KR102254632B1 - Novel Phytoene desaturase variant and a method for producing IMP using the same - Google Patents

Abstract

The present application relates to a novel phytoene desaturase variant, a Corynebacterium stationis strain containing the variant, and a method for producing 5'-inosine monophosphate (IMP). According to the present invention, it is possible to produce IMP of high yield.

Description

Novel Phytoene desaturase variant and a method for producing IMP using the same}

The present application relates to a novel Phytoene desaturase variant, a Corynebacterium stationis strain including the variant, and a method for producing IMP (5'-inosine monophosphate) using the strain. will be.

In order to produce IMP (5'-inosine monophosphate) and other useful substances, various studies have been conducted to develop highly efficient microorganisms and fermentation process technology. For example, a target substance-specific approach such as increasing the expression of a gene encoding an enzyme involved in IMP biosynthesis or removing a gene unnecessary for biosynthesis is mainly used (EP 3722430 A1, US 2020-0347346 A1).

However, as the demand for IMP increases, research is still needed to increase the production capacity of effective IMP.

One object of the present application is a phytoental, consisting of the amino acid sequence shown in SEQ ID NO: 1, in which Aspartic acid, an amino acid corresponding to the 425th position of the amino acid sequence of SEQ ID NO: 3, is substituted with Asparagine. It is to provide a saturation enzyme (Phytoene desaturase) variant.

Another object of the present application is to provide a polynucleotide encoding the variant of the present application.

Another object of the present application is a variant of the present application or a polynucleotide encoding the variant, and having the ability to produce 5'-inosine monophosphate (IMP), Corynebacterium stationis strain Is to provide.

Another object of the present application is to provide a method for producing IMP, including a variant or a polynucleotide encoding the variant, and culturing a Corynebacterium stasis strain in a medium having an IMP-producing ability. To provide.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present application. In addition, it cannot be considered that the scope of the present application is limited by the specific description described below. In addition, throughout this specification, a number of papers and patent documents are referenced and citations thereof are indicated. The disclosure contents of the cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

In one aspect of the present application, aspartic acid, an amino acid corresponding to the 425th position of the amino acid sequence of SEQ ID NO: 3, is substituted with Asparagine, consisting of the amino acid sequence shown in SEQ ID NO: 1, phytoental Provides a saturating enzyme variant.

Specifically, the variant of the present application may have, include, consist of, or consist essentially of the amino acid sequence described in SEQ ID NO: 1.

In addition, in the variant of the present application, the amino acid corresponding to position 425 based on the amino acid sequence of SEQ ID NO: 3 in the amino acid sequence described in SEQ ID NO: 1 is asparagine, and at least 70%, 75 from the amino acid sequence of SEQ ID NO: 1 %, 425%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more of homology or identity. In addition, if an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. Is self-explanatory.

For example, addition or deletion of a sequence that does not alter the function of the variant of the present application in the amino acid sequence N-terminus, C-terminus and/or inside, a naturally occurring mutation, a silent mutation, or conservation This is the case with enemy substitution.

The "conservative substitution" means the substitution of one amino acid for another amino acid having similar structural and/or chemical properties. Such amino acid substitutions can generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.

In the present application, the term "variant" means that one or more amino acids are conservative substitution and/or modification to be different from the amino acid sequence before mutation of the variant, but functions or properties Refers to the retained polypeptide. Such variants can generally be identified by modifying one or more amino acids of the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide prior to the mutation. In addition, some variants may include variants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other variants may include variants in which a portion of the mature protein has been removed from the N- and/or C-terminus. The term "variant" can be used interchangeably with terms such as variant, modified, variant polypeptide, mutated protein, mutant and variant (in English, modified, modified polypeptide, modified protein, mutant, mutein, divergent, etc.). And, if it is a term used in a mutated meaning, it is not limited thereto. For the purposes of the present application, the variant may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 in which aspartic acid, an amino acid corresponding to position 425 of the amino acid sequence of SEQ ID NO: 3, is substituted with asparagine.

In addition, variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence involved in protein translocation may be conjugated to the N-terminus of the variant co-translationally or post-translationally. In addition, the variant may be conjugated with another sequence or linker so that it can be identified, purified, or synthesized.

In the present application, the term'homology' or'identity' refers to a degree of similarity between two given amino acid sequences or base sequences, and may be expressed as a percentage. The terms homology and identity can often be used interchangeably.

The sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, and the default gap penalty established by the program used can be used together. Substantially homologous or identical sequences are generally capable of hybridizing with all or part of the sequence in medium or high stringent conditions. It is apparent that hybridization also includes hybridization of a polynucleotide with a polynucleotide containing a codon or a codon in consideration of codon degeneracy.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity is determined, for example, in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: Can be determined using a known computer algorithm such as the "FASTA" program using default parameters as in 2444. Alternatively, as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine (GCG program package (Devereux, J., et al, Nucleic Acids). Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop , [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073), for example, BLAST of the National Center for Biotechnology Information, or ClustalW can be used to determine homology, similarity or identity.

The homology, similarity or identity of a polynucleotide or polypeptide is described, for example, in Smith and Waterman, Adv. Appl. As known in Math (1981) 2:482, for example, Needleman et al. (1970), J Mol Biol. It can be determined by comparing sequence information using a GAP computer program such as 48:443. In summary, the GAP program can be defined as the total number of symbols in the shorter of the two sequences, divided by the number of similarly aligned symbols (ie, nucleotides or amino acids). Default parameters for the GAP program are (1) binary comparison matrix (contains values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. As disclosed by 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); And (3) no penalty for end gaps.

As an example of the present application, the variant of the present application may have phytoene desaturase activity. In addition, the variant of the present application may have an activity to increase IMP (5'-inosine monophosphate) production ability compared to a wild-type polypeptide having phytoene desaturase activity.

In the present application, the term "Phytoene desaturase" refers to a colorless 15-cis-phytoene in a biochemical pathway called a poly-trans pathway. It is a polypeptide that converts to lycopene. Specifically, the phytoene desaturase of the present application may be used in combination with phytoene desaturase or CrtI. In the present application, the sequence of the phytoene desaturation enzyme can be obtained from GenBank of NCBI, a known database. Specifically, it may be a polypeptide having a phytoene desaturase activity encoded by crtI, but is not limited thereto.

In the present application, the term "corresponding to" refers to an amino acid residue at a position listed in a polypeptide, or similar, identical or homologous to a residue listed in a polypeptide. Identifying the amino acid at the corresponding position may be determining a specific amino acid in a sequence that refers to a specific sequence. As used herein, “corresponding region” generally refers to a related protein or similar or corresponding position in a reference protein.

For example, an arbitrary amino acid sequence is aligned with SEQ ID NO: 3, and based on this, each amino acid residue in the amino acid sequence can be numbered with reference to the numerical position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 3 . For example, a sequence alignment algorithm as described in the present application can determine the position of an amino acid or a position at which modification, such as substitution, insertion, or deletion, occurs compared to a query sequence (also referred to as a “reference sequence”).

Such alignments include, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. , 2000), Trends Genet. 16: 276-277) may be used, but the present disclosure is not limited thereto, and a sequence alignment program known in the art, a pairwise sequence comparison algorithm, and the like may be appropriately used.

Another aspect of the present application is to provide a polynucleotide encoding the variant of the present application.

In the present application, the term "polynucleotide" refers to a polymer of nucleotides in which a nucleotide unit is connected in a long chain by covalent bonds, and is a DNA or RNA strand having a certain length or more, and more specifically, encoding the variant. Refers to a polynucleotide fragment.

The polynucleotide encoding the variant of the present application may include a nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 1. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 2. In addition, the polynucleotide of the present application may consist of or consist essentially of the sequence of SEQ ID NO: 2.

The polynucleotide of the present application is various in the coding region within a range that does not change the amino acid sequence of the variant of the present application in consideration of the codon degeneracy or the preferred codon in the organism to express the variant of the present application. Transformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 425% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% of homology or identity with the sequence of SEQ ID NO: 2 Or more, 98% or more, and less than 100% of the nucleotide sequence, or has or has homology or identity with the sequence of SEQ ID NO: 2 is 70% or more, 75% or more, 425% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and may consist of or consist essentially of a base sequence of less than 100%, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the position 425 of SEQ ID NO: 1 may be one of the codons encoding asparagine.

In addition, the polynucleotide of the present application is a probe that can be prepared from a known gene sequence, for example, a complementary sequence to all or part of the polynucleotide sequence of the present application and a sequence capable of hydride under stringent conditions without limitation. Can be included. The "stringent condition" means a condition that enables specific hybridization between polynucleotides. These conditions are described in 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, 9.50-9.51, 11.7-11.8). For example, polynucleotides with high homology or identity, 70% or more, 75% or more, 425% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, Alternatively, polynucleotides with 99% or more homology or identity are hybridized, and polynucleotides with lower homology or identity are not hybridized, or at 60° C., which is a washing condition for common southern hybridization, 1 ХSSC, 0.1% SDS, specifically 60° C., 0.1 ХSSC, 0.1% SDS, more specifically 68° C., 0.1 ХSSC, at a salt concentration and temperature equivalent to 0.1% SDS, once, specifically 2 to 3 times washing Conditions to do can be enumerated.

Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments that are complementary to the entire sequence.

Specifically, a polynucleotide having homology or identity to the polynucleotide of the present application can be detected using a hybridization condition including a hybridization step at a Tm value of 55°C and using the above-described conditions. In addition, the Tm value may be 60°C, 63°C or 65°C, but is not limited thereto and may be appropriately adjusted by a person skilled in the art according to the purpose.

The appropriate stringency to hybridize the polynucleotide depends on the length and degree of complementarity of the polynucleotide, and the parameters are well known in the art (eg, J. Sambrook et al., homologous).

Another aspect of the present application is to provide a vector comprising the polynucleotide of the present application. The vector may be an expression vector for expressing the polynucleotide in a host cell, but is not limited thereto.

The vector of the present application includes a DNA preparation comprising a nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. I can. The expression control region may include a promoter capable of initiating transcription, an arbitrary operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling 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.

The vector used in the present application is not particularly limited, 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. , pBluescriptII system, pGEM system, pTZ system, pCL system, pET system, etc. can be used. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, and the like can be used.

For example, a polynucleotide encoding a target polypeptide 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. The selection marker is used to select cells transformed with a vector, that is, to confirm whether a nucleic acid molecule of interest is inserted, and select a selectable phenotype such as drug resistance, nutritional demand, resistance to cytotoxic agents, or expression of a surface polypeptide. Markers to give 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.

In the present application, the term "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or a microorganism so that the polypeptide 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/or RNA encoding the polypeptide of interest. 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.

In addition, the term "operably linked" in the above means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target variant of the present application.

Another aspect of the present application is to provide a strain of Corynebacterium stationis , comprising the variant of the present application or the polynucleotide of the present application.

The strain of the present application may include a variant polypeptide of the present application, a polynucleotide encoding the polypeptide, or a vector including the polynucleotide of the present application.

In the present application, the term "strain (or microorganism)" includes all wild-type microorganisms or microorganisms that have undergone natural or artificial genetic modification, and causes such as insertion of an external gene or enhancement or inactivation of an endogenous gene. As a result, a specific mechanism is weakened or enhanced, and may be a microorganism including a genetic modification for the production of a desired polypeptide, protein, or product.

The strain of the present application includes a strain including any one or more of the variant of the present application, the polynucleotide of the present application, and the vector including the polynucleotide of the present application; A strain modified to express the variant of the present application or the polynucleotide of the present application; A variant of the present application or a strain expressing the polynucleotide of the present application (eg, a recombinant strain); Alternatively, it may be a strain having the mutant activity of the present application (eg, a recombinant strain), but is not limited thereto.

The strain of the present application may be a strain having IMP-producing ability.

The strain of the present application is a microorganism having a natural phytoene desaturase or IMP-producing ability, or a mutant of the present application or a polynucleotide encoding the same in a parent strain without phytoene desaturase or IMP-producing ability (or the polynucleotide A vector comprising a) may be introduced and/or an IMP-producing ability is imparted, but is not limited thereto.

For example, the strain of the present application is transformed with a vector containing the polynucleotide of the present application or the polynucleotide encoding the variant of the present application, and is a cell or microorganism expressing the variant of the present application. The strain of the application may include all microorganisms capable of producing IMP, including the variant of the present application. For example, the strain of the present application is a recombinant strain with increased IMP production ability by introducing a polynucleotide encoding a variant of the present application into a natural wild-type microorganism or a microorganism producing IMP, thereby expressing a phytoene desaturation enzyme variant. I can. The recombinant strain with increased IMP-producing ability is a natural wild-type microorganism or a phytoene desaturation enzyme-unmodified microorganism (ie, a microorganism expressing a wild-type phytoene desaturation enzyme (SEQ ID NO: 3) or a mutant (SEQ ID NO: 1) protein). Microorganisms that do not express) may be microorganisms with increased IMP production capacity, but are not limited thereto. As an example, the phytoene desaturation enzyme-unmodified microorganism, which is a target strain for comparing the increase in IMP production ability, may be the CJI2332 strain (KCCM12277P, KR 10-1950141 B1), but is not limited thereto.

For example, the recombinant strain with increased production capacity is about 1% or more, specifically about 2% or more, about 4% or more, about 6% or more, about 8% compared to the IMP production capacity of the parent strain or unmodified microorganism before mutation. Or more, about 10% or more, about 12% or more, about 14% or more, about 16% or more, about 18% or more, or about 20% or more (the upper limit is not particularly limited, e.g., about 200% or less, about 150% or less , It may be about 100% or less, about 50% or less, about 40% or less, about 30% or less, about 25% or less), but the amount of increase in + value compared to the production capacity of the parent strain or unmodified microorganism before mutation As long as it has, it is not limited thereto. In another example, the recombinant strain having increased production capacity has an IMP production capacity of about 1.01 times or more, about 1.02 times or more, about 1.03 times or more, about 1.05 times or more, about 1.06 times or more compared to the parent strain or unmodified microorganism before mutation. , About 1.07 times or more, about 1.08 times or more, about 1.09 times or more, about 1.10 times or more, about 1.11 times or more, about 1.12 times or more, about 1.13 times or more, about 1.14 times or more, about 1.15 times or more, about 1.16 times or more , About 1.17 times or more, about 1.18 times or more, about 1.19 times or more, or about 1.20 times or more (the upper limit is not particularly limited, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less. May) may be increased, but is not limited thereto. The term “about” is a range that includes all of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values in the range equal to or similar to the value following the term about, but Not limited.

In the present application, the term "unmodified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, and is either a wild-type strain or a natural-type strain itself, or is characterized by genetic mutations caused by natural or artificial factors. It may mean a strain before being changed. For example, the unmodified microorganism may mean a strain in which the phytoene desaturase variant described herein has not been introduced or has been introduced. The "unmodified microorganism" may be used interchangeably with "pre-modified strain", "pre-modified microorganism", "non-mutated strain", "unmodified strain", "non-modified microorganism" or "reference microorganism".

In another example of the present application, the microorganism of the present application is Corynebacterium stationis , Corynebacterium glutamicum , Corynebacterium crudilactis , Corynebacterium crudilactis, Corynebacterium Nebacterium deserti , Corynebacterium efficiens , Corynebacterium callunae , Corynebacterium singulare , Corynebacterium halo Toledo lance (Corynebacterium halotolerans), Corynebacterium registry Atum (Corynebacterium striatum), Corynebacterium ammoniagenes to Ness (Corynebacterium ammoniagenes), Corynebacterium pole Ruti Solid (Corynebacterium pollutisoli), Corynebacterium already Tansu (Corynebacterium imitans ), Corynebacterium testudinoris or Corynebacterium flavescens .

In the present application, the term "weakening" of a polypeptide is a concept that includes both a decrease in activity or no activity compared to intrinsic activity. The attenuation may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, decrease, and attenuation.

When the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide of the original microorganism due to mutation of the polynucleotide encoding the polypeptide, the attenuation is inhibiting the expression of the gene of the polynucleotide encoding the polypeptide or translation into the polypeptide. When the overall polypeptide activity degree and/or concentration (expression amount) in the cell is lower than that of the native strain due to (translation) inhibition, etc., when the polynucleotide is not expressed at all, and/or the expression of the polynucleotide is Even if there is no activity of the polypeptide, it may also be included. The "intrinsic activity" refers to the activity of a specific polypeptide originally possessed by the parental strain, wild-type or unmodified microorganism before the trait change, when the trait is changed due to genetic variation caused by natural or artificial factors. It can be used interchangeably with "active before modification". When the activity of the polypeptide is "inactivated, deficient, decreased, downregulated, decreased, attenuated" compared to the intrinsic activity, it means that the activity of the specific polypeptide originally possessed by the parent strain or unmodified microorganism before the transformation of the trait is lowered.

The attenuation of the activity of such a polypeptide may be performed by any method known in the art, but is not limited thereto, and may be achieved by application of various methods well known in the art (eg, Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing.Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

Specifically, the attenuation of the polypeptide of the present application is

1) deletion of all or part of the gene encoding the polypeptide;

2) modification of the expression control region (or expression control sequence) to reduce the expression of the gene encoding the polypeptide;

3) modification of the amino acid sequence constituting the polypeptide such that the activity of the polypeptide is eliminated or weakened (eg, deletion/replacement/addition of one or more amino acids on the amino acid sequence);

4) modification of the gene sequence encoding the polypeptide such that the activity of the polypeptide is eliminated or attenuated (e.g., one or more nucleotides on the nucleotide sequence of the polypeptide gene to encode the modified polypeptide such that the activity of the polypeptide is eliminated or attenuated. Deletion/replacement/addition of);

5) modification of the nucleotide sequence encoding the 5'-UTR region or the start codon of the gene transcript encoding the polypeptide;

6) introduction of an antisense oligonucleotide (eg, antisense RNA) that complementarily binds to the transcript of the gene encoding the polypeptide;

7) Addition of a sequence complementary to a sinusoidal-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide in order to form a secondary structure in which the ribosome cannot be attached;

8) addition of a promoter transcribed in the opposite direction to the 3'end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE); or

9) It may be a combination of two or more selected from 1) to 8), but is not particularly limited thereto.

for example,

The deletion of a part or all of the gene encoding the 1) polypeptide may be removal of the entire polynucleotide encoding the target polypeptide intrinsic to the chromosome, replacement with a polynucleotide in which some nucleotides have been deleted, or replacement with a marker gene.

In addition, the 2) modification of the expression control region (or expression control sequence) is caused by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. It may be a replacement with an active sequence. The expression control region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence controlling termination of transcription and translation.

In addition, the 3) base sequence modification encoding the initiation codon or the 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a base encoding another initiation codon having a lower polypeptide expression rate compared to the intrinsic initiation codon. It may be substituted with a sequence, but is not limited thereto.

In addition, the modification of the amino acid sequence or polynucleotide sequence of 4) and 5) is a deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so as to weaken the activity of the polypeptide. Alternatively, a change in sequence may occur due to a combination thereof, or replacement of an amino acid sequence or polynucleotide sequence improved to have weaker activity, or an amino acid sequence or polynucleotide sequence improved to have no activity, but is not limited thereto. For example, by introducing a mutation in the polynucleotide sequence to form a stop codon, the expression of the gene may be inhibited or attenuated, but is not limited thereto.

The introduction of an antisense oligonucleotide (eg, antisense RNA) that complementarily binds to the transcript of the gene encoding the 6) polypeptide is described in, for example, Weintraub, H. et al., Antisense-RNA as a molecular tool. for genetic analysis, Reviews-Trends in Genetics, Vol. 1(1) 1986] may be referred to.

In order to form a secondary structure in which the 7) ribosome cannot be attached, the addition of a sequence complementary to the sinusoidal-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide is used for mRNA translation. It could be something that makes it impossible or slows it down.

The addition of a promoter transcribed in the opposite direction to the 3'end of the 8) open reading frame (ORF) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE) is an antisense complementary to the transcript of the gene encoding the polypeptide. It may be to weaken the activity by making nucleotides.

In the present application, the term "enhancement" of a polypeptide activity means that the activity of the polypeptide is increased compared to the intrinsic activity. The enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Herein, activation, enhancement, upregulation, overexpression, and increase may include all of exhibiting an activity that was not originally present, or exhibiting an intrinsic or enhanced activity compared to the pre-modification activity. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain or non-modified microorganism before the change in the trait when the trait is changed due to genetic variation caused by natural or artificial factors. The activity of a polypeptide "enhanced", "upregulated", "overexpressed" or "increased" compared to its intrinsic activity means that the activity of a specific polypeptide originally possessed by the parental strain or unmodified microorganism before transformation And/or concentration (expression amount).

The enrichment can be achieved through the introduction of a foreign polypeptide, or by enhancing the activity and/or concentration (expression amount) of the intrinsic polypeptide. Whether or not the activity of the polypeptide is enhanced can be confirmed from an increase in the level of activity, the expression level, or the amount of the product excreted from the polypeptide.

The enhancement of the activity of the polypeptide can be applied by various methods well known in the art, and is not limited as long as the activity of the target polypeptide can be enhanced than that of the microorganism before modification. Specifically, genetic engineering and/or protein engineering well known to those skilled in the art, which is a routine method of molecular biology, may be used, but is not limited thereto (eg, Sitnicka et al. Functional Analysis of Genes. Advances in Cell. Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

Specifically, the enhancement of the polypeptide of the present application is

1) increasing the intracellular copy number of the polynucleotide encoding the polypeptide;

2) replacing the gene expression control region on the chromosome encoding the polypeptide with a sequence with strong activity;

3) modification of the nucleotide sequence encoding the 5'-UTR region or the start codon of the gene transcript encoding the polypeptide;

4) modification of the amino acid sequence of the polypeptide to enhance the activity of the polypeptide;

5) modification of the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide (eg, modification of the polynucleotide sequence of the polypeptide gene to encode the modified polypeptide to enhance the activity of the polypeptide);

6) introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same;

7) codon optimization of the polynucleotide encoding the polypeptide;

8) By analyzing the tertiary structure of the polypeptide, select the exposed site to modify or chemically modify it; or

9) It may be a combination of two or more selected from 1) to 8), but is not particularly limited thereto.

More specifically,

The increase in the intracellular copy number of the polynucleotide encoding the 1) polypeptide is achieved by introducing a vector capable of replicating and functioning independently of the host into the host cell, to which the polynucleotide encoding the polypeptide is operably linked. It can be. Alternatively, the polynucleotide encoding the polypeptide may be achieved by introducing one copy or two or more copies into a chromosome in a host cell. The introduction into the chromosome may be performed by introducing a vector capable of inserting the polynucleotide into the chromosome in the host cell, but is not limited thereto. The vector is as described above.

The 2) replacement of the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence having a strong activity, for example, deletion, insertion, non-conservative or A conservative substitution or a combination thereof may result in a mutation in the sequence, or may be a replacement with a sequence having a stronger activity. The expression control region is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence controlling the termination of transcription and translation. For example, it may be to replace the original promoter with a strong promoter, but is not limited thereto.

Examples of known strong promoters include CJ1 to CJ7 promoters (US Patent US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US patent US 10584338 B2), O2 promoter (US patent US 10273491 B2), tkt promoter, yccA promoter, and the like, but are not limited thereto.

3) The nucleotide sequence modification encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a nucleotide sequence encoding another initiation codon having a higher expression rate of the polypeptide compared to the intrinsic start codon. It may be substituted, but is not limited thereto.

Modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above may include deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so as to enhance the activity of the polypeptide. The combination thereof may be a change in sequence, or replacement with an amino acid sequence or polynucleotide sequence improved to have stronger activity, or an amino acid sequence or polynucleotide sequence improved to increase activity, but is not limited thereto. The replacement may be specifically performed by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto. The vector used at this time may additionally include a selection marker for confirming whether a chromosome is inserted. The selection marker is as described above.

The introduction of a foreign polynucleotide exhibiting the activity of the 6) polypeptide may be the introduction of a foreign polynucleotide encoding a polypeptide exhibiting the same/similar activity as the polypeptide into a host cell. The foreign polynucleotide is not limited in its origin or sequence as long as it exhibits the same/similar activity as the polypeptide. The method used for 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 a polypeptide and increasing its activity.

The 7) codon optimization of the polynucleotide encoding the polypeptide is codon-optimized so that the endogenous polynucleotide increases transcription or translation in the host cell, or the foreign polynucleotide optimizes transcription and translation in the host cell. It may be that the codon of its codon is optimized so that it is set.

8) Analyzing the tertiary structure of the polypeptide, selecting and modifying the exposed site, or chemically modifying it is, for example, comparing the sequence information of the polypeptide to be analyzed with a database in which the sequence information of the known proteins is stored, to determine the degree of sequence similarity. Accordingly, a candidate template protein may be determined, a structure is confirmed based on this, and an exposed site to be modified or chemically modified may be selected and modified or modified.

Such enhancement of the polypeptide activity is that the activity or concentration expression level of the corresponding polypeptide is increased based on the activity or concentration of the polypeptide expressed in the wild type or microbial strain before modification, or the amount of the product produced from the polypeptide is increased. However, it is not limited thereto.

In the microorganism of the present application, the modification of part or all of the polynucleotide is (a) homologous recombination using a vector for chromosome insertion in the microorganism, or genome editing using engineered nuclease (eg, CRISPR-Cas9) and/or (b) It may be induced by treatment of light and/or chemicals such as ultraviolet rays and radiation, but is not limited thereto. The method for modifying part or all of the gene may include a method by DNA recombination technology. For example, by injecting a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism to cause homologous recombination, a part or all of the gene may be deleted. The injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.

In the microorganism of the present application, variants, polynucleotides, IMPs, and the like are as described in the other embodiments.

Another aspect of the present application provides a method for producing IMP, comprising the step of culturing in a medium a Corynebacterium stasis strain comprising the variant of the present application or the polynucleotide of the present application.

The IMP production method of the present application may include the step of culturing in a medium a Corynebacterium stasis strain containing the variant of the present application or the polynucleotide of the present application or the vector of the present application.

In the present application, the term "culture" means growing the Corynebacterium staionis strain of the present application under appropriately controlled environmental conditions. 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 strain. Specifically, the culture may be a batch type, continuous type and/or fed-batch type, but is not limited thereto.

In the present application, the term "medium" refers to a substance obtained by mixing as a main component a nutrient required to cultivate the Corynebacterium staionis strain of the present application, and nutrients including water essential for survival and development And development factors. Specifically, any medium and other culture conditions used for cultivation of the Corynebacterium stasis strain of the present application may be used without particular limitation as long as it is a medium used for cultivation of ordinary microorganisms, but Corynebacterium of the present application The Leeum stationary strain can be cultured under aerobic conditions in a conventional medium containing an appropriate carbon source, nitrogen source, personnel, inorganic compounds, amino acids and/or vitamins, while controlling temperature, pH, and the like.

Specifically, the culture medium for the strain of the genus Corynebacterium can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D.C., USA, 1981)].

In the present application, the carbon source includes carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, and the like; Sugar alcohols such as mannitol and sorbitol, organic acids such as pyruvic acid, lactic acid, and citric acid; Amino acids such as glutamic acid, methionine, and lysine may be included. In addition, natural organic nutrients such as starch hydrolyzate, molasses, black strap molasses, rice winter, cassava, sugarcane residue and corn steep liquor can be used, specifically glucose and sterilized pretreated molasses (i.e., converted to reducing sugar) Carbohydrates such as molasses) can be used, and other appropriate amounts of carbon sources can be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, etc., organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, skim soybean cake or its degradation products, etc. Can be used. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

The personnel may include first potassium phosphate, second potassium phosphate, or a sodium-containing salt corresponding thereto. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like may be used, and amino acids, vitamins, and/or suitable precursors may be included. These constituents or precursors may be added to the medium batchwise or continuously. However, it is not limited thereto.

In addition, the pH of the medium may be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the medium in an appropriate manner during the cultivation of the Corynebacterium stasis strain of the present application. In addition, during cultivation, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, in order to maintain the aerobic state of the medium, oxygen or oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen, or carbon dioxide gas may be injected without the injection of gas to maintain the anaerobic and microaerobic state. It is not.

In the culture of the present application, the culture temperature may be maintained at 20 to 45°C, specifically 25 to 40°C, and may be cultured for about 10 to 160 hours, but is not limited thereto.

The IMP produced by the culture of the present application may be secreted into the medium or may remain in the cells.

The IMP production method of the present application comprises the steps of preparing the Corynebacterium stationary strain of the present application, preparing a medium for culturing the strain, or a combination thereof (regardless of the order, in any order). , For example, prior to the culturing step, it may be further included.

The IMP production method of the present application may further include the step of recovering the IMP from the culture medium (cultured medium) or the Corynebacterium stasis strain. The recovering step may be further included after the culturing step.

The recovery may be to collect the desired IMP using a suitable method known in the art according to the method of culturing the microorganism of the present application, for example, a batch, continuous, or fed-batch culture method. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salt-in method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity Various types of chromatography such as road chromatography, HPLC, or a combination thereof may be used, and a desired IMP may be recovered from a medium or microorganism using a suitable method known in the art.

In addition, the IMP production method of the present application may additionally include a purification step. The purification can be carried out using a suitable method known in the art. In one example, when the IMP production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step are performed continuously or discontinuously regardless of order, or simultaneously or integrated into one step. It may be, but is not limited thereto.

In the method of the present application, variants, polynucleotides, vectors, strains, and the like are as described in the other embodiments.

Another aspect of the present application is a variant of the present application, a polynucleotide encoding the variant, a vector containing the polynucleotide, or a Corynebacterium stationary strain comprising the polynucleotide of the present application; Culture medium; Or to provide a composition for IMP production comprising a combination of two or more of these.

The composition of the present application may further include any suitable excipients commonly used in the composition for producing amino acids, and such excipients may be, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizers, isotonic agents, and the like. However, it is not limited thereto.

In the composition of the present application, variants, polynucleotides, vectors, strains, media, and IMP are as described in the other embodiments.

In the case of culturing a strain of Corynebacterium stationis , including a variant of the phytoene desaturase of the present application, a high yield of IMP (5 '-inosine monophosphate) production is possible.

1 is a schematic diagram of the pDCM2 plasmid.

Hereinafter, the present application will be described in more detail by examples. However, the following examples are only preferred embodiments for illustrating the present application, and therefore, are not intended to limit the scope of the present application thereto. On the other hand, technical matters not described in the present specification can be sufficiently understood and easily implemented by a person skilled in the art or similar technical field of the present application.

Example 1: Construction of plasmid

A plasmid (pDCM2, Fig. 1, SEQ ID NO: 5) for the insertion and replacement of genes in the Corynebacterium chromosome was designed, and a plasmid was synthesized using the Gene-synthesis service of Bionics. The plasmid was designed to contain a restriction enzyme that is easy to use for cloning by referring to the commonly known sacB system related paper [Gene, 145 (1994) 69-73]. The pDCM2 plasmid synthesized in this way has the following characteristics.

1) Since it has a replication origin that only works in E. coli, self-replication is possible within E. coli, but it is not possible to self-replicate in Corynebacterium.

2) It has a kanamycin resistance gene as a selection marker.

3) It has the Levan sucrase gene (sacB) as a secondary positive-selection marker.

4) No gene information derived from the pDCM2 plasmid is left in the final produced strain.

Example 2: Phytoene desaturase in microorganisms Vector construction for variant expression

To confirm the effect of aspartic acid at position 425 of the phytoene desaturase amino acid sequence (SEQ ID NO: 3) with asparagine (D425N; SEQ ID NO: 1) on the production of 5'-inosine monophosphate (IMP) The vector for was constructed as follows.

PCR was performed using a primer pair of sequences of SEQ ID NOs: 6 and 7 and a primer pair of sequences of SEQ ID NOs: 8 and 9 as a template using wild-type Corynebacterium stationis ATCC6872 gDNA (genomic DNA) I did. The mixture of the two fragments obtained above was again subjected to overlapping PCR using a primer pair of the sequence of SEQ ID NO: 6 and SEQ ID NO: 9 as a template to obtain a fragment. PCR was performed 30 times at 94°C for 5 minutes, followed by 30 seconds at 94°C, 30 seconds at 55°C, and 1 minute 30 seconds at 72°C, followed by 5 minutes at 72°C. The pDCM2 vector was treated with smaI, and the PCR product obtained above was fusion cloned. Fusion cloning was performed using In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-crtI (D425N).

Example 3: Evaluation of IMP production ability of microorganisms expressing phytoene desaturation enzyme variants

3-1. Phytoene desaturation enzyme Production of variant expression strains

The vector prepared in Example 2 was transformed into a Corynebacterium stationary CJI2332 strain (KCCM12277P, KR 10-1950141 B1).

From the strain in which homologous recombination took place, the strain into which the variant was introduced was selected using SEQ ID NOs: 10 and 11. The selected strain was named CJI2332_crtI_D425N.

3-2. Comparison of IMP production ability of strains expressing phytoene desaturation enzyme variants

IMP production ability was analyzed through flask fermentation titer evaluation of each strain prepared in Example 3-1 and the control parent strain.

First, each strain was inoculated into a 18 mm diameter test tube containing 2 ml of seed medium, and cultured with shaking at 30° C. for 24 hours to be used as a seed culture solution. A 250 ml corner-baffle flask containing 29 ml of production medium (24 ml of main medium + 5 ml of separate sterilization medium) was inoculated with 2 ml of a seed culture solution, followed by incubation at 30° C. for 72 hours and at 170 rpm. After completion of the culture, the production capacity of IMP was measured by HPLC. IMP concentration and concentration increase rate in the culture medium for each of the tested strains are shown in Table 1 below.

<Seed medium (pH 7.5)>

Glucose 1%, peptone 1%, meat juice 1%, yeast extract 1%, sodium chloride 0.25%, adenine 100mg/l, guanine 100mg/l (based on 1 liter of distilled water)

<Production medium-Main medium (pH 7.5)>

Yeast extract 0.2%, sodium glutamate 0.1%, magnesium sulfate 1.2%, calcium chloride 0.01%, iron sulfate 20mg/l, manganese sulfate 20mg/l, zinc sulfate 20mg/l, copper sulfate 5mg/l, L-cysteine 23mg/l, beta Alanine 24mg/l, nicotinic acid 8mg/l, biotin 45㎍/l, thiamine hydrochloric acid 5mg/l, adenine 30mg/l, glucose 2.55%, fructose 1.45% (based on 1 liter of distilled water)

<Production medium-Separate sterilization medium (pH 7.5)>

Phosphoric acid (85%) 2.3%, potassium hydroxide (45%) 2.55% (based on 1 liter of distilled water)

The experiment was repeated three times, and the average values of the analysis results are shown in Table 1 below.

IMP production capacity comparison Strain IMP concentration (g/L) IMP concentration increase rate (%) CJI2332 1.74 - CJI2332_crtI_D425N 2.1 20.7

As shown in Table 1, the CJI2332_crtI_D425N strain exhibited an increase in IMP production capacity by 20.7% compared to the control group.

The CJI2332_crtI_D425N was named CN01-2649, and was deposited with the Korea Microbial Conservation Center, a trust organization under the Budapest Treaty, on November 30, 2020, and was given the accession number KCCM12844P.

From the above description, those skilled in the art to which the present application belongs will understand that the present application may be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present application should be construed as including all changes or modified forms derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof.

Korea Microorganism Conservation Center (overseas) KCCM12844P 20201130

<110> CJ CheilJedang Corporation <120> Novel Phytoene desaturase variant and a method for producing IMP using the same <130> KPA201571-KR <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 488 <212> PRT <213> Artificial Sequence <220> <223> variant AA <400> 1 Met Lys Thr Thr Val Ile Ile Ile Gly Ala Gly Val Ala Gly Leu Ala 1 5 10 15 Thr Ala Gly Leu Leu Ala Arg Asn Gly Tyr Gln Val Thr Val Val Glu 20 25 30 Lys Asn Ala Thr Phe Gly Gly Arg Ala Gly Asn Leu Glu Ala Gln Gly 35 40 45 Phe Arg Trp Asp Thr Gly Pro Ser Trp Tyr Leu Met Pro Asp Ala Phe 50 55 60 Glu His Phe Phe Gln Gln Met Gly Thr Ser Thr Lys Asp Val Leu Asp 65 70 75 80 Val Val Arg Leu Ser Glu Pro Ala Tyr Arg Val Phe Pro Glu His His 85 90 95 Pro Pro Leu Asp Ile Pro Ala Gly Arg Glu Asn Val Val Lys Leu Phe 100 105 110 Glu Ser Ile Glu Glu Gly Ala Gly Lys Gln Leu Glu Ala Tyr Leu Asp 115 120 125 Ser Ala Glu Phe Ala Tyr Arg Thr Ala Leu Asp His Phe Leu Tyr Thr 130 135 140 Thr Phe Ser Ser Pro Leu Val Phe Leu Arg Arg Asp Ile Leu Thr Asn 145 150 155 160 Leu Ala Phe Leu Ile Ser Leu Leu Thr Thr Ser Leu Asp Lys Tyr Val 165 170 175 Ala Lys Arg Phe Thr Asp Thr Arg Leu Arg Gln Ile Leu Ala Tyr Pro 180 185 190 Ala Val Phe Leu Ser Ser Arg Pro Glu Lys Thr Pro Ala Leu Tyr His 195 200 205 Leu Met Ser His Thr Asp Leu Thr Gln Gly Val Ser Tyr Pro Ile Gly 210 215 220 Gly Phe His Ala Val Met Lys Val Leu Tyr Lys Leu Ala Leu Asp Asn 225 230 235 240 Gly Val Lys Phe Arg Phe Ser Ser Pro Val Ser Ser Ile Thr Thr Ser 245 250 255 Asp Gly Arg Ala Thr Gly Ile Val Leu Ser Glu Glu Arg Leu Gln Ala 260 265 270 Asp Val Val Val Ser Cys Ala Asp Leu His His Thr Glu Thr Gln Leu 275 280 285 Leu Pro Glu Asn Leu Arg Ser Tyr Ser Glu Asn Trp Phe Asp Lys Arg 290 295 300 Ser Pro Gly Leu Gly Thr Val Leu Leu Met Leu Gly Ile Ser Gly Glu 305 310 315 320 Leu Pro Gln Leu Ala His His Asn Leu Phe Phe Ser Glu Asn Trp Thr 325 330 335 Val Asp Phe Asp Ala Val Phe Glu Asp Thr Gly Trp Ser Gln Ser Ile 340 345 350 Tyr Val Ser Lys Pro Ser Ala Thr Asp Pro Glu Val Ala Pro Ala Gly 355 360 365 His Glu Asn Leu Phe Val Leu Val Pro Val Lys Ala Asp Pro Ala Phe 370 375 380 Glu Arg Glu Glu Glu Val Ala Glu Glu Ala Leu Arg Leu Ile Ser Glu 385 390 395 400 Arg Ala Glu Ile Pro Asp Leu Ala Ser Arg Ile Ala Phe Gln His Ile 405 410 415 Leu Gly Pro Ala Asp Phe Ala Arg Asn Tyr His Ala Trp Ser Gly Gly 420 425 430 Ser Ile Gly Pro Ser His Ser Leu Lys Gln Ser Ala Phe Leu Arg Gly 435 440 445 Ser Asn Lys Ser Lys Lys Ile Ala Gly Leu Tyr Tyr Ala Gly Ala Thr 450 455 460 Thr Val Pro Gly Val Gly Val Pro Leu Cys Leu Ile Ser Ala Glu Asn 465 470 475 480 Val Leu Ile Arg Leu Arg Glu Glu 485 <210> 2 <211> 1467 <212> DNA <213> Artificial Sequence <220> <223> variant NT <400> 2 gtgaaaacta ctgtgataat tatcggcgcc ggggtcgctg gtctcgcgac ggctggctta 60 ctagcccgta atggctacca agtcacggtg gtggagaaaa atgctacttt cggaggaaga 120 gcggggaatt tagaagctca aggctttcga tgggatacgg gtccctcctg gtatctcatg 180 cctgatgctt tcgagcattt tttccaacaa atgggcacct cgaccaagga tgtactcgac 240 gtagttcggt tgagcgaacc cgcttatagg gtcttcccag agcaccaccc tcctctagac 300 atcccagctg gcagggaaaa tgttgtaaag ctttttgagt ccattgagga aggcgctgga 360 aaacaactag aagcctatct agatagcgct gaatttgcct accggactgc tcttgaccac 420 ttcctttata caacgttttc ctccccgcta gttttcctcc gccgtgatat tttaaccaat 480 ctcgccttcc ttataagctt gctaacgacg tcactcgata agtatgtggc caagagattc 540 actgacactc gcttgcgcca aatacttgct taccctgcag tttttttgtc ttcccgccct 600 gaaaaaacac ctgcgctcta tcatctaatg agtcatactg atttaactca aggtgtgagc 660 taccctatcg gcggcttcca cgcagtgatg aaagtcttgt acaagctcgc cctcgataat 720 ggtgttaaat tccgtttttc ctcgcctgtc tcatccatca cgacgtccga cggccgagcg 780 acaggtatcg tcctgtcgga agagcgttta caggctgatg tcgtggtcag ctgcgctgat 840 ctacaccaca ccgaaaccca gcttttaccg gaaaacctac gttcttattc cgaaaactgg 900 ttcgacaaac gcagccctgg cttgggaact gtacttctaa tgctgggtat cagcggggaa 960 ttaccccaac tagcccatca caatcttttt ttcagcgaga actggacggt agattttgac 1020 gcagttttcg aggacaccgg ttggtcccag tcgatctatg tgtctaaacc ctctgcaacg 1080 gatcccgaag tagccccagc tggtcatgaa aatctttttg ttctagtccc ggttaaggct 1140 gaccccgcgt tcgagcgcga ggaggaagtc gcagaagagg ccctgcggct aatttctgaa 1200 cgggcggaaa ttcctgatct cgcttcccgc attgccttcc aacatattct aggccccgca 1260 gacttcgccc gaaattacca cgcttggagt ggcggttcga tcggaccgag tcattcgctc 1320 aaacaatccg catttttgcg gggaagcaat aagtcgaaga agatagcagg gctttactat 1380 gctggagcaa ccaccgtccc aggggtcgga gtaccacttt gccttatttc agcggaaaac 1440 gttctcatcc gactgcgtga agaataa 1467 <210> 3 <211> 488 <212> PRT <213> Artificial Sequence <220> <223> WT AA <400> 3 Met Lys Thr Thr Val Ile Ile Ile Gly Ala Gly Val Ala Gly Leu Ala 1 5 10 15 Thr Ala Gly Leu Leu Ala Arg Asn Gly Tyr Gln Val Thr Val Val Glu 20 25 30 Lys Asn Ala Thr Phe Gly Gly Arg Ala Gly Asn Leu Glu Ala Gln Gly 35 40 45 Phe Arg Trp Asp Thr Gly Pro Ser Trp Tyr Leu Met Pro Asp Ala Phe 50 55 60 Glu His Phe Phe Gln Gln Met Gly Thr Ser Thr Lys Asp Val Leu Asp 65 70 75 80 Val Val Arg Leu Ser Glu Pro Ala Tyr Arg Val Phe Pro Glu His His 85 90 95 Pro Pro Leu Asp Ile Pro Ala Gly Arg Glu Asn Val Val Lys Leu Phe 100 105 110 Glu Ser Ile Glu Glu Gly Ala Gly Lys Gln Leu Glu Ala Tyr Leu Asp 115 120 125 Ser Ala Glu Phe Ala Tyr Arg Thr Ala Leu Asp His Phe Leu Tyr Thr 130 135 140 Thr Phe Ser Ser Pro Leu Val Phe Leu Arg Arg Asp Ile Leu Thr Asn 145 150 155 160 Leu Ala Phe Leu Ile Ser Leu Leu Thr Thr Ser Leu Asp Lys Tyr Val 165 170 175 Ala Lys Arg Phe Thr Asp Thr Arg Leu Arg Gln Ile Leu Ala Tyr Pro 180 185 190 Ala Val Phe Leu Ser Ser Arg Pro Glu Lys Thr Pro Ala Leu Tyr His 195 200 205 Leu Met Ser His Thr Asp Leu Thr Gln Gly Val Ser Tyr Pro Ile Gly 210 215 220 Gly Phe His Ala Val Met Lys Val Leu Tyr Lys Leu Ala Leu Asp Asn 225 230 235 240 Gly Val Lys Phe Arg Phe Ser Ser Pro Val Ser Ser Ile Thr Thr Ser 245 250 255 Asp Gly Arg Ala Thr Gly Ile Val Leu Ser Glu Glu Arg Leu Gln Ala 260 265 270 Asp Val Val Val Ser Cys Ala Asp Leu His His Thr Glu Thr Gln Leu 275 280 285 Leu Pro Glu Asn Leu Arg Ser Tyr Ser Glu Asn Trp Phe Asp Lys Arg 290 295 300 Ser Pro Gly Leu Gly Thr Val Leu Leu Met Leu Gly Ile Ser Gly Glu 305 310 315 320 Leu Pro Gln Leu Ala His His Asn Leu Phe Phe Ser Glu Asn Trp Thr 325 330 335 Val Asp Phe Asp Ala Val Phe Glu Asp Thr Gly Trp Ser Gln Ser Ile 340 345 350 Tyr Val Ser Lys Pro Ser Ala Thr Asp Pro Glu Val Ala Pro Ala Gly 355 360 365 His Glu Asn Leu Phe Val Leu Val Pro Val Lys Ala Asp Pro Ala Phe 370 375 380 Glu Arg Glu Glu Glu Val Ala Glu Glu Ala Leu Arg Leu Ile Ser Glu 385 390 395 400 Arg Ala Glu Ile Pro Asp Leu Ala Ser Arg Ile Ala Phe Gln His Ile 405 410 415 Leu Gly Pro Ala Asp Phe Ala Arg Asp Tyr His Ala Trp Ser Gly Gly 420 425 430 Ser Ile Gly Pro Ser His Ser Leu Lys Gln Ser Ala Phe Leu Arg Gly 435 440 445 Ser Asn Lys Ser Lys Lys Ile Ala Gly Leu Tyr Tyr Ala Gly Ala Thr 450 455 460 Thr Val Pro Gly Val Gly Val Pro Leu Cys Leu Ile Ser Ala Glu Asn 465 470 475 480 Val Leu Ile Arg Leu Arg Glu Glu 485 <210> 4 <211> 1467 <212> DNA <213> Artificial Sequence <220> <223> WT NT <400> 4 gtgaaaacta ctgtgataat tatcggcgcc ggggtcgctg gtctcgcgac ggctggctta 60 ctagcccgta atggctacca agtcacggtg gtggagaaaa atgctacttt cggaggaaga 120 gcggggaatt tagaagctca aggctttcga tgggatacgg gtccctcctg gtatctcatg 180 cctgatgctt tcgagcattt tttccaacaa atgggcacct cgaccaagga tgtactcgac 240 gtagttcggt tgagcgaacc cgcttatagg gtcttcccag agcaccaccc tcctctagac 300 atcccagctg gcagggaaaa tgttgtaaag ctttttgagt ccattgagga aggcgctgga 360 aaacaactag aagcctatct agatagcgct gaatttgcct accggactgc tcttgaccac 420 ttcctttata caacgttttc ctccccgcta gttttcctcc gccgtgatat tttaaccaat 480 ctcgccttcc ttataagctt gctaacgacg tcactcgata agtatgtggc caagagattc 540 actgacactc gcttgcgcca aatacttgct taccctgcag ttttcttgtc ttcccgccct 600 gaaaaaacac ctgcgctcta tcatctaatg agtcatactg atttaactca aggtgtgagc 660 taccctatcg gcggcttcca cgcagtgatg aaagtcttgt acaagctcgc cctcgataat 720 ggtgttaaat tccgtttttc ctcgcctgtc tcatccatca cgacgtccga cggccgagcg 780 acaggtatcg tcctgtcgga agagcgttta caggctgatg tcgtggtcag ctgcgctgat 840 ctacaccaca ccgaaaccca gcttttaccg gaaaacctac gttcttattc cgaaaactgg 900 ttcgacaaac gcagccctgg cttgggaact gtacttctaa tgctgggtat cagcggggaa 960 ttaccccaac tagcccatca caatcttttt ttcagcgaga actggacggt agattttgac 1020 gcagttttcg aggacaccgg ttggtcccag tcgatctatg tgtctaaacc ctctgcaacg 1080 gatcccgaag tagccccagc tggtcatgaa aatctttttg ttctagtccc ggttaaggct 1140 gaccccgcgt tcgagcgcga ggaggaagtc gcagaagagg ccctgcggct aatttctgaa 1200 cgggcggaaa ttcctgatct cgcttcccgc attgccttcc aacatattct aggccccgca 1260 gacttcgccc gagattacca cgcttggagt ggcggttcga tcggaccgag tcattcgctc 1320 aaacaatccg catttttgcg gggaagcaat aagtcgaaga agatagcagg gctttactat 1380 gctggagcaa ccaccgtccc aggggtcgga gtaccacttt gccttatttc agcggaaaac 1440 gttctcatcc gactgcgtga agaataa 1467 <210> 5 <211> 5803 <212> DNA <213> Artificial Sequence <220> <223> pDCM2 <400> 5 gttcgcttgc tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag 60 ctacctgctt tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat 120 tcatccgggg tcagcaccgt ttctgcggac tggctttcta cgtgttccgc ttcctttagc 180 agcccttgcg ccctgagtgc ttgcggcagc gtgaagctag cttttatcgc cattcgccat 240 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc 300 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt 360 cacgacgttg taaaacgacg gccagtgaat tcgagctcgg tacccgggga tcctctagag 420 tcgacctgca ggcatgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 480 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 540 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 600 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 660 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 720 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 780 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 840 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 900 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 960 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 1020 tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg 1080 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 1140 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 1200 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 1260 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 1320 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 1380 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 1440 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 1500 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttgggg 1560 tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc ctgatagaaa 1620 cagaagccac tggagcacct caaaaacacc atcatacact aaatcagtaa gttggcagca 1680 tcacccgacg cactttgcgc cgaataaata cctgtgacgg aagatcactt cgcagaataa 1740 ataaatcctg gtgtccctgt tgataccggg aagccctggg ccaacttttg gcgaaaatga 1800 gacgttgatc ggcacgtaag aggttccaac tttcaccata atgaaataag atcactaccg 1860 ggcgtatttt ttgagttatc gagattttca ggagctgata gaaacagaag ccactggagc 1920 acctcaaaaa caccatcata cactaaatca gtaagttggc agcatcaccc gacgcacttt 1980 gcgccgaata aatacctgtg acggaagatc acttcgcaga ataaataaat cctggtgtcc 2040 ctgttgatac cgggaagccc tgggccaact tttggcgaaa atgagacgtt gatcggcacg 2100 taagaggttc caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt 2160 tatcgagatt ttcaggagct ctttggcatc gtctctcgcc tgtcccctca gttcagtaat 2220 ttcctgcatt tgcctgtttc cagtcggtag atattccaca aaacagcagg gaagcagcgc 2280 ttttccgctg cataaccctg cttcggggtc attatagcga ttttttcggt atatccatcc 2340 tttttcgcac gatatacagg attttgccaa agggttcgtg tagactttcc ttggtgtatc 2400 caacggcgtc agccgggcag gataggtgaa gtaggcccac ccgcgagcgg gtgttccttc 2460 ttcactgtcc cttattcgca cctggcggtg ctcaacggga atcctgctct gcgaggctgg 2520 ccggctaccg ccggcgtaac agatgagggc aagcggatgg ctgatgaaac caagccaacc 2580 aggaagggca gcccacctat caaggtgtac tgccttccag acgaacgaag agcgattgag 2640 gaaaaggcgg cggcggccgg catgagcctg tcggcctacc tgctggccgt cggccagggc 2700 tacaaaatca cgggcgtcgt ggactatgag cacgtccgcg agggcgtccc ggaaaacgat 2760 tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgtg atggcaggtt 2820 gggcgtcgct tggtcggtca tttcgaaaaa ggttaggaat acggttagcc atttgcctgc 2880 ttttatatag ttcantatgg gattcacctt tatgttgata agaaataaaa gaaaatgcca 2940 ataggatatc ggcattttct tttgcgtttt tatttgttaa ctgttaattg tccttgttca 3000 aggatgctgt ctttgacaac agatgttttc ttgcctttga tgttcagcag gaagctcggc 3060 gcaaacgttg attgtttgtc tgcgtagaat cctctgtttg tcatatagct tgtaatcacg 3120 acattgtttc ctttcgcttg aggtacagcg aagtgtgagt aagtaaaggt tacatcgtta 3180 ggcggatcaa gatccatttt taacacaagg ccagttttgt tcagcggctt gtatgggcca 3240 gttaaagaat tagaaacata accaagcatg taaatatcgt tagacgtaat gccgtcaatc 3300 gtcatttttg atccgcggga gtcagtgaac aggtaccatt tgccgttcat tttaaagacg 3360 ttcgcgcgtt caatttcatc tgttactgtg ttagatgcaa tcagcggttt catcactttt 3420 ttcagtgtgt aatcatcgtt tagctcaatc ataccgagag cgccgtttgc taactcagcc 3480 gtgcgttttt tatcgctttg cagaagtttt tgactttctt gacggaagaa tgatgtgctt 3540 ttgccatagt atgctttgtt aaataaagat tcttcgcctt ggtagccatc ttcagttcca 3600 gtgtttgctt caaatactaa gtatttgtgg cctttatctt ctacgtagtg aggatctctc 3660 agcgtatggt tgtcgcctga gctgtagttg ccttcatcga tgaactgctg tacattttga 3720 tacgtttttc cgtcaccgtc aaagattgat ttataatcct ctacaccgtt gatgttcaaa 3780 gagctgtctg atgctgatac gttaacttgt gcagttgtca gtgtttgttt gccgtaatgt 3840 ttaccggaga aatcagtgta gaataaacgg atttttccgt cagatgtaaa tgtggctgaa 3900 cctgaccatt cttgtgtttg gtcttttagg atagaatcat ttgcatcgaa tttgtcgctg 3960 tctttaaaga cgcggccagc gtttttccag ctgtcaatag aagtttcgcc gactttttga 4020 tagaacatgt aaatcgatgt gtcatccgca tttttaggat ctccggctaa tgcaaagacg 4080 atgtggtagc cgtgatagtt tgcgacagtg ccgtcagcgt tttgtaatgg ccagctgtcc 4140 caaacgtcca ggccttttgc agaagagata tttttaattg tggacgaatc aaattcagaa 4200 acttgatatt tttcattttt ttgctgttca gggatttgca gcatatcatg gcgtgtaata 4260 tgggaaatgc cgtatgtttc cttatatggc ttttggttcg tttctttcgc aaacgcttga 4320 gttgcgcctc ctgccagcag tgcggtagta aaggttaata ctgttgcttg ttttgcaaac 4380 tttttgatgt tcatcgttca tgtctccttt tttatgtact gtgttagcgg tctgcttctt 4440 ccagccctcc tgtttgaaga tggcaagtta gttacgcaca ataaaaaaag acctaaaata 4500 tgtaaggggt gacgccaaag tatacacttt gccctttaca cattttaggt cttgcctgct 4560 ttatcagtaa caaacccgcg cgatttactt ttcgacctca ttctattaga ctctcgtttg 4620 gattgcaact ggtctatttt cctcttttgt ttgatagaaa atcataaaag gatttgcaga 4680 ctacgggcct aaagaactaa aaaatctatc tgtttctttt cattctctgt attttttata 4740 gtttctgttg catgggcata aagttgcctt tttaatcaca attcagaaaa tatcataata 4800 tctcatttca ctaaataata gtgaacggca ggtatatgtg atgggttaaa aaggatcacc 4860 ccagagtccc gctcagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc 4920 gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc 4980 agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac ccagccggcc 5040 acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc 5100 gccatgggtc acgacgagat cctcgccgtc gggcatccgc gccttgagcc tggcgaacag 5160 ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga caagaccggc 5220 ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga atgggcaggt 5280 agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata ctttctcggc 5340 aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata gcagccagtc 5400 ccttcccgct tcagtgacaa cgtcgagaca gctgcgcaag gaacgcccgt cgtggccagc 5460 cacgatagcc gcgctgcctc gtcttggagt tcattcaggg caccggacag gtcggtcttg 5520 acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg 5580 attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct 5640 gcgtgcaatc catcttgttc aatcatgcga aacgatcctc atcctgtctc ttgatcagat 5700 cttgatcccc tgcgccatca gatccttggc ggcaagaaag ccatccagtt tactttgcag 5760 ggcttcccaa ccttaccaga gggcgcccca gctggcaatt ccg 5803 <210> 6 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 1F <400> 6 tcgagctcgg tacccctgtc tcatccatca cgacg 35 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 2R <400> 7 tccaagcgtg gtaatttcgg gcgaagtctg 30 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 3F <400> 8 cagacttcgc ccgaaattac cacgcttgga 30 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 4R <400> 9 ctctagagga tcccctgcag gataagctat ggaag 35 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5F <400> 10 ctgtctcatc catcacgacg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 6R <400> 11 tgcaggataa gctatggaag 20

Claims (5)

A phytoene desaturation enzyme variant consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which aspartic acid, which is an amino acid corresponding to position 425 of SEQ ID NO: 3, is substituted with asparagine.
A polynucleotide encoding the variant of claim 1.
The variant of claim 1 or comprising a polynucleotide encoding the variant, Corynebacterium staionis strain.
The strain according to claim 3, wherein the strain has an increased IMP production ability compared to Corynebacterium stasis comprising the polypeptide of SEQ ID NO: 3 or a polynucleotide encoding the same.
A method for producing IMP comprising the step of culturing the variant of claim 1 or a Corynebacterium stasis strain comprising a polynucleotide encoding the variant in a medium.

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