Specification
Title of the invention: microorganism producing mycosporine-like amino acid and method for producing mycosporine-like amino acid using the same
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]
The ultraviolet rays emitted from the sun are composed 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). have. Of the sun's rays, about 6% of ultraviolet rays A and B reach the surface, whereas ultraviolet rays C are absorbed and scattered from the ozone layer and the atmosphere on the ground and do not reach the surface.
[4]
These ultraviolet rays have advantages such as synthesis of vitamin D in the body, treatment of skin diseases, and bactericidal effects, but there are also harmful aspects such as sunburn, skin cancer, skin aging, photosensitive skin disease, and mutagenesis. Ultraviolet A penetrates to the dermis layer, mainly causing 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.
[5]
Attempts have been made to block sunlight to prevent this side effect of sunlight. Types of sunscreens include chemical sunscreen agents and physical sunscreen agents. Chemical sunscreens mainly absorb sunlight, and physical sunscreens prevent sunlight penetration through reflection and scattering. Block.
[6]
[7]
Chemical sunscreens contain at least one component that absorbs ultraviolet rays, PABA, PABA esters (Amyl dimethyl PABA, octyl dimethyl PABA), Cinnamates: Cinoxate, and salicylate ( Salicylate: Homomenthyl salicylate), Camphor, etc., benzophenone (Oxybenzone, Dioxybenzone, Suliso benzene), dibenzoyl methane, and 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.
[8]
[9]
Physical sunscreens are ingredients that exist in nature and protect the skin by reflecting and scattering ultraviolet rays that penetrate the skin. For example, physical sunscreens such as titanium dioxide, talc (magnesium silicate), magnesium oxide, zinc oxide, kaolin, etc. Blocking effect can be realized. 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.
[10]
[11]
Mycosporine-like amino acids (MAAs) are substances that exist in natural organisms, and are known to absorb UVA (320-400nm) and UVB (290-320mm) effectively. It is known that more than 35 types of MAAs exist in nature depending on the type of amino acid, cyclohexanone, or cyclohexenimine ring as a precursor (Mar. Biol., 1991, 108: 157-166; Planta Med. ., 2015, 81: 813-820). Recently, various sugar-attached forms of MAAs exist in microalgae, which have been reported to 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).
[12]
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.
[13]
[14]
[Prior technical literature]
[15]
[Non-patent literature]
[16]
(Non-Patent Document 1) Comp. Biochem. Physiol. B 1995, 112: 105-114.
[17]
(Non-Patent Document 2) FEMS Microbiol Lett. 2007, 269: 1-10.
[18]
(Non-Patent Document 3) Ann. Rev. Physiol. 2002, 64: 223-262.
[19]
(Non-Patent Document 4) Mar. Biol. 1991, 108: 157-166.
[20]
(Non-Patent Document 5) Journal of Photochemistry and Photobiology B: Biology. 2015, 142: 154-168
[21]
(Non-Patent Document 6) Biol. Rev. 1999, 74: 311-345.
[22]
(Non-Patent Document 7) Mol. Biol. Evol. 2006, 23: 1437-1443.
[23]
(Non-Patent Document 8) Science, 2010, 329: 1653-1656.
[24]
(Non-Patent Document 9) Genomics 2010, 95: 120-128.
[25]
(Non-Patent Document 10) Geomicrobiol. J. 1997. 14: 231-241.
[26]
(Non-Patent Document 11) Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2007. 146: 60-78.
[27]
(Non-Patent Document 12) Can. J. Bot. 2003. 81: 131-138.
[28]
(Non-Patent Document 13) J. Photochem. Photobiol. B. 2007, 89: 29-35.
[29]
(Non-Patent Document 14) J. Bacteriol. 2011. 193(21): 5923-5928.
[30]
(Non-Patent Document 15) Planta Med. 2015. 81: 813-820
[31]
(Non-Patent Document 16) ACS Appl. Mater. Interfaces. 2015. 7: 16558-16564
[32]
(Non-Patent Document 17) Appl Environ Microbiol. 2016, 82(20): 6167-6173
[33]
(Non-Patent Document 18) ChemBioChem. 2015, 16: 320-327
[34]
(Non-Patent Document 19) Methods Mol Biol. 2013, 1073: 43-7
[35]
(Non-Patent Document 20) Enzyme Microb Technol., 2016, Jan, 82: 96-104
[36]
(Non-Patent Document 21) Nature Review, 2011, 9: 791-802
[37]
Detailed description of the invention
Technical challenge
[38]
As a result of the present inventors' diligent efforts to increase the production of MAAs in microorganisms, the present inventors have conducted various studies to inactivate the activity of 3-dehydroquinate dehydratase protein in microorganisms that produce MAAs. By confirming that the production was increased, the present invention was completed.
[39]
Means of solving the task
[40]
One object of the present application is to provide a microorganism that produces a mycosporine-like amino acid whose activity of 3-dehydroquinate dehydratase protein is inactivated compared to an unmodified microorganism.
[41]
Another object of the present application is to cultivate the microorganism; And recovering mycosporine-like amino acids from the cultured microorganism or medium.
[42]
Effects of the Invention
[43]
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.
[44]
Best mode for carrying out the invention
[45]
Hereinafter, the present application will be described in more detail.
[46]
Meanwhile, each description and embodiment disclosed in the present application 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.
[47]
[48]
One aspect of the present application for achieving the above object is a microorganism producing a mycosporine-like amino acid, in which the 3-dehydroquinate dehydratase protein activity is inactivated compared to the unmodified microorganism. to provide.
[49]
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.
[50]
[Reaction Scheme]
[51]
3-dehydroquinate 3-dehydroshikimate + H 2 O
[52]
[53]
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; No protein is 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 the expression of the gene encoding the enzyme or inhibition of translation, or the like; Part or all of the gene encoding the enzyme is deleted; And a combination thereof, and is not limited thereto. "Unmodified microorganism" refers to a microorganism that has the activity of a specific protein originally possessed by the parent strain before the transformation when a specific protein of a microorganism to be compared is genetically mutated due to a natural or artificial factor. Say. In the present application, "unmodified microorganism" may be used interchangeably with "a microorganism having intrinsic activity" in which genetic variation does not occur.
[54]
[55]
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.
[56]
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 is 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.
[57]
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.
[58]
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 of the gene sequence, or a combination thereof 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.
[59]
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.
[60]
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.
[61]
[62]
In addition, the microorganisms of the present invention include 2-dihydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase, and transketolase ( transketolase I/II), and 3-dehydroquinate synthase (3-dehydroquinate synthase) at least one activity, specifically, at least 1, at least 2, at least 3, or the activity of all enzymes is unmodified It can be fortified compared to microorganisms.
[63]
[64]
The 2-dehydro-3-deoxyphosphoheptonate aldolase (2-dehydro-3-deoxyphosphoheptonate aldolase) refers to an enzyme that catalyzes the reversible reaction of the following reaction formula, specifically, 3-deoxy-ara It is possible to synthesize 3-deoxy-arabino-heptulosonate 7-phosphate, but is not limited thereto.
[65]
[Reaction Scheme]
[66]
phosphoenolpyruvate + D-erythrose-4-phosphate + H2O 3-deoxy-D-arabino-heptulosonate-7-phosphate + phosphate
[67]
[68]
The phosphoenolpyruvate synthetase (phosphoenolpyruvate synthetase) refers to an enzyme that catalyzes the reversible reaction of the following reaction formula, and specifically, phosphoenolpyruvate may be synthesized, but is not limited thereto.
[69]
[Reaction Scheme]
[70]
ATP + pyruvate + H2O AMP + phosphoenolpyruvate + phosphate
[71]
[72]
The transketolase (transketolase I/II) refers to an enzyme that catalyzes the reversible reaction of the following scheme.
[73]
[Reaction Scheme]
[74]
Sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate = D-ribose 5-phosphate + D-xylulose 5-phosphate
[75]
[76]
The 3-dehydroquinate synthase (3-dehydroquinate synthase) means 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.
[77]
[Reaction Scheme]
[78]
3-deoxy-arabino-heptulosonate 7-phosphate 3-dehydroquinate + phosphate
[79]
[80]
As used herein, the term "enhancing activity" means that the activity of the enzyme protein is introduced, or the activity is improved compared to the intrinsic activity of the microorganism or the activity before modification. "Introduction" of the above activity means that the activity of a specific protein that the microorganism originally did not have is naturally or artificially manifested. For example, the activity enhancement is foreign 2-dihydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase, transketolase and/or 3-dihydroquinate synthase. To strengthen by introducing; Or all that enhance the activity of intrinsic 2-dihydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase, transketolase and/or 3-dihydroquinate synthase. can do. Specifically, as a method of enhancing activity herein,
[81]
1) increase the copy number of the polynucleotide encoding the enzymes,
[82]
2) modification of the expression control sequence to increase the expression of the polynucleotide,
[83]
3) modification of the polynucleotide sequence on the chromosome to enhance the activity of the enzymes, or
[84]
4) It may be performed by a method of transforming to be strengthened by a combination thereof, but is not limited thereto.
[85]
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 may be expressed in a host cell, thereby generating an enzyme, thereby increasing its activity.
[86]
Next, 2) modification of the expression control sequence to increase the 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 the termination of transcription and translation, and the like.
[87]
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 is operably linked to the lysCP1 promoter (WO2009/096689) or the CJ7 promoter (WO2006/065095), which is a promoter derived from the genus Corynebacterium, and can improve the expression rate of the polynucleotide encoding the enzyme. Not limited.
[88]
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 an improved polynucleotide sequence to have a stronger activity.
[89]
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 a foreign polynucleotide or a codon-optimized variant polynucleotide thereof exhibiting the activity of the enzyme may be performed by applying one or more methods together.
[90]
As used herein, the term "vector" refers to a DNA preparation containing the nucleotide 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 a suitable host cell and then replicated or function independently of the host genome, and can be integrated into the genome itself.
[91]
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 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 vectors, etc. may be used, but are not limited thereto.
[92]
The vector usable herein is not particularly limited, and a known expression vector may be used. In addition, a polynucleotide encoding a protein of interest 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, 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, that is, to confirm the insertion of a nucleic acid molecule of interest, and impart a selectable phenotype such as drug resistance, nutrient 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.
[93]
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. Transformed polynucleotides 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 in any form as long as it can be introduced into a host cell and 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 can be performed by selecting a suitable standard technique as known in the art according to the host cell. For example, electroporation, calcium phosphate (CaPO 4) Precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method, but are not limited thereto.
[94]
In addition, the term "operably linked" in the above means that a promoter sequence for initiating and mediating transcription of a polynucleotide encoding a target protein of the present application is functionally linked to the polynucleotide sequence. 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.
[95]
[96]
The 3-dihydroquinate dihydratase, 2-dihydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase, transketolase and 3-dihydroquinate synthase Genetic information of can be obtained from a known database, for example, GenBank of the National Center for Biotechnology Information (NCBI), but is not limited thereto.
[97]
The 3-dihydroquinate dihydratase, 2-dihydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase, transketolase, and 3-dihydroquinate synthase 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.
[98]
Specifically, the 3-dihydroquinate dehydratase may be a protein comprising the amino acid sequence of SEQ ID NO: 72 or 80, and 2-dihydro-3-deoxyphosphoheptonate aldolase, phospho Enolpyruvate synthetase, transketolase, and 3-dihydroquinate synthase may be proteins including amino acid sequences of SEQ ID NOs: 74, 76, 78, and 84, respectively, but are not limited thereto. In the present application, "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".
[99]
In addition, in the present application, the enzymes are 80% or more, specifically 90% or more, more specifically 95% or more of the amino acid sequence, as well as the sequence number described, as long as they have the same or corresponding biological activity as each enzyme, More specifically, it may include a protein exhibiting 99% or more homology.
[100]
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 to the above sequence, some sequences have a deleted, modified, substituted, or added amino acid sequence. It is obvious that it is included in the scope of the application.
[101]
3-dihydroquinate dihydratase, 2-dihydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase, transketolase, and 3-dihydroquinate synthetase of the present application The polynucleotide encoding the agent is 80% or more, specifically 90% or more, more specifically 95% or more of the amino acid sequence of the described sequence number or the sequence as long as it has the same biological activity as the respective enzymes. , More specifically, it may include a polynucleotide encoding a protein exhibiting 99% or more homology.
[102]
In addition, polynucleotides encoding 2-dihydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase, transketolase, and 3-dihydroquinate synthase are codon degenerate. In consideration of codons preferred in organisms to express the protein due to (degeneracy), various modifications can be made to the coding region within a range that does not change the amino acid sequence of the protein expressed from the coding region. Accordingly, the polynucleotide may be included without limitation as long as it is a polynucleotide sequence encoding each enzyme protein.
[103]
In addition, a probe that can be prepared from a known gene sequence, for example, a complementary sequence for all or a part of the polynucleotide sequence and hydride under stringent conditions, the 3-dihydroquinate dehydratase, If a sequence encoding a protein having the activity of 2-dihydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase, transketolase and 3-dihydroquinate synthase enzyme protein Can be included without limitation.
[104]
As used herein, the term “homology” refers to the degree to which a given amino acid sequence or nucleotide sequence matches and can 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 indicated as "% homology". 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). As used herein, 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).
[105]
[106]
As used herein, the term "mycosporine-like amino acids (MAAs)" refers to a cyclic compound that absorbs ultraviolet rays. Herein, the mycosporine-like amino acid is not limited as long as it can absorb ultraviolet rays, but specifically, a compound having a central ring of cyclohexenone or cyclohexenimine; Alternatively, 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-methylamine-threonine, Mycosporine-glycine-valine, Palythine, Asterina-330 (Asterina-330), Shinorine, Porphyra-334, Euhalotes-362, Mycosporine-glycine, Mycosporine-Ornithine -ornithine), mycosporine-lysine, mycosporine-glutamic acid-glycine, mycosporine-methylamine-serine, mycosporine-taurine -taurine), Palythene,
[107]
Mycosporine-like amino acids herein may be used interchangeably with MAA and MAAs.
[108]
[109]
As used herein, the term "a microorganism producing a mycosporine-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. In addition, as used herein, the term "mycosporin-like amino acid biosynthesis gene" refers to a gene encoding an enzyme involved in mycosporin-like amino acid biosynthesis, and includes clusters of the genes. 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.
[110]
The mycosporine-like amino acid biosynthesis gene is not limited to the microorganism 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 acid. , 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 ), etc., but is not limited thereto.
[111]
According to an embodiment, a microorganism producing a mycosporine-like amino acid of the present application includes a mycosporine-like amino acid biosynthesis gene.
[112]
Specifically, 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 CN ligase; Or it may include a gene encoding an enzyme protein having the same and/or similar activity.
[113]
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.
[114]
In addition, a microorganism producing a 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 (non-ribosomal peptide synthetase: NRPS), non-ribosomal peptide synthetase-like enzyme (NRPS-like enzyme) and D-alanine D-alanine ligase (D-Ala D -Ala ligase: DDL) at least one selected from the group consisting of, specifically, at least 1, at least 2, at least 3, or all enzyme proteins; Or it may include a gene encoding an enzyme protein having the same and/or similar activity. 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-ribosome peptide synthetase-like enzyme, and D-alanine D-alanine ligase may attach a second amino acid residue to mycosporine-glycine.
[115]
According to an embodiment, a microorganism producing a 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 derived microorganism species.
[116]
For example, non-ribosome peptide synthetase-like enzymes (Ava_3855) in Anabaena variabilis or D-alanine D-alanine ligase (NpF5597) in Nostoc punctiforme are mycotic. 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 may 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.
[117]
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 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 microbial species or sequence homology 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.
[118]
[119]
In addition, the mycosporine-like amino acid biosynthesis gene may be a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NOs: 2, 4, 86, 88, 90, 92, 94, 96, 98, 100, 102, or 104. , Is not limited thereto.
[120]
In addition, the mycosporine-like amino acid biosynthesis gene is 50%, 60%, or 70% or more of the amino acid sequence of SEQ ID NO: 2, 4, 86, 88, 90, 92, 94, 96, 98, 100, 102, or 104, Specifically, it may include a nucleotide sequence encoding a protein comprising an amino acid sequence having homology of 80% or more, more specifically 90% or more, even more specifically 95% or more, and even more specifically 99% or more. In addition, as long as the microorganism can produce mycosporine-like amino acids, a nucleotide sequence encoding a protein out of the homology may be included without limitation. Specifically, the mycosporine-like amino acid biosynthesis gene may include the nucleotide sequence of SEQ ID NOs: 1, 3, 85, 84, 89, 91, 93, 95, 97, 99, 101, 103, but is not limited thereto.
[121]
In addition, if an amino acid sequence having biological activity substantially identical to or corresponding to the protein of the sequence number described as a sequence having homology with the above sequence, some sequences have a deleted, modified, substituted or added amino acid sequence. It is obvious to be included in the category of.
[122]
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.
[123]
Or, a probe that can be prepared from a known gene sequence, for example, a sequence encoding a protein involved in the biosynthesis of mycosporine-like amino acids by hydride under stringent conditions with a complementary sequence for all or part of the nucleotide sequence. If so, it may be included without limitation herein.
[124]
[125]
According to an embodiment, a microorganism producing a mycosporin-like amino acid may include genes for biosynthetic mycosporin-like amino acids that are different from each other.
[126]
[127]
In the present application, the inactivation of the protein, the enhancement of the activity of the protein and/or the introduction of the gene may be performed simultaneously, sequentially, or in the reverse order regardless of the order.
[128]
[129]
As used herein, the term "a microorganism producing a mycosporin-like amino acid" has the mycosporin-like amino acid biosynthetic gene internally and/or externally introduced to produce a mycosporin-like amino acid, and additionally, the endogenous 3- By inactivating dihydroquinate dihydratase activity, it may be a microorganism having an increased ability to produce mycosporine-like amino acids. The introduction of the mycosporine-like amino acid biosynthesis gene and the inactivation of 3-dihydroquinate dihydratase may be performed simultaneously, sequentially, or in reverse order regardless of the order.
[130]
In addition, the microorganism of the present application is a natural type microorganism originally having the mycosporine-like amino acid biosynthesis gene; And a heterogeneous and/or homogeneous mycosporine-like amino acid biosynthetic gene may be introduced, but is not limited thereto.
[131]
In addition, the microorganism of the present application may be a microorganism in which the activity of an enzyme encoded by a gene related to mycosporine-like amino acid biosynthesis is enhanced, but is not limited thereto.
[132]
In addition, the microorganism of the present application is not limited as long as it has 3-dihydroquinate dihydratase protein activity before modification, specifically, it may be a microorganism of the genus Corynebacterium, a microorganism of the genus Escherichia, or a yeast.
[133]
The microorganisms of the genus Corynebacterium are specifically, Corynebacterium glutamicum , Corynebacterium ammoniagenes , Brevibacterium lactofermentum , Brevibacterium Solarium Plastic pan ( Brevibacterium flavu m), Corynebacterium thermo amino to Ness ( Corynebacterium thermoaminogenes ), Corynebacterium epi syeonseu ( Corynebacterium efficiens , and the like), more specifically, Corynebacterium can however kumil , Is not limited thereto.
[134]
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 vulneris may be, but is not limited thereto.
[135]
Specifically, the yeast is a number of microorganisms belonging to the Ascomycota, Saccharomycotina, Taphrinomycotina, or Basidiomycota, Agaricomycotina, Pucciniomycotina, etc. And, more specifically, Saccharomyces genus microorganism, Schizosaccharomyces genus microorganism, Papia genus microorganism, Kluyveromyces genus microorganism, Pichia genus microorganism, It may be a microorganism of the genus Candida , and more specifically, it may be Saccharomyces cerevisiae, but is not limited thereto.
[136]
[137]
In the present invention, the yeast producing the mycosporine-like amino acid may be introduced with a gene encoding the 3-dihydroquinate synthase or the 3-dihydroquinate synthase activity may be enhanced. For example, in the case of partially or all deletion of ARO1 to inactivate 3-dihydroquinate dihydratase activity in the yeast , 3-dehydroquinate synthase enzyme function is lost. It can be difficult to synthesize 3-DHQ. Therefore, when the ARO1 gene in yeast is partially or completely deleted, a gene encoding 3-dihydroquinate synthase (eg, aroB gene) may be introduced, but is not limited thereto.
[138]
[139]
Another aspect of the present application is culturing the microorganism of the present application; And it provides a method for producing a mycosporine-like amino acid comprising the step of recovering the mycosporine-like amino acid from the cultured microorganism or medium.
[140]
“Microbial” and “mycosporine-like amino acids” are as described above.
[141]
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, but specifically, the microorganisms of the present application are suitable carbon sources, nitrogen sources, personnel, inorganic compounds, 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 may be maintained at 20 to 45, specifically 25 to 40, and may be cultured for about 10 to 160 hours, but is not limited thereto. Also,
[142]
In addition, the culture medium used is a carbon source such as sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molase, starch and cellulose), fats and fats (e.g., 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 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.
[143]
MAAs produced by the culture may be secreted into the medium or may remain in the cells.
[144]
As used herein, the term "medium" refers to a product obtained after culturing the microorganism of the present application. The medium is a concept including both a form containing microorganisms and a form in which microorganisms are removed by centrifugation or filtration from a culture solution containing the microorganisms.
[145]
[146]
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 culturing 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.
[147]
Mode for carrying out the invention
[148]
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.
[149]
[150]
< Production of recombinant microorganisms producing MAAs derived from E. coli and production of MAAs using the same >
[151]
[152]
Example 1: Construction of a vector overexpressing a microalgal-derived sinorin biosynthesis gene
[153]
A. variabilis- based synorin biosynthesis gene cluster is 2-dimethyl 4-deoxygadusol synthase, O-methyltransferase, CN ligase , And non-ribosomal peptide synthetase (non-ribosomal peptide synthetase) consists of four genes, a kind of blue-green algae Nostoc punctiforme can also be used to produce sinorine . Amps . variabilis ATCC29413 and N. punctiforme ATCC29133 genomic DNA was used to identify the synorin biosynthetic gene cluster. Using two vectors pECCG 117_Ptrc_GFP_terminator, and pECCG 117_Pcj1_GFP_terminator, A. variabilis ATCC29413 and N. punctiforme Four vectors were constructed each containing the synorin biosynthesis genes (Ava_ABCD and Npr_ABCD) derived from ATCC29133. The names of the four types of sinorine biosynthetic gene expression vectors and respective templates and primers used to construct the vector are shown in Table 1 below.
[154]
[Table 1]
[155]
[156]
After obtaining a gene fragment using the template and primer, each of the gene fragments were bound to pECCG 117_Ptrc_GFP_terminator treated with EcoRV/XbaI restriction enzyme, and pECCG 117_Pcj1_GFP_terminator vector using In-Fusion R HD cloning kit (clontech) ( ligation). Each expression vector is referred to as pECCG117_Ptrc_Ava_ABCD, pECCG117_Pcj1_Ava_ABCD, pECCG117_Ptrc_Npr_ABCD, and pECCG117_Pcj1_Npr_ABCD. Each expression vector was all confirmed by sequencing. The nucleotide and amino acid sequences of Ava_ABCD and Npr_ABCD are specified in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.
[157]
[158]
Example 2: Shino Lin biosynthesis of a gene over-expression vector is introduced strain Sino Lin producing ability evaluation
[159]
In order to confirm the MAAs-producing ability in E. coli, the four plasmids prepared in Example 1 were introduced into the wild-type E. coli W3110 strain to produce a strain enhancing the biosynthesis of sinorine. The thus-produced strain was plated on LB solid medium containing kanamycine, and then cultured overnight in an incubator at 37°C. 25 ml titer medium of the strain cultured overnight in LB solid medium [Medium 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 inoculation, it was incubated for 48 hours in a 37°C, 200 rpm incubator, and the results are shown in [Table 2].
[160]
[Table 2]
Strain name OD (600nm) Sinorine concentration (mg/L)
W3110 22.3 -
W3110/ pECCG117_Ptrc_Ava_ABCD 20.1 121
W3110/ pECCG117_PCJ1_Ava_ABCD 19.8 382
W3110/ pECCG117_Ptrc_Npr_ABCD 21.0 96
W3110/ pECCG117_PCJ1_Npr_ABCD 20.2 332
[161]
As shown in [Table 2], it was confirmed that when the sinorin biosynthesis gene was introduced into W3110, it was possible to produce sinorine. In addition, by increasing the strength of the promoter (introducing the promoter PCJ1), it was confirmed that the amount of synorin production increased through the enhancement of the biosynthetic pathway.
[162]
[163]
Example 3: 3 - to hydroquinone carbonate having hydrazide hydratase (3- dehydroquinate dehydratase) is an inert production strain
[164]
Ava-A, the first gene of MAAs biosynthesis in microalgae, shares DHQ (3-dehydroquinate) in the shikimate pathway and SH-7P (sedoheptulose 7-phosphate) in the Pentose Phosphate pathway and is used as a substrate. In order to prepare a strain in which the aroD gene was deleted and 3-dehydroquinate dehydratase was inactivated, a homologous recombination method using lambda red recombinase was used. The chloramphenicol resistance gene of pKD3 was used as the 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 PCR using primers of SEQ ID NOs: 9 (forward) and 10 (reverse). Was produced through. After preparing a strain (wild-type Escherichia coli W3110) to delete the aroD gene (SEQ ID NOs: 71 and 72), transforming the pKD46 plasmid containing the lambda red recombinase gene into the strain, and inducing the expression of the gene using arabinose Thus, a competent cell was produced. AroD in the competent cells After the deletion cassette was introduced using an electroporation method, it was plated on an LB solid medium containing 30 mg/L of chloramphenicol. The thus obtained strain was PCR using primers of SEQ ID NOs: 11 (forward) and 12 (reverse), and aroD gene deletion was confirmed through observation of a 1300bp amplified fragment .
[165]
[166]
Example 4: 3 - to hydroquinone carbonate having hydrazide I other inert strains Shino Lin producing ability evaluation
[167]
In the strain in which the aroD gene was deleted prepared in Example 3, two plasmids whose expression was regulated by the PCJ1 promoter among the four plasmids prepared in Example 1 were introduced, and then into LB solid medium containing kanamycin. Smeared (W3110ㅿ aroD / pECCG117_PCJ1_Ava_ABCD, and W3110ㅿ aroD /pECCG117_PCJ1_Npr_ABCD). aroD is deficient strain and aroD After overnight culture the strains, respectively is not deficient in a 37 ℃ incubator, 25 mL titer medium [medium 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 2O 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] by inoculating one platinum at 37°C and 200 rpm. Incubated for 48 hours in an incubator, and the results are shown in [Table 3].　
[168]
[Table 3]
Strain name OD (600nm) Sinorine concentration (mg/L)
W3110/ pECCG117_PCJ1_Ava_ABCD 20.3 352
W3110ㅿ aroD / pECCG117_PCJ1_Ava_ABCD 18.7 683
W3110/ pECCG117_PCJ1_Npr_ABCD 18.9 331
W3110ㅿ aroD / pECCG117_PCJ1_Npr_ABCD 17.9 601
[169]
The table 3, as shown in aroD the Sino lean concentration produced in the deletion strain, aroD were each increased 194%, and 182% compared to the levels produced in the Sino Lynn strain is not deficient. The aroD -deficient strain, the W3110ㅿ aroD / pECCG117_PCJ1_Ava_ABCD strain, and the W3110ㅿ aroD /pECCG117_PCJ1_Npr_ABCD strain, were named CB06-0017 and CB06-0018, respectively, and under the Treaty of Budapest (Korean Microbiology Conservation Center as of June 26, 2017. Center of Microorganisms, KCCM), and were given accession numbers KCCM12044P and KCCM12045P, respectively, in the order described above.
[170]
[171]
Example 5: 2 - dehydro- 3- deoxyphosphoheptonate aldolase / phosphoenol pyruvate synthetase / transketolase I/II activity enhancing strain production
[172]
In order to increase the MAAs-producing ability of the MAAs-producing microorganism, the activity of 2-dehydro-3-deoxyphosphoheptonate aldolase/phosphoenol pyruvate synthetase/transketolase I/II was enhanced. Specifically, three kinds of genes derived from Escherichia coli W3110 aroG (2-dehydro-3-deoxyphosphoheptonate aldolase; SEQ ID NO: 73 and 74), ppsA (phosphoenolpyruvate synthetase; SEQ ID NO: 75 and 76), tktA (transketolase I/II; SEQ ID NO: 77 and 78) was further introduced. To enhance the aroG , ppsA , and tktA genes, the pSKH130-ㅿfhuA- Pn- aroG -Pn- ppsA -Pn- tktA plasmid was constructed. The pSKH130-ㅿfhuA- Pn- aroG -Pn- ppsA -Pn- tktA Templates and primers used in the construction of the plasmid are shown in Table 4 below.
[173]
[Table 4]
[174]
Using the above template and primers, aroG , ppsA , and tktA gene fragments were amplified through PCR, and then introduced into pSKH130-fhuA vectors digested with BamH1-Pst1 restriction enzymes, respectively. After confirming the cloning of the vector and the gene sequence of the vector through sequencing, the wild-type E. coli W3110 and aroD -deficient E. coli W3110ㅿ aroD strain were transformed 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 E. coli transformant strains having completed the secondary recombination were confirmed to introduce aroG , ppsA, and tktA genes using primers of SEQ ID NOs: 19 (forward) and 20 (reverse) .
[175]
[176]
Example 6: 2 - dehydro -3- deoxyphosphoheptonate aldolase / phosphoenol pyruvate synthetase / transketolase activity enhanced strain of the I / II Shino Lin producing ability evaluation
[177]
To the aroG , ppsA, and tktA gene-transducing strain prepared in Example 5, two plasmids whose expression was regulated by the PCJ1 promoter among the four produced in Example 1 were introduced, respectively, and then plated on LB solid medium. The strain was cultured overnight in a 37° C. incubator, and then one platinum was inoculated into the 25 mL titer medium of Example 4 and cultured for 48 hours in an incubator at 37° C. and 200 rpm, and the results are shown in [Table 5]. Done.　
[178]
[Table 5]
Strain name OD (600nm) Sinorine concentration (mg/L)
W3110 / pECCG117_PCJ1_Ava_ABCD 19.8 352
W3110 / pECCG117_PCJ1_Npr_ABCD 19.6 344
W3110ㅿ aroD / pECCG117_PCJ1_Ava_ABCD 17.3 688
W3110ㅿ aroD / pECCG117_PCJ1_Npr_ABCD 17.8 652
W3110ㅿfhuA:: Pn- aroG -Pn- ppsA -pn- tktA /pECCG117_PCJ1_Ava_ABCD 18.9 1163
W3110ㅿfhuA:: Pn- aroG -Pn- ppsA -pn- tktA /pECCG117_PCJ1_Npr_ABCD 18.6 989
W3110ㅿ aroD ㅿfhuA:: Pn- aroG -Pn- ppsA -pn- tktA /pECCG117_PCJ1_Ava_ABCD 17.3 1928
W3110ㅿ aroD ㅿfhuA:: Pn- aroG -Pn- ppsA -pn- tktA /pECCG117_PCJ1_Npr_ABCD 17.7 1889
[179]
As shown in [Table 5] , the concentration of synorin produced in the strain enriched with three genes ( aroG , ppsA , tktA ) increased by about 300% compared to the control group.
[180]
[181]
Example 7: ava _ ABCD chromosome insertion vector and strain construction
[182]
In order to introduce the synorin biosynthesis gene into E. coli, a pSKH130ㅿpinR::Ava-ABCD plasmid was constructed. Using pECCG117_Ptrc_Ava_ABCD as a template, Ava_ABCD was PCR performed using a primer pair of SEQ ID NO: 21 (forward) and 22 (reverse). A PCR fragment of about 7 kb was ligated to pSKH130pinR vector treated with BamHI and PstI restriction enzymes infusion (clontech) to produce pSKH130pinR::Ava_ABCD . Then, to control the expression amount of Ava-ABCD, Ptrc, PCJ1, and promoter fragments were PCR, respectively, with forward and reverse primer pairs of SEQ ID NOs: 23 and 24, SEQ ID NOs: 25 and 26, and SEQ ID NOs: 25 and 27. Restriction enzyme-treated pSKH130pinR::Ava_ABCD vector was ligated with Infusion (clontech) to produce pSKH130pinR::Ptrc-Ava-ABCD, pSKH130pinR::PCJ1-Ava-ABCD. W3110 ㅿ aroD ㅿfhuA:: Pn- aroG -Pn- ppsA -Pn- tktA prepared in Example 5 of the above recombinant plasmid The strain was transformed by electroporation and introduced into the chromosome by primary recombination (crossover), and then the vector part except the target gene was excised from the chromosome through secondary recombination (crossover).
[183]
The introduction of the Ava_ABCD gene was confirmed by PCR using the primers of SEQ ID NOs: 28 (forward) and 29 (reverse) targeting the E. coli transformed lines for which the secondary recombination was completed .
[184]
[185]
Example 8: ava _ ABCD of chromosomal insertion strains Shino Lin producing ability evaluation
[186]
The strain prepared in Example 7 was plated on LB solid medium, and then cultured overnight in an incubator at 37°C. Thereafter, one platinum was inoculated into the 25 mL titer medium of Example 4, and then cultured for 48 hours in an incubator at 37° C. and 200 rpm, and the results are shown in [Table 6].　
[187]
[Table 6]
Strain name OD (600nm) Sinorine concentration (mg/L)
W3110ㅿ aroD ㅿfhuA:: Pn- aroG -Pn- ppsA -Pn- tktA 18.5 -
W3110ㅿ aroD ㅿfhuA:: Pn- aroG -Pn- ppsA -Pn- tktA /pECCG117_PCJ1_Ava_ABCD 17.8 1928
W3110ㅿ aroD ㅿfhuA:: Pn- aroG -Pn- ppsA -Pn- tktA ㅿpinR::Ptrc-Ava-ABCD 18.2 483
W3110ㅿ aroD ㅿfhuA:: Pn- aroG -Pn- ppsA -Pn- tktA ㅿpinR::PCJ1-Ava-ABCD 17.9 832
[188]
As shown in [Table 6], when Ava-ABCD was introduced onto the chromosome, it was confirmed that synorin was produced, and the concentration increased with the strength of the promoter. However, it was confirmed that the amount of synorin production decreased compared to the strain whose biosynthesis was enhanced with the plasmid. When the pECCG117_PCJ1_Ava_ABCD plasmid was additionally introduced into the strain into which Ava-ABCD was introduced on the chromosome, it was observed that the amount of synorin production increased by 353% and 152%, respectively, compared to the strain introduced only into the chromosome (based on the CJ1 promoter) and the strain into which only the plasmid was introduced. .
[189]
[190]
Example 9: MAAs gene overexpression vector construction and MAAs production ability evaluation
[191]
4-Deoxygadusol and Mycosporine glycine are MAAs that are intermediate products produced in the process of synorin biosynthesis and have UV-blocking effects. A vector was constructed to confirm whether these substances can be produced in a strain in which E. coli AroD is defective, which is shown in [Table 7].
[192]
Using pECCG117_Ptrc_Ava_ABCD as a template, Ptrc_Ava_AB and Ptrc_Ava_ABC were PCR using primer pairs of SEQ ID NOs: 30 and 31 and SEQ ID NOs: 30 and 32. The PCR fragment was ligated to pECCG117 Prc GFP vector treated with BamHI and SpeI restriction enzymes to produce pECCG117_Ptrc_Ava_AB and pECCG117_Ptrc_Ava_ABC. In the same way, a PCR fragment using pECCG117_PCJ1_Ava_ABCD as a template and primer pairs of SEQ ID NOs: 30 and 31 and SEQ ID NOs: 30 and 32 was ligated to the pECCG117 Pcj1 GFP vector treated with BamHI and SpeI restriction enzymes, and pECCG117_PCJ1_Ava_AB and pECCG117_PCJ1_Ava_PC117 Was produced. The nucleotide sequence and amino acid sequence of Ava_AB and Ava_ABC are shown in SEQ ID NOs: 85 to 88.
[193]
[Table 7]
[194]
The resulting vector was transformed by the electric pulse method commonly used in the W3110ㅿ aroD ㅿfhuA:: Pn- aroG -Pn- ppsA -Pn- tktA strain used in Example 8, and each strain was transformed into LB solid medium. After smearing, it was incubated overnight in an incubator at 37°C. The strain cultured overnight in LB solid medium was inoculated into the 25 mL titer medium of Example 4, and then cultured for 48 hours in an incubator at a temperature of 37° C. and 200 rpm, and after completion of the culture, liquid high-speed chromatography was performed. The production amount of MAAs was measured using, and the concentration of MAAs in the culture medium for each strain tested is shown in Table 8 below.
[195]
[Table 8]
Strain name OD (600nm) 4-Deoxygadusol concentration (mg/L) Mycosporine glycine concentration (mg/L)
W3110ㅿ aroD ㅿfhuA:: Pn- aroG -Pn- ppsA -Pn- tktA 19.2 - -
W3110ㅿ aroD ㅿfhuA:: Pn- aroG -Pn- ppsA -Pn- tktA / pECCG117_Ptrc_Ava_AB 18.2 12.0 -
W3110ㅿ aroD ㅿfhuA:: Pn- aroG -Pn- ppsA -Pn- tktA / pECCG117_Pcj1_Ava_AB 17.6 25.3 -
W3110ㅿ aroD ㅿfhuA:: Pn- aroG -Pn- ppsA -Pn- tktA / pECCG117_Ptrc_Ava_ABC 18.7 2.0 9.3
W3110ㅿ aroD ㅿfhuA:: Pn- aroG -Pn- ppsA -Pn- tktA / pECCG117_Pcj1_Ava_ABC 18.1 2.9 19.7
[196]
As shown in [Table 8] above, when the Ava_AB and Ava_ABC genes were introduced, it was confirmed that 4-Deoxygadusol and Mycosporine glycine were produced, and the amount was also increased as the promoter strength was strengthened.
[197]
[198]
< Corynebacterium glutamicum - derived MAAs production recombinant microorganism production and MAAs production using the same >
[199]
[200]
Example 10: Sinorin biosynthetic gene overexpression Vector-introduced strain of synorin production ability evaluation
[201]
In order to confirm the ability to produce MAAs in Corynebacterium glutamicum, the four plasmids prepared in Example 1 were introduced into the Corynebacterium glutamicum 13032 strain to create a strain that enhanced synorin biosynthesis and included Kanamycine. After spreading on BHIS solid medium, it was incubated overnight in 30 incubators. 25 mL titer medium of the strain cultured overnight in BHIS solid 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 5 g/L, NaCl 5 g/L, yeast extract 5 g/L, calcium carbonate 30 g/L: pH7.0], and then inoculated each platinum in an incubator at 37°C and 200 rpm for 48 hours And the results are shown in [Table 9].　
[202]
[Table 9]
Strain name OD (600nm) Sinorine concentration (mg/L)
c.gl 13032 72.1 -
c.gl 13032/ pECCG117_Ptrc_Ava_ABCD 71.5 132
c.gl 13032/ pECCG117_PCJ1_Ava_ABCD 69.8 496
c.gl 13032/ pECCG117_Ptrc_Npr_ABCD 70.9 103
c.gl 13032/ pECCG117_PCJ1_Npr_ABCD 71.4 421
[203]
As shown in [Table 9], it was confirmed that when the synorin biosynthesis gene was introduced into Corynebacterium glutamicum 13032, it was confirmed that synorin production was possible, and the production amount could be increased up to 375% depending on the strength of the promoter. I did.
[204]
[205]
Example 11: Construction of synorin biosynthesis gene chromosome introduction vector and strain production
[206]
The pDC ΔN1021_Ava_ABCD plasmid was constructed to introduce the synorin biosynthesis gene into Corynebacterium glutamicum. Using pECCG117_Ptrc_Ava_ABCD as a template, Ava_ABCD was PCR performed using a primer pair of SEQ ID NOs: 33 (forward) and 34 (reverse). A PCR fragment of about 7 kb was infusion (clonteh) ligated to a pDC ΔN1021 vector treated with NdeI restriction enzyme to produce pDC ΔN1021_Ava_ABCD. Then, to control the expression amount of Ava_ABCD, CJ7, Lysc8, and O2 promoter fragments were PCR with the forward and reverse primer pairs of SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, and SEQ ID NOs: 39 and 40, and pDC ΔN1021_Ava_ABCD was NdeI Infusion (clonteh) ligation to the restriction enzyme-treated vector was performed to prepare pDC ΔN1021_Pcj7_Ava_ABCD, pDC ΔN1021_Plysc8_Ava_ABCD, and pDC ΔN1021_PO2_Ava_ABCD.
[207]
The recombinant plasmid was transformed into wild Corynebacterium glutamicum 13032 by electroporation (van der Rest et al. 1999), and the plasmid was introduced into the chromosome by primary recombination (crossover), and 2 Plasmids were excised from chromosomes through secondary recombination (crossover).
[208]
The introduction of the Ava_ABCD gene was confirmed by PCR using each gene-specific primer pair, SEQ ID NO: 33 and 34, targeting the Corynebacterium glutamicum transformants having completed the secondary recombination.
[209]
[210]
Example 12: Evaluation of synorin production ability of the strain introduced with the chromosome of the synorin biosynthesis gene
[211]
In order to confirm the synorin production ability, all strains were plated on BHIS solid medium and cultured overnight in 30 incubators. The strain cultured overnight in BHIS solid medium was inoculated overnight in the 25 mL titer medium of Example 11, and then cultured for 48 hours in an incubator at 37° C. and 200 rpm. The results are shown in [Table 10].
[212]
[Table 10]
Strain name OD (600nm) Sinorine concentration (mg/L)
c.gl 13032 70.2 -
c.gl 13032 △N1021_PCJ7_Ava_ABCD 76.1 36
c.gl 13032 △N1021_Plysc8_Ava_ABCD 79.8 75
c.gl 13032 △N1021_PO2_Ava_ABCD 72.5 173
[213]
As shown in the result of [Table 10], it was confirmed that when 1 copy of the synorin biosynthesis gene was introduced into wild-type Corynebacterium, it could produce up to 36 mg to 173 mg.
[214]
[215]
Example 13: Corynebacterium aroD (3- dehydroquinate dehydratase ) deletion vector and strain construction
[216]
As mentioned in Example 3, a defective strain was prepared to confirm whether the amount of synorin production could be increased through the deletion of aroD (3-dehydroquinate dehydratase). Specific - Corynebacterium glutamicum portion of aroD gene (SEQ ID NO: 79 and 80), deletions in order to produce a strain aroD open reading frame of the (open reading frame) is an internally deleted pDC-Δ aroD prepare a plasmid . The internal gene loss of the pDC-Δ aroD is a gene generated by cross-PCR with a pair of forward and reverse primers of SEQ ID NOs: 41 and 42, and SEQ ID NOs: 43 and 44 using Corynebacterium glutamicum ATCC 13032 genomic DNA as a template. The fragment was constructed by introducing it into a pDC vector. The recombinant plasmid was transformed into Corynebacterium glutamicum 13032 ΔN1021_PO2_Ava_ABCD by electroporation (van der Rest et al. 1999), and the plasmid was introduced into the chromosome by primary recombination (crossover), and 2 Plasmids were excised from chromosomes through secondary recombination (crossover).
[217]
The deletion of the aroD gene was confirmed by PCR using each gene-specific primer pair, SEQ ID NO: 41 and 44, targeting the Corynebacterium glutamicum transformants having completed the secondary recombination .
[218]
[219]
Example 14: Corynebacterium aroD (3- dehydroquinate dehydratase ) defect evaluation
[220]
Corynebacterium glutamicum 13032 ΔN1021_PO2_Ava_ABCD strain of the dihydroquinate dehydratase deficient and expected to accumulate DHQ was plated on BHIS solid medium and cultured overnight in 30 incubators. 25 mL titer medium of the strain cultured overnight in BHIS solid 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 5g/L, yeast extract 5g/L, calcium carbonate 30g/L: pH7.0] was inoculated with one platinum at a time, and then incubated for 48 hours in an incubator at 37°C and 200 rpm. , The results are shown in [Table 11].　
[221]
[Table 11]
Strain name OD (600nm) Sinorine concentration (mg/L)
c.gl 13032 △N1021_PO2_Ava_ABCD 71.3 182
c.gl 13032 △N1021_PO2_Ava_ABCD_△ aroD 74.1 435
c.gl 13032 △N1021_PO2_Ava_ABCD_△ aroD /pECCG117_PCJ1_Ava_ABCD 73.2 1162
[222]
As shown in [Table 11], when aroD was deleted, the concentration of sinorine was improved by 239% compared to the control group, and it was confirmed that the concentration of sinorine increased as the biosynthesis was further enhanced with pECCG117_PCJ1_Ava_ABCD. The aroD -deficient strain, c.gl 13032 N1021_PO2_Ava_ABCD_△aroD, was named CB06-0019, and the deposit number was deposited with the Korean Culture Center of Microorganisms (KCCM) on June 26, 2017 under the Budapest Treaty. Received KCCM12046P.
[223]
[224]
< Production of recombinant microorganisms producing MAAs derived from Yeast and production of MAAs using the same >
[225]
[226]
Example 15: Microalgae-derived sinorine biosynthesis gene overexpression yeast vector construction
[227]
A. variabilis ATCC29413 and N. punctiforme ATCC29133 genomic DNA was used to construct a S. cerevisiae vector into which a synorin biosynthesis gene was introduced . A vector was constructed using the ADH, TEF, and GPD promoters of S. cerevisiae , and each template and primer used to produce a total of 24 types of sinorin biosynthetic gene expression vectors are shown in Table 12 below. The nucleotide sequences and amino acid sequences of Ava_A, Ava_B, Ava_C, Ava_D, Npr_A, Npr_B, Npr_C, and Npr_D are shown in SEQ ID NOs: 89 to 104 in the enzyme order.
[228]
[Table 12]
Vector people Used mold Primer used (forward, reverse)
p413-pADH-Ava_A A. variabilis ATCC29413 genomic DNA SEQ ID NO: 45, SEQ ID NO: 46
p413-pADH-Ava_B SEQ ID NO: 47, SEQ ID NO: 48
p413-pADH-Ava_C SEQ ID NO: 49, SEQ ID NO: 50
p413-pADH-Ava_D SEQ ID NO: 51, SEQ ID NO: 52
p413-pTEF-Ava_A SEQ ID NO: 45, SEQ ID NO: 46
p413-pTEF-Ava_B SEQ ID NO: 47, SEQ ID NO: 48
p413-pTEF-Ava_C SEQ ID NO: 49, SEQ ID NO: 50
p413-pTEF-Ava_D SEQ ID NO: 51, SEQ ID NO: 52
p413-pGPD-Ava_A SEQ ID NO: 45, SEQ ID NO: 46
p413-pGPD-Ava_B SEQ ID NO: 47, SEQ ID NO: 50
p413-pGPD-Ava_C SEQ ID NO: 49, SEQ ID NO: 50
p413-pGPD-Ava_D SEQ ID NO: 51, SEQ ID NO: 52
p413-pADH-Npr_A N. punctiforme ATCC29133 genomic DNA SEQ ID NO: 53, SEQ ID NO: 54
p413-pADH-Npr_B SEQ ID NO: 55, SEQ ID NO: 56
p413-pADH-Npr_C SEQ ID NO: 57, SEQ ID NO: 58
p413-pADH-Npr_D SEQ ID NO: 59, SEQ ID NO: 60
p413-pTEF-Npr_A SEQ ID NO: 53, SEQ ID NO: 54
p413-pTEF-Npr_B SEQ ID NO: 55, SEQ ID NO: 56
p413-pTEF-Npr_C SEQ ID NO: 57, SEQ ID NO: 58
p413-pTEF-Npr_D SEQ ID NO: 59, SEQ ID NO: 60
p413-pGPD-Npr_A SEQ ID NO: 53, SEQ ID NO: 54
p413-pGPD-Npr_B SEQ ID NO: 55, SEQ ID NO: 56
p413-pGPD-Npr_C SEQ ID NO: 57, SEQ ID NO: 58
p413-pGPD-Npr_D SEQ ID NO: 59, SEQ ID NO: 60
[229]
By matching the template and primer combination, the gene fragment obtained through the PCR technique and the p413/414/415/416-pADH/pTEF/pGPD-CYC1_terminator vector treated with BamH1/XhoI restriction enzyme were ligated using T4 ligase enzyme (NEB). , p413/414/415/416-pADH/pTEF/pGPD-A,B,C,D 24 vectors were prepared. The presence or absence of each expression vector and gene sequence were all confirmed by sequencing technique. The prepared expression vector was introduced into the wild-type S.cerevisiae CEN.PK-1D strain to produce a strain capable of producing sinorine.
[230]
[231]
Example 16: Sinorin biosynthetic gene overexpression vector-introduced strain Sinorin production ability evaluation
[232]
In order to confirm the ability to produce MAAs in yeast, the 24 plasmids prepared in Example 15 were introduced into CEN.PK-1D ( S. cerevisiae CEN.PK-1D) strains to Cynolin biosynthesis to Cyclomyces cerevisiae. A strain enhanced with was prepared, and then plated on a SC (synthetic complete) solid medium excluding Leu, Trp, Ura, and His, and cultured overnight in 30 incubators. Thereafter, the overnight cultured strain was inoculated into the 25 mL titer medium of [Table 13], and then inoculated for 24 hours in an incubator at 30 and 150 rpm, and the results are shown in [Table 14].　
[233]
[Table 13]
Composition Use concentration (g/L)
YNB (Yeast nitrogen base) without amino acids 6.7
Amino acid mixtures (without Leucine, Tryptophan, Histidine, Uracil) 2
Glucose 20
[234]
[Table 14]
Plasmid 24hr Sinorine concentration (mg/L)
OD 600 Residual
pADH-Ava_A,B,C,D 11.0 0.0 107
pTEF-Ava_A,B,C,D 11.1 0.0 215
pGPD-Ava_A,B,C,D 11.5 0.0 302
pADH-Npr_A,B,C,D 20.1 0.0 234
pTEF-Npr_A,B,C,D 20.4 0.0 387
pGPD-Npr_A,B,C,D 20.5 0.0 521
[235]
Through the above results, it was confirmed that the activity of the Npr A, B, C, and D genes was higher in CEN.PK-1D in CEN.PK-1D, which is a yeast strain, compared to Ava A, B, C, D. In addition, it was confirmed that the amount of gene expression was regulated according to the strength of the promoter, and the amount of synorin production was changed accordingly. In particular, when introducing Npr A, B, C, and D vectors based on the Glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, it was confirmed that the production of sinorine was the highest at 521 mg/l.
[236]
[237]
Example 17: S. cerevisiae ARO1 deletion and E. coli aroB introduction to increase shinorine production
[238]
In Yeast, in order to confirm that the ability to produce sinorine was improved by inactivation of dehydroquinate dehydratase , the ARO1 gene was deleted from CEN.PK -1D in Cyaromyces cerevisiae . The wrap of the Caro My process serenity busy ARO1 gene is a gene with five functions, ARO1 defect when E. coli aroB the 3-dihydro-quinolyl carbonate synthetase (3-dehydroquinate synthase) enzyme function corresponding to the loss of 3-DHQ Becomes impossible to synthesize. Therefore , after the deletion of the ARO1 gene (SEQ ID NOs: 81 and 82) into the Escherichia coli aroD homologue on the chromosome, the E. coli aroB gene (SEQ ID NOs: 83 and 84) was inserted in the same position based on the GPD promoter . Each of the templates and primers used are shown in Table 15 below. 24 kinds of plasmids prepared in Example 15 were ARO1 deleted and E. coli aroB The gene was introduced, Cyclomyces cerevisiae was introduced into the CEN.PK-1D strain, plated on SC (synthetic complete) solid medium excluding Leu, Trp, Ura, and His, and cultured overnight in 30 incubators. Thereafter, the overnight cultured strain was inoculated into the 25 mL titer medium of [Table 13], and then incubated in an incubator at 30 and 150 rpm for 24 hours, and the results are shown in [Table 16].　
[239]
[Table 15]
Cassette name Amplified DNA template Sequence number (forward, reverse)
ARO1 △ pGPD-aroB cassette pGPD S. cerevisiae gDNA SEQ ID NO: 61, SEQ ID NO: 62
LoxP(Ura) pUG28 vector SEQ ID NO: 63, SEQ ID NO: 64
aroB W3110 gDNA SEQ ID NO: 65, SEQ ID NO: 66
ARO1 Fragment1 S. cerevisiae gDNA SEQ ID NO: 67, SEQ ID NO: 68
ARO1 Fragment2 S. cerevisiae gDNA SEQ ID NO: 69, SEQ ID NO: 70
[240]
[Table 16]
[241]
[242]
Through the above [Table 16], it was confirmed that the amount of synorin production increased by 3 times or more compared to WT in the strain in which the ARO1 was deleted and the E. coli aroB gene was introduced and the 3-DHQ production ability was enhanced. It was additionally confirmed that the activity of the Npr A, B, C, and D genes was higher than that of Ava A, B, C, and D, and it was confirmed that the amount of synorin production increased as the strength of the promoter increased. In particular, when introducing Npr A, B, C, and D vectors based on the GPD promoter, it was confirmed that the production of sinorine was the highest at 1.6 g/l.
[243]
[244]
From the above description, those skilled in the art to which the present application pertains will appreciate that the present application may be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present application should be construed that all changes or modified forms derived from the meaning and scope of the claims to be described later, and equivalent concepts thereof, rather than the detailed description, are included in the scope of the present application.
[245]

[246]
[247]

[248]
[249]

Claims
[Claim 1]
3-dehydroquinate dehydratase (3-dehydroquinate dehydratase) A microorganism that produces a mycosporine-like amino acid whose protein activity is inactivated compared to an unmodified microorganism.
[Claim 2]
The method of claim 1, wherein the microorganism is 2-dimethyl 4-deoxygadusol synthase, O-methyltransferase, and CN ligase. Microorganisms that produce mycosporine-like amino acids, including genes encoding one or more proteins selected from the group consisting of.
[Claim 3]
The method of claim 1, wherein the microorganism is a non-ribosomal peptide synthetase, a non-ribosomal peptide synthetase-like enzyme: NRPS-like enzyme, and D- Alanine D- alanine ligase (D-Ala D-Ala 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 1, wherein the microorganism is additionally 2-dihydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase, Transketolase (transketolase I/II) and 3-dehydroquinate synthase microbe.
[Claim 5]
The microorganism of claim 1, wherein the microorganism is a microorganism of the genus Corynebacterium, a microorganism of the genus Escherichia, or a yeast.
[Claim 6]
The microorganism according to claim 5, wherein the yeast has introduced a gene encoding 3-dehydroquinate synthase.
[Claim 7]
The method of claim 1, wherein the mycosporine-like amino acid is Mycosporine-2-glycine, Palythinol, Palythenic acid, deoxygadusol, Mycosporine-methylamine-threonine, Mycosporine-glycine-valine, Palythine, Asterina-330 (Asterina-330), Sinorine ( Shinorine), Porphyra-334, Euharotes-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 8]
Culturing the microorganism of any one of claims 1 to 7; And recovering mycosporine-like amino acid from the cultured microorganism or medium.