifically, a tkt (transketolase; SEQ ID NOs: 95 and 96) or pps (phosphoenolpyruvate synthetase; SEQ ID NOs: 97 and 98) genes based on Corynebacterium glutamicum ATCC13032 were further added. Introduced. The templates and primers used in the construction of the plasmid are shown in Table 13 below.
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[Table 13]
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The gene fragment obtained through the PCR technique by matching the template and primer combination and the EcoRV/XbaI restriction enzyme-treated pECCG117, pECCG117_Ptrc_GFP_terminator, and pECCG 117_Pcj7_GFP_terminator vector were prepared by ligation using In-Fusion R HD cloning kit (clontech). The prepared vectors were named pECCG117-Pn-tkt/pECCG117-Pcj7-tkt and pECCG117-Ptrc-pps/pECCG117-Pcj7-pps, respectively. After confirming the cloning of the vectors and the gene sequence of the vector through sequencing, the vectors were transformed into Corynebacterium glutamicum 13032 ΔN1021_PO2_Ava_ABCD strain by electroporation. The prepared strain cultured overnight in BHIS solid medium containing Kanamycin was inoculated with one platinum each in the 25 mL titer medium of Example 12, and then cultured for 48 hours in an incubator at 37° C. and 200 rpm, and the results are shown in [Table 14].
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[Table 14]
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As shown in [Table 14], when enhancing the genes tkt and pps , it was confirmed that synorin production was improved by up to 57% or 72%, respectively.
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Example 14: 2 - dihydro -3- deoxy-phosphonate hept carbonate aldolase / play pyruvate in phosphorylation bait activity enhanced strain Production and evaluation of the synthetase / trans-ketol cyclase
[271]
In order to increase the MAAs production capacity of microorganisms, Corynebacterium glutamicum with enhanced activity of aroG , pps and tkt was prepared.In order to confirm higher MAAs production, additionally 3-dihydroquinate dihydratase ( aroD) was also inactivated. Specifically, aroG , pps , tktA pDZ- DELTA aroD-Pcj7- to enhance the genes aroG -Pcj7- pps -Pcj7- tktA The plasmid was produced. The pDZ- DELTA aroD-Pcj7- aroG -Pcj7- pps -Pcj7- tktA the same as the template and primers used in the production of plasmid to [Table 15].
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[Table 15]
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First, a pDZ-ΔaroD plasmid in which an open reading frame of aroD was internally deleted was constructed to prepare a strain having a deletion of the aroD gene (SEQ ID NOs: 89 and 90) of Corynebacterium glutamicum. The internal gene loss of the pDZ-ΔaroD is a gene fragment generated by cross-PCR with a pair of forward and reverse primers of SEQ ID NOs: 91 and 92 and SEQ ID NOs: 93 and 94 using Corynebacterium glutamicum ATCC 13032 genomic DNA as a template. It was produced by introducing it into the pDZ vector.
[276]
Next, the gene fragments of aroG , pps , and tkt were amplified through PCR using the template and primers of Table 15 , and then introduced into pDZ-peroD vectors cut with SpeI restriction enzymes. The two vectors were transformed into Corynebacterium glutamicum 13032ΔN1021 _PO2_Ava_ABCD strain by electroporation after confirming the cloning status and gene sequence of the vector through sequencing. The prepared strain cultured overnight in BHIS solid medium containing Kanamycin was inoculated with one platinum each in the 25 mL titer medium of Example 12, and then cultured for 48 hours in an incubator at 37°C and 200 rpm, and the results were shown in [Table 16]. ].
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[Table 16]
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As shown in [Table 16], the concentration of synorin produced in the strain enriched with three genes ( aroG , pps , tktA ) increased by about 25% compared to the control group. From this, it was confirmed that even in the strain having increased synorin-producing ability through aroD deletion, synorin can be produced at a high concentration when the three kinds of genes are combined. In addition, it can be interpreted that when the aroD gene is further inactivated in a strain in which the enhancement of the three kinds of genes is combined, synorin can be produced at a higher concentration.
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Example 15: the Sino Lin producing saccharide my process to the celebrity busy (S. cerevisiae) strain produced
[285]
My process in serenity busy as Saccharomyces ( S. cerevisiae a) for use as lean Sino-producing strain A. variabilis ATCC29413 was introduced Shino Lin biosynthesis genes derived from a yeast vector for expression. Ava_A and Ava_B genes were inserted into the pRS-413 vector using the GPD promoter. Specifically, after linking the pGPD-Ava_A and pGPD-Ava_B sites using overlapping PCR, the vector and the PCR product were treated with restriction enzymes BamH I, Sal I, and then linked using T4 ligase, pRS-413 -pGPD-Ava_A-pGPD-Ava_B vector was constructed.
[286]
Next, the Ava_C and Ava_D genes were inserted into the pRS-414 vector using the GPD promoter. Specifically, after linking the pGPD-Ava_C, pGPD-Ava_D sites using overlapping PCR, the vector and the PCR product were treated with restriction enzymes BamH I, Sal I, and then linked using T4 ligase, pRS-414 -pGPD-Ava_C-pGPD-Ava_D vector was constructed. Primers and template DNA used for vector construction are shown in Table 17 below.
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[Table 17]
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Lithium pRS-413-pGPD-Ava_A-pGPD-Ava_B vector and pRS-414-pGPD-Ava_C-pGPD-Ava_D vector were added to Saccharomyces cerevisiae CEN.PK-1D ( S. cerevisiae CEN.PK-1D). After introduction through the acetate transformation method, it was confirmed whether or not the production of sinorine. After spreading on SC (synthetic complete) solid medium excluding Trp and His, which are auxotrophic markers, were incubated overnight in an incubator at 30°C. The strains cultured overnight in SC (synthetic complete) solid medium excluding Trp and His were inoculated with one platinum each in 25 mL titer medium shown in [Table 18], and then cultured for 24 hours in an incubator at 30° C. and 150 rpm. , The results are shown in [Table 19].
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[Table 18]
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[Table 19]
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As a result of the experiment, as a result of introducing the synorin biosynthesis gene into the wild-type Saccharomyces cerevisiae strain that does not produce sinorine, it was confirmed that 331 mg/l of sinorine was produced.
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[298]
Example 16: Saccharomyces cerevisiae TKL1 ( transketolase ) enhancement of synorin production
[299]
Saccharomyces cerevisiae strain with enhanced TKL1 activity was constructed to increase MAAs production capacity . To this end, pRS-415-pGPD, pRS -415-pADH, the vector pRS-415-pTEF TKL1 by cloning a gene (SEQ ID NO: 110 and 123) TKL1 was enhanced expression of the gene.
[300]
The GPD promoter included in the pRS-415-pGPD vector is a promoter of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), isozyme 3 (TDH3) gene, and the start of the THD3 gene ORF It contains the -674bp to -1bp sequence from the codon.
[301]
The ADH promoter included in the pRS-415-pADH vector is a promoter of an alcohol dehydrogenase (ADH1) gene, and includes a -1500bp to -1bp sequence from the start codon of the ORF of the ADH1 gene.
[302]
The TEF promoter included in the pRS-415-pTEF vector is a promoter of a translational elongation factor EF-1 alpha (TEF1) gene, and includes a -500bp to -1bp sequence from the start codon of the ORF of the TEF1 gene.
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[304]
Specifically, after PCR of the TKL1 gene using the primers of the following [Table 20] , the PCR product and pRS-415-pGPD, pRS-415-pADH, pRS-415-pTEF vector were used with restriction enzymes BamH I and Sal I. After treatment, a T4 ligase was used to ligate to prepare pRS-415-pGPD- TKL1 , pRS-415-pADH- TKL1 , pRS-415-pTEF- TKL1 vectors.
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[306]
[Table 20]
[307]
[308]
Next, the synorin biosynthetic plasmid prepared in Example 15 was CEN.PK-1D to Saccharomyces cerevisiae together with pRS-415-pGPD- TKL1 , pRS-415-pADH- TKL1 , pRS-415-pTEF- TKL1. Introduced into the strain, Trp, Ura, His was plated on a solid medium excluding SC (synthetic complete) and cultured overnight in a 30 ℃ incubator. The strains cultured overnight in SC (synthetic complete) solid medium excluding Trp, Ura, and His were inoculated with one platinum each in 25 mL titer medium, and then incubated in an incubator at 30°C and 150 rpm for 24 hours, and the result It is shown in the following [Table 21].
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[Table 21]
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As shown in [Table 21], it was confirmed that the amount of synorin production increased compared to WT in the strain in which the expression of the TKL1 gene was enhanced using the GPD promoter . In addition, it was additionally confirmed that as the strength of the promoter increased (pGPD>pTEF>pADH), the amount of synorin production increased.
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[314]
Example 17: a saccharide My process of the celebrity busy ARO4 (3- deoxy -D- arabino - bovine carbonate in heptul -7-phosphate (DAHP) synthase) Shino Lin production increased by strengthening
[315]
Saccharomyces cerevisiae strain with enhanced ARO4 activity was constructed to increase MAAs production capacity . To this end, pRS-415-pGPD, pRS -415-pADH, the vector pRS-415-pTEF ARO4 by cloning a gene (SEQ ID NO: 111 and 124) ARO4 was used as a strategy to enhance the expression of the gene. Specifically, after PCR of the ARO4 gene using the primers in Table 22 , the ARO4 PCR product and pRS-415-pGPD, pRS-415-pADH, pRS-415-pTEF vector were used with restriction enzymes BamH I and Sal I. After the treatment, it was ligated using T4 ligase to prepare pRS-415-pGPD- ARO4 , pRS-415-pADH- ARO4 , pRS-415-pTEF- ARO4 vectors.
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[Table 22]
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Next, the synorin biosynthesis plasmid prepared in Example 15 was combined with pRS-415-pGPD- ARO4 , pRS-415-pADH- ARO4 , pRS-415-pTEF- ARO4 and Saccharomyces cerevisiae CEN.PK-1D strain. Introduced to, Trp, Ura, His was plated on a solid medium excluding SC (synthetic complete) and then cultured overnight in a 30 ℃ incubator. The strain cultured overnight in SC (synthetic complete) solid medium excluding Trp, Ura, and His was inoculated with one platinum each in 25 mL titer medium, and then incubated for 24 hours in an incubator at 30° C. and 150 rpm, and the results were obtained. It is shown in [Table 23].
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[Table 23]
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As shown in [Table 23], it was confirmed that the amount of synorin production increased by 187% compared to WT in the strain in which the expression of the ARO4 gene was enhanced using the GPD promoter .
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[325]
Example 18: saccharide with my process to the celebrity busy in pps ( phosphorylation play pyruvate in the bait synthetase Shino Lin production increased by strengthening)
[326]
Saccharomyces cerevisiae strain with enhanced pps activity was constructed to improve MAAs production capacity . To this end, a strategy was used to enhance the expression of the pps gene by cloning the pps gene of E. coli in the pRS-415-pGPD, pRS-415-pADH, and pRS-415-pTEF vectors .
[327]
Specifically, after PCR of the pps gene using the primers of [Table 24] , the pps PCR product and pRS-415-pGPD, pRS-415-pADH, pRS-415-pTEF vector were used with restriction enzymes BamH I and Sal I. After the treatment, T4 ligase was used to connect, pRS-415-pGPD- pps , pRS-415-pADH- pps , pRS-415-pTEF- pps vectors were prepared.
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[329]
[Table 24]
[330]
[331]
Next, the synorin biosynthesis plasmid prepared in Example 15 was prepared with pRS-415-pGPD- pps , pRS-415-pADH- pps , and pRS-415-pTEF- pps together with Saccharomyces cerevisiae CEN.PK-1D. Introduced into the strain, Trp, Ura, His was plated on a solid medium excluding SC (synthetic complete) and cultured overnight in a 30 ℃ incubator. The strains cultured overnight in SC (synthetic complete) solid medium excluding Trp, Ura, and His were inoculated with one platinum each in 25 mL titer medium, and then incubated in an incubator at 30°C and 150 rpm for 24 hours. It is shown in [Table 25].
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[Table 25]
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[335]
As shown in [Table 25], it was confirmed that the amount of synorin production increased by 70% compared to WT in the strain overexpressing the pps gene. In addition, it was additionally confirmed that as the strength of the promoter increases (pGPD>pTEF>pADH), the amount of synorin production increases.
[336]
[337]
Example 19: In Saccharomyces cerevisiae strain, TKL1 enhancement, ARO4 enhancement, and increase of synorin production through introduction of pps gene
[338]
Examples 16, 17 and 18 based on the results in my process serenity busy with saccharide as effective factors affecting the Sino Lin biosynthesis in TKL1 , ARO4 , pps (E. Coli ) selecting a gene, and the same time of the three genes It was intended to increase the biosynthesis of sinorine through strengthening. In order to introduce three effective genes, pRS-415-pGPD- TKL1 -pGPD- ARO4 and pRS-416-pGPD- pps vectors were constructed. Specifically, the pGPD- TKL1 , pGPD- ARO4 sites were ligated using overlapping PCR, and then the vector and the PCR product were treated with restriction enzymes BamH I, Sal I, and then ligated using T4 ligase, pRS-415 -pGPD- TKL1 -pGPD- ARO4 vector was constructed.
[339]
Next, after PCR of the pps gene derived from E. coli , the pps PCR product and pRS-416-pGPD vector were treated with restriction enzymes BamH I and Sal I, and then ligated using T4 ligase, pRS-416-pGPD- A pps vector was constructed. Primers and template DNA used for vector construction are shown in Table 26.
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[Table 26]
[342]
[343]
Next, the synorin biosynthesis plasmid prepared in Example 15 was introduced into the Saccharomyces cerevisiae CEN.PK-1D strain together with pRS-415-pGPD- TKL1 -pGPD- ARO4 and pRS-416-pGPD- pps vectors. Then, after spreading on SC (synthetic complete) solid medium excluding Leu, Trp, Ura, and His, the cells were cultured overnight in an incubator at 30°C. The strain cultured overnight in SC (synthetic complete) solid medium excluding Leu, Trp, Ura, and His was inoculated with one platinum each in 25 mL titer medium, and then incubated for 24 hours in an incubator at 30°C and 150 rpm, The results are shown in [Table 27].　
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[345]
[Table 27]
[346]
[347]
As shown in [Table 27] , it was confirmed that in the strain in which the three effective genes ( pps , TKL1 , ARO4 ) were overexpressed , the amount of synorin production compared to WT was significantly increased to 230%.
[348]
[349]
In the present specification, details that can be sufficiently recognized and inferred by those of ordinary skill in the technical field of the present invention have been omitted, and the technical spirit or essential configuration of the present invention other than the specific examples described in the present specification is changed. More various modifications are possible within the range that does not. Accordingly, the present invention may be implemented in a manner different from that specifically described and illustrated in the present specification, which is a matter that can be understood by those of ordinary skill in the technical field of the present invention.
[350]

Claims
[Claim 1]
From the group consisting of 2-dehydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase and transketolase Microorganisms that produce mycosporine-like amino acids with enhanced activity of one or more selected proteins.
[Claim 2]
The microorganism according to claim 1, wherein the microorganism further comprises a mycosporine-like amino acid biosynthesis gene cluster.
[Claim 3]
The method of claim 2, wherein the mycosporin-like amino acid biosynthesis gene cluster is 2-dimethyl 4-deoxygadusol synthase, O-methyltransferase, and CN ligase (CN ligase) containing a gene encoding one or more proteins selected from the group consisting of, a microorganism producing a mycosporine-like amino acid.
[Claim 4]
The method of claim 2, wherein the mycosporin-like amino acid biosynthesis gene cluster is non-ribosomal peptide synthetase, non-ribosomal peptide synthetase-like enzyme: NRPS-like enzyme) and D-alanine D-alanine ligase (D-Ala D-Ala ligase).
[Claim 5]
The microorganism according to claim 1, wherein the microorganism is a microorganism of the genus Corynebacterium, a microorganism of the genus Escherichia, or a yeast, a mycosporine-like amino acid producing microorganism.
[Claim 6]
The method of claim 1, wherein the mycosporine-like amino acid is Mycosporine-2-glycine, Palythinol, Palythenic acid, deoxygadusol, Mycosporine-methylamine-threonine (Mycosporine-methylamine-threonine), Mycosporine-glycine-valine, Palythine, Asterina-330 (Asterina-330), Sinorine ( Shinorine), Porphyra-334, Euhalotes-362 (Euhalothece-362), Mycosporine-glycine, Mycosporine-ornithine, Mycosporine-Lysine ( Mycosporine-lysine), Mycosporine-glutamic acid-glycine, Mycosporine-methylamine-serine, Mycosporine-taurine, Palythene ), Palythine-serine, Palythine-serine-sulfate, Palythinol, and at least one selected from the group consisting of Usujirene, Microorganisms that produce mycosporine-like amino acids.
[Claim 7]
Culturing the microorganism of any one of claims 1 to 6 in a medium; And recovering mycosporine-like amino acid from the cultured microorganism or medium.