KR20240118943A - Microorganism having enhanced activity of pantoate-beta-alanine ligase from glutamicibacter halophytocola and uses thereof - Google Patents

Abstract

The present application discloses a microorganism with enhanced activity of pantoate-beta-alanine ligase derived from Glutamicibacter halophytocola, a composition for producing panthenol containing the microorganism, and a method for producing panthenol comprising culturing the microorganism. It has excellent production capacity of panthenol.

Description

Microorganism having enhanced activity of pantoate-beta-alanine ligase from glutamicibacter halophytocola and uses thereof}

Provided are microorganisms with enhanced activity of pantoate-beta-alanine ligase derived from Glutamicibacter halophytocola, a composition for producing panthenol containing the microorganisms, and a method for producing panthenol comprising culturing the microorganisms. .

Panthenol is a provitamin form of vitamin B5 and is one of the commercially important substances with various applications in cosmetics, food, and beverages. Panthenol has excellent moisturizing properties, so it is used in skin care products such as cosmetics and personal care products, and is mainly manufactured by chemical synthesis.

During the process of chemically producing panthenol, wastewater such as formaldehyde and cyanide is generated during the process of producing DL-pantolactone and separating the D-form, and also the D-form of pantolactone and 3-aminopropanol are generated. In the condensation step, a large amount of organic solvents such as methane and dichloromethane are used, and waste water is generated, causing environmental problems.

When the D-form panthenol produced by chemical synthesis is fermented and produced using microorganisms, bioactive D-form panthenol can be produced without the process of separating DL-pantolactone, so the large amount required for chemical synthesis can be produced. It has the advantage of not causing wastewater treatment problems because it does not use organic solvents.

Accordingly, there is a need for the development of microorganisms that have an advantageous effect in biotechnologically producing panthenol and technologies for producing panthenol with high efficiency using them.

W.O. 2001-021772 A2

An example of the present application provides a microorganism with enhanced pantoate-beta-alanine ligase activity and improved panthenol production ability.

Another example of the present application provides a method for producing panthenol, including culturing the microorganism in a medium.

Another example of the present application provides a composition for producing panthenol containing the above microorganism.

Another example of the present application provides the use of said microorganism for panthenol production.

Another example of the present application provides the use of the microorganism for the production of a composition for producing panthenol.

The present application relates to panthenol production and/or production ability of pantoate-beta-alanine ligase, such as pantoate-beta-alanine ligase derived from Glutamicibacter halophytocola. It is about use for improvement.

One aspect of the present application provides a microorganism (strain, or recombinant cell) with enhanced pantoate-beta-alanine ligase activity.

In the present application, pantoate-beta-alanine ligase (pantoate-beta-alanine ligase, or PanC protein) may be included without limitation as long as it is a protein having pantoate-beta-alanine ligase activity. You can. The pantoate-beta-alanine ligase is a microorganism of the genus Corynebacterium (e.g., Corynebacterium glutamicum ), a microorganism of the genus Escherichia (e.g., Escherichia coli ), Microorganisms of the genus Coraliomargarita (e.g., Coraliomargarita akajimensis ), microorganisms of the genus Actinobacteria (e.g., Actinobacteria bacterium ), microorganisms of the genus Nitrosospira (e.g., Nitrosospira) Nitrosospira lacus , a microorganism of the genus Nitrosococcus (e.g., Nitrosococcus watsonii ), or a microorganism of the genus Glutamicibacter (e.g., Glutamicibacter halophytocola ) It may be possible, but it is not limited to this.

In one example, the pantoate-beta-alanine ligase may be from Glutamicibacter halopytocola. The sequence of the pantoate-beta-alanine ligase derived from Glutamicibacter halophytocola can be obtained from NCBI, a known database (NCBI Reference Sequence: WP_060700217.1).

In one example, the pantoate-beta-alanine ligase derived from Glutamicibacter halopytocola has, includes, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 43. It can be accomplished (essentially consisting of).

In one example, the pantoate-beta-alanine ligase derived from Glutamicibacter halopytocola has at least 70%, 75%, 80%, 85%, or 90% of the amino acid sequence of SEQ ID NO: 43. , 94.44% or higher, 94.79% or higher, 95% or higher, 95.14% or higher, 95.49% or higher, 95.83% or higher, 96% or higher, 96.18% or higher, 96.53% or higher, 96.88% or higher, 97% or higher, 97.22% or higher, 97.57 % or more, 97.92% or more, 98% or more, 98.26% or more, 98.61% or more, 98.96% or more, 99% or more, 99.31% or more, 99.5% or more, 99.65% or more, 99.7% or more, or 99.9% or more homology or identity. It may contain or consist of an amino acid sequence having the above sequence.

In addition, if the amino acid sequence has such homology or identity and shows efficacy corresponding to pantoate-beta-alanine ligase, a variant having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added may also be used in the pantoate-beta-alanine ligase. It may be included in ate-beta-alanine ligase. For example, addition or deletion of sequences at the N-terminus, C-terminus and/or within the amino acid sequence without altering the pantoate-beta-alanine ligase activity may occur naturally. It may be a case of an existing mutation, a silent mutation, or a conservative substitution.

The “conservative substitution” means replacing one amino acid with another amino acid having similar structural and/or chemical properties. These amino acid substitutions may generally occur based on similarities 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 pantoate-beta-alanine ligase may be a polypeptide having pantoate-beta-alanine ligase activity encoded by the pantoate-beta-alanine ligase gene. The pantoate-beta-alanine ligase gene is a microorganism of the genus Corynebacterium (e.g., Corynebacterium glutamicum ), a microorganism of the genus Escherichia (e.g., Escherichia coli ). , microorganisms of the genus Coraliomargarita (e.g., Coraliomargarita akajimensis ), microorganisms of the genus Actinobacteria (e.g., Actinobacteria bacterium ), microorganisms of the genus Nitrosospira (e.g., nitrosource) Nitrosospira lacus , a microorganism of the genus Nitrosococcus (e.g., Nitrosococcus watsonii ) or a microorganism of the genus Glutamicibacter (e.g., Glutamicibacter halophytocola ) It may be possible, but it is not limited to this.

In one example, the pantoate-beta-alanine ligase gene may be from Glutamicibacter halopytocola. The sequence of the pantoate-beta-alanine ligase gene can be obtained from NCBI, a known database (NCBI Reference Sequence: CP012750.1).

In one example, the pantoate-beta-alanine ligase gene may have, include, consist of, or consist essentially of the nucleic acid sequence of SEQ ID NO: 42. .

In one example, the pantoate-beta-alanine ligase gene is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and the nucleic acid sequence of SEQ ID NO: 42. It may contain or consist of a nucleic acid sequence having more than 99%, 99.5%, 99.7%, or 99.9% homology or identity.

In the present application, the term "polynucleotide or polypeptide" refers to "having, comprising, consisting of, or consisting essentially of a specific nucleic acid sequence (base sequence) or amino acid sequence". This may mean that a nucleotide or polypeptide essentially includes the specific nucleic acid sequence (base sequence) or amino acid sequence, and the specific nucleic acid sequence (base sequence) or amino acid sequence may be used to It can be interpreted as including (or not excluding) a “substantially equivalent sequence” in which mutations (deletions, substitutions, modifications, and/or additions) are made to the nucleic acid sequence (base sequence) or amino acid sequence. . In one example, a polynucleotide or polypeptide "has, includes, consists of, or consists essentially of a specific nucleic acid sequence (base sequence) or amino acid sequence" means that the polynucleotide or polypeptide is (i) essentially comprising the specific nucleic acid sequence (base sequence) or amino acid sequence, or (ii) at least 70%, 80%, 85%, or 90% of the specific nucleic acid sequence (base sequence) or amino acid sequence. or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more. It may mean that it consists of or essentially includes a nucleic acid sequence or amino acid sequence having homology or identity and maintains its original function and/or desired function.

In one example, the desired function may refer to a function that grants or increases (improves) the panthenol production ability of microorganisms.

In this application, ‘homology’ or ‘identity’ refers to the degree of similarity between two given amino acid sequences or base sequences and can 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 may be used with a default gap penalty established by the program used. Substantially homologous or identical sequences are generally capable of hybridizing to all or part of a sequence under moderate or high stringent conditions. It is obvious that hybridization also includes hybridization of polynucleotides with polynucleotides containing common codons or codons taking codon degeneracy into account.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: It can be determined using a known computer algorithm such as the "FASTA" program using default parameters as in 2444. Or, as performed in the Needleman program in 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), It can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) (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), or BLAST from the National Center for Biotechnology Information Database. ClustalW can be used to determine homology, similarity, or identity.

Homology, similarity or identity of polynucleotides or polypeptides is defined in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, for example, Needleman et al. (1970), J Mol Biol. This can be determined by comparing sequence information using a GAP computer program such as 48:443. In summary, a GAP program can be defined as the number of similarly aligned symbols (i.e. nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The default parameters for the GAP program are (1) a binary comparison matrix (containing values 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: Weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) permutation 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 and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

The polynucleotide encoding the pantoate-beta-alanine ligase can be introduced into a microorganism using a recombinant vector, but is not limited thereto. The recombinant vector can be used as an insertion vector or an expression vector. Expressing the pantoate-beta-alanine ligase in a microorganism can be performed by culturing a recombinant microorganism containing a polynucleotide encoding the pantoate-beta-alanine ligase, or a recombinant vector containing the same. there is.

Introduction of the polynucleotide encoding the pantoate-beta-alanine ligase or a recombinant vector containing the same into a microorganism can be performed by appropriately selecting a known transformation method by a person skilled in the art. The transformation method can be performed by any method of introducing a nucleic acid into a host cell (microorganism), and can be performed by appropriately selecting transformation techniques known in the art depending on the host cell. The known transformation methods include electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) precipitation (polyethylene glycol-mediated uptake), Examples may include the DEAE-dextran method, cationic liposome method, lipofection, and lithium acetate-DMSO method, but are not limited thereto.

As long as the transformed polynucleotide can be expressed within the host cell, it may be inserted into and located within the chromosome of the host cell or may be located outside the chromosome. As long as the polynucleotide can be introduced and expressed into a host cell, the form in which it is introduced is not limited. For example, the polynucleotide can be introduced into a host cell in the form of an expression cassette, which is a genetic structure containing all elements necessary for self-expression. The expression cassette may typically include expression control elements such as a promoter, transcription termination signal, ribosome binding site, and/or translation termination signal that are operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replication. Additionally, the polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence required for expression in the host cell. As used herein, the term "operably linked" may mean that an expression control element (e.g., promoter) and a polynucleotide are functionally connected to perform transcriptional regulation (e.g., transcription initiation) of the polynucleotide. . Operable linkage can be accomplished using genetic recombination techniques known in the art.

As used in this application, the term “vector” refers to a DNA preparation for delivering a polynucleotide of interest into a suitable host or host cell. As an example, it may include a base 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. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating termination of transcription and translation. After transformation into a suitable host cell, the vector can replicate or function independently of the host genome and can be integrated into the genome itself. For example, a polynucleotide of interest can be inserted into a chromosome through a vector for chromosome insertion. Insertion of the polynucleotide into the chromosome may be accomplished by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker may be additionally included to confirm whether the chromosome has been inserted. The selection marker is used to select cells transformed with a vector, that is, to confirm the insertion of the target nucleic acid molecule, and is used to determine selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides. Markers that give may be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other expression traits, so transformed cells can be selected.

The vector used in this application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ-, pDC-, and pBR-based plasmid vectors can be used. , pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, pET-based, etc. can be used. For example, pDZ, pDC, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. can be used.

In this application, the term “enhancement” of 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. Here, activation, enhancement, upregulation, overexpression, and increase may include showing an activity that it did not originally have, or showing improved activity compared to the intrinsic activity or activity before modification. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the change in trait when the trait changes due to genetic mutation caused by natural or artificial factors. This can be used interchangeably with “activity before modification.” “Enhanced,” “upregulated,” “overexpressed,” or “increased” in the activity of a polypeptide compared to its intrinsic activity means that the activity and/or concentration of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the transformation. It means improved compared to (expression amount).

The enhancement can be achieved by introducing an exogenous polypeptide, enhancing the activity and/or increasing the concentration (expression amount) of the intrinsic polypeptide. Whether the activity of the polypeptide is enhanced can be confirmed from the level of activity of the polypeptide, the expression level, or an increase in the amount of products due to the activity of the polypeptide.

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

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

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

2) Replacement of the gene expression control region on the chromosome encoding the polypeptide with a highly active sequence;

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

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

5) modification of the polynucleotide sequence encoding the polypeptide to enhance the polypeptide activity (e.g., modification of the polynucleotide sequence of the polypeptide gene to encode a polypeptide modified 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) Analyzing the tertiary structure of the polypeptide, select exposed sites and modify or chemically modify them; or

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

More specifically,

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

2) Replacement of the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a highly active sequence, for example, deletion, insertion, or non-conservative use to further enhance the activity of the expression control region. Alternatively, it may be a mutation in the sequence due to conservative substitution or a combination thereof, or replacement with a sequence with 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 that regulates the termination of transcription and translation. As an example, the original promoter may be replaced with a strong promoter, but the method 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, etc., but is not limited thereto.

3) The base sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a base encoding another start codon with a higher polypeptide expression rate than the internal start codon. It may be a substitution by sequence, but is not limited thereto.

The modification of the amino acid sequence or polynucleotide sequence of 4) and 5) includes deletion, insertion, non-conservative or conservative amino acid sequence of the polypeptide or polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide. It may be a mutation in the sequence due to a substitution or a combination thereof, or a 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 limited thereto. no. 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 to check whether chromosome insertion has occurred.

6) Introduction of a foreign polynucleotide showing the activity of the polypeptide may be introduction into the host cell of a foreign polynucleotide encoding a polypeptide showing the same/similar activity as the polypeptide. There are no restrictions on the origin or sequence of the foreign polynucleotide as long as it exhibits the same/similar activity as the polypeptide. The method used for the introduction can be performed by appropriately selecting a known transformation method by a person skilled in the art, and by expressing the introduced polynucleotide in the host cell, a polypeptide can be produced and its activity can be increased.

7) Codon optimization of the polynucleotide encoding the polypeptide is codon optimization of the native polynucleotide to increase transcription or translation within the host cell, or codon optimization of the foreign polynucleotide to increase transcription or translation within the host cell. The codon may have been optimized to achieve this.

Above 8) Analyzing the tertiary structure of a polypeptide and selecting exposed sites to modify or chemically modify the sequence information, for example, by comparing the sequence information of the polypeptide to be analyzed with a database storing the sequence information of known proteins to determine sequence similarity. Depending on the degree, a template protein candidate may be determined, the structure may be confirmed based on this, and an exposed site to be modified or chemically modified may be selected and modified or modified.

Such enhancement of polypeptide activity means that the activity or concentration of the corresponding polypeptide is increased based on the activity or concentration of the polypeptide expressed in the wild type or unmodified microbial strain, or the amount of product produced from the polypeptide is increased. may be increased, but is not limited to this.

In this application, the term "microorganism (or strain)" includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, due to causes such as insertion of foreign genes or strengthening or weakening of the activity of intrinsic genes. As a result, it is a microorganism in which a specific mechanism is strengthened or weakened, and may be a microorganism that includes genetic modification for the production of a desired polypeptide, protein, or product (e.g., panthenol).

The microorganism of the present application may be a microorganism (e.g., a recombinant microorganism) that has been genetically modified to enhance the activity of pantoate-beta-alanine ligase or a polynucleotide encoding it, but is not limited thereto.

The microorganism (or strain, recombinant cell) of the present application may be a microorganism that has panthenol production ability or has improved panthenol production ability (or production volume).

The microorganism of the present application may be a microorganism that does not naturally have the ability to produce panthenol, or a microorganism that has the ability to produce panthenol or is given or enhanced the ability to produce panthenol by introducing or enhancing the activity of pantoate-beta-alanine ligase in a microorganism that has the ability to produce panthenol, but is not limited thereto. No. The microorganism endowed or improved with the ability to produce panthenol may be a microorganism into which pantoate-beta-alanine ligase derived from Glutamicibacter halophytocola has been introduced.

The fact that the microorganism (or strain, recombinant cell) has an improved panthenol production capacity (or production capacity) or beta-alanine or panthenol production capacity means that the microorganism (or strain, recombinant cell) is an unmodified microorganism, a cell before recombination, or a parent cell. Strains, wild-type strains, and/or microorganisms with improved panthenol production ability than microorganisms into which pantoate-beta-alanine ligase derived from other microorganisms has been introduced, or unmodified microorganisms without panthenol production ability, cells before recombination, parent strain, and/or wild-type strains. Unlike this, it may mean that the ability to produce panthenol is granted.

In one example, microorganisms with enhanced pantoate-beta-alanine ligase activity may have improved (increased) panthenol production ability compared to microorganisms before enhancement, that is, unmodified microorganisms of the same type. In this application, “unmodified microorganism” does not exclude strains containing mutations that may occur naturally in microorganisms, and is a wild-type strain or a natural strain itself, or whose characteristics change due to genetic mutation caused by natural or artificial factors. It may refer to a strain before becoming a strain. For example, the unmodified microorganism is a strain in which the activity of pantoate-beta-alanine ligase is not enhanced or is not enhanced (or a mutation that induces enhancement of the activity of pantoate-beta-alanine ligase is present). It may mean a strain that is not introduced or before being introduced. The “non-modified microorganism” may be used interchangeably with “pre-transformed strain”, “pre-transformed microorganism”, “non-mutated strain”, “non-modified strain”, “non-mutated microorganism” or “reference microorganism”. As described above, the activity of the pantoate-beta-alanine ligase is enhanced. In one example, the unmodified microorganism, which is the target strain to compare whether the panthenol production ability is increased, may be a wild-type Corynebacterium glutamicum ATCC13032 strain.

According to one example, the microorganism with enhanced activity of pantoate-beta-alanine ligase may be a microorganism into which pantoate-beta-alanine ligase derived from Glutamicibacter halophytocola has been introduced, and the microorganism may be derived from another microorganism. Compared to microorganisms into which pantoate-beta-alanine ligase was introduced, the panthenol production ability may be further improved (increased). The pantoate-beta-alanine ligase derived from the other microorganisms is a microorganism of the genus Corynebacterium (e.g., Corynebacterium glutamicum ), a microorganism of the genus Escherichia (e.g., Escherichia coli) coli ), microorganisms of the genus Coraliomargarita (e.g., Coraliomargarita akajimensis ), or microorganisms of the genus Actinobacteria (e.g., Actinobacteria bacterium ), microorganisms of the genus Nitrosospira ( For example, it may be from Nitrosospira lacus, or a microorganism of the genus Nitrosococcus (e.g., Nitrosococcus watsonii ).

The microorganism (or strain, recombinant cell) may additionally contain a mutation that increases panthenol production, and the location of the mutation and/or the type of gene and/or protein subject to the mutation are such that panthenol production is increased. May be included without limitation. The recombinant cells can be used without limitation as long as they are cells capable of transformation.

The panthenol may be DL-panthenol (CAS No. 16485-10-2) or D-panthenol (CAS No. 81-13-0), and in one example, it may be D-panthenol.

In one example, the microorganism with improved panthenol production ability has a panthenol production ability of about 10% or more, about 10% or more, compared to the parent strain before mutation, an unmodified microorganism, or a microorganism into which pantoate-beta-alanine ligase from another microorganism has been introduced. 20% or more, about 30% or more, about 50% or more, about 100% or more, about 150% or more, about 200% or more, about 250% or more, about 300% or more, about 350% or more, about 400% or more, about It may be increased by more than 450%, more than about 500%, more than about 600%, more than about 700%, more than about 800%, more than about 900%, or more than about 1000%, but is not limited thereto.

In another example, the microorganism with improved panthenol production ability has a panthenol production ability of about 1.1 times more, about 1.1 times or more, compared to the parent strain before mutation, an unmodified microorganism, or a microorganism into which pantoate-beta-alanine ligase from another microorganism has been introduced. 1.2 times or more, approximately 1.3 times or more, approximately 1.5 times or more, approximately 2 times or more, approximately 2.5 times or more, approximately 3 times or more, approximately 3.5 times or more, approximately 4 times or more, approximately 4.5 times or more, approximately 5 times or more, approximately It may be an increase of 6 times or more, about 7 times or more, about 8 times or more, about 9 times or more, or about 10 times or more, but is not limited thereto.

In another example, the microorganism with improved panthenol production ability has a panthenol production ability of about 0.1 g/l or more compared to the parent strain before mutation, an unmodified microorganism, or a microorganism into which pantoate-beta-alanine ligase from another microorganism has been introduced. , about 0.2 g/l or more, about 0.3 g/l or more, about 0.4 g/l or more, about 0.5 g/l or more, about 0.6 g/l or more, about 0.7 g/l or more, about 0.8 g/l or more, About 0.9 g/l or more, about 1.0 g/l or more, about 1.1 g/l or more, about 1.2 g/l or more, about 1.3 g/l or more, about 1.4 g/l or more, about 1.5 g/l or more, about An increase of more than 1.6 g/l, more than about 1.7 g/l, more than about 1.8 g/l, more than about 1.9 g/l, more than about 2.0 g/l, more than about 2.5 g/l, or more than about 3.0 g/l It may be possible, but it is not limited to this.

The term “about” is a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values in a range that are equivalent or similar to the value that appears after the term “about.” Not limited.

In one example, the microorganism with enhanced pantoate-beta-alanine ligase activity may be a Corynebacterium sp. microorganism. The microorganisms in the Corynebacterium genus include Corynebacterium glutamicum , Corynebacterium crudilactis , Corynebacterium deserti , and Corynebacterium eph. Corynebacterium efficiens , Corynebacterium callunae , Corynebacterium stationis , Corynebacterium singulare , Corynebacterium halotolerans ), Corynebacterium striatum , Corynebacterium ammoniagenes, Corynebacterium pollutisoli , Corynebacterium imitans , Corynebacterium It may be one or more types of microorganisms selected from the group consisting of Corynebacterium testudinoris and Corynebacterium flavescens.

Another aspect of the present application provides a method (or production method) for producing panthenol, including the step of culturing the microorganism of the present application in a medium.

The panthenol production method of the present application may include culturing the microorganism of the present application in a medium. The microorganisms, panthenol, etc. of the present application are as described above.

In the present application, “culturing” means growing a microorganism with enhanced activity of the panthoate-beta-alanine ligase of the present application, such as a Corynebacterium glutamicum strain, under appropriately controlled environmental conditions. The culture process of the present application can be carried out according to appropriate media and culture conditions known in the art. This culture process can be easily adjusted and used by a person skilled in the art depending on the strain selected. Specifically, the culture may be batch, continuous, and/or fed-batch, but is not limited thereto.

In the present application, “medium” refers to a material mixed as a main ingredient with the nutrients necessary for cultivating microorganisms with enhanced pantoate-beta-alanine ligase activity, such as Corynebacterium glutamicum strains of the present application. It supplies water, nutrients and growth factors that are essential for survival and development. Specifically, the medium and other culture conditions used for cultivating the microorganisms of the present application can be any medium used for cultivating ordinary microorganisms without particular restrictions, but the microorganisms of the present application can be grown with an appropriate carbon source, nitrogen source, personnel, and inorganic substances. It can be cultured under aerobic conditions in a typical medium containing compounds, amino acids, and/or vitamins while controlling temperature, pH, etc.

Specifically, culture media for microorganisms of the present application, such as strains 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, etc.; Sugar alcohols such as mannitol, sorbitol, etc., organic acids such as pyruvic acid, lactic acid, citric acid, etc.; Amino acids such as glutamic acid, methionine, lysine, etc. may be included. Additionally, natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice bran, cassava, bagasse and corn steep liquor can be used, specifically glucose and sterilized pre-treated molasses (i.e. converted to reducing sugars). Carbohydrates such as molasses) can be used, and various other carbon sources in an appropriate amount can be used without limitation. These carbon sources may be used alone or in combination of two or more types, but are not limited thereto.

The nitrogen source includes inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. can be used These nitrogen sources may be used alone or in combination of two or more types, but are not limited thereto.

The agent may include monopotassium phosphate, dipotassium phosphate, or a corresponding sodium-containing salt. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and/or appropriate precursors may be included. These components or precursors can be added to the medium batchwise or continuously. However, it is not limited to this.

In addition, during the cultivation of the microorganism of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium. Additionally, during culturing, foam generation can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, to maintain the aerobic state of the medium, oxygen or oxygen-containing gas can be injected into the medium, or to maintain the anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection, and is limited thereto. That is not the case.

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

Panthenol produced by the culture of the present application may be secreted into the medium or remain within the cells.

The panthenol production method of the present application includes preparing a microorganism (strain) of the present application, preparing a medium for culturing the microorganism, or a combination thereof (in any order), for example , may be additionally included before the culturing step.

The method for producing panthenol of the present application may further include the step of recovering panthenol from the culture medium (medium in which the culture was performed) or microorganisms (e.g., strains of the genus Corynebacterium). The recovering step may be additionally included after the culturing step.

The recovery may be to collect the desired panthenol using a suitable method known in the art according to the microorganism culture method of the present application, such as a batch, continuous, or fed-batch culture method. For example, centrifugation, filtration, crystallization, treatment with protein precipitants (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity. Various chromatography such as chromatography, HPLC, or a combination of these methods can be used, and the desired panthenol can be recovered from the medium or microorganism using a suitable method known in the art.

In addition, the panthenol production method of the present application may additionally include a purification step. The purification can be performed using a suitable method known in the art. In one example, when the panthenol 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 the order, or are performed simultaneously or integrated into one step. It may be, but is not limited to this.

Another aspect of the present application is to provide a composition for producing panthenol containing the microorganism of the present application, a medium culturing the microorganism, or a combination thereof.

Another aspect of the present application is to provide a use of the microorganism for producing panthenol.

Another aspect of the present application is to provide a use for using the microorganism in the production of a composition for producing panthenol.

The composition of the present application may further comprise any suitable excipients commonly used in compositions for the production of panthenol, such excipients may be, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents or isotonic agents, etc. However, it is not limited to this.

In the composition of the present application, the microorganism (strain), medium, panthenol, etc. are as described in the other aspects above.

The present application provides a microorganism with enhanced activity of pantoate-beta-alanine ligase derived from Glutamicibacter halopytocola, and the microorganism has an excellent ability to produce panthenol.

Hereinafter, the present application will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present application is not limited by these examples, and will be clear to those skilled in the art to which this application pertains.

Example 1. Construction of vector for enhancing panC gene expression

In this example, an enhancement vector was created to compare the production ability of panthenol when the expression of the panC gene, known as one of the panthenol biosynthetic genes, was enhanced.

Specifically, a recombinant plasmid vector was constructed using the following method to additionally insert six types of panC genes derived from Corynebacterium glutamicum and six foreign species into the Corynebacterium glutamicum ATCC13032 chromosome.

First, a vector was created to remove the NCgl2284 (Transposase) gene in order to insert the panC gene into the chromosome. Based on the Corynebacterium glutamicum NCgl2284 gene and surrounding sequence (sequence number 1) provided by the U.S. National Center for Biotechnology Information (NCBI), Corynebacterium glue was used to construct a vector for deleting the NCgl2284 gene on the chromosome. Using the chromosomal DNA of Tamicum ATCC13032 as a template, PCR was performed using primers of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively. The PCR was performed using PfuUltraTM high-reliability DNA polymerase (Stratagene), and the PCR conditions were 30 cycles of denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute. After repeating, the polymerization reaction was performed at 72°C for 5 minutes. As a result, DNA fragments of 1 Kb each were obtained. The pDCM2 vector (Korean Patent No. 10-2278000), which cannot replicate in Corynebacterium glutamicum treated with the restriction enzyme smaI, and the amplified NCgl2284 gene fragment were mixed at a molar concentration (M) of 2:1:1 (pDCM2). Vector: NCgl2284 fragment 1: NCgl2284 fragment 2) was cloned using TaKaRa's Infusion Cloning Kit according to the provided manual to obtain a plasmid, and was named pDCM2-△NCgl2284.

The following panC genes from six species derived from Corynebacterium glutamicum and foreign species were obtained. Specifically, the panC gene derived from Corynebacterium glutamicum or Escherichia coli is extracted from Corynebacterium glutamicum ATCC13032 strain or Escherichia coli K-12 substr MG1655 strain. Genomic DNA was obtained by performing PCR using primers of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12, and SEQ ID NO: 13, respectively, as a template. Coraliomargarita akajimensis , Actinobacteria bacterium, Nitrosospira lacus, Nitrosococcus watsonii , and Glutamicibacter halophytocola. halophytocola )-derived panC gene was created by synthesizing the PanC protein coding sequence based on the sequences in NCBI's Genbank database (Accession: CP001998.1, CP011489.1, CP021106.3, CP002086.1, and CP012750.1) and using this as a template. PCR was performed using primers of SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 20 and SEQ ID NO: 21, SEQ ID NO: 24 and SEQ ID NO: 25, SEQ ID NO: 28 and SEQ ID NO: 29, SEQ ID NO: 32, and SEQ ID NO: 33, respectively. Acquired. PCR was performed using PfuUltraTM high-reliability DNA polymerase (Stratagene), and the PCR conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, repeated 30 times. Afterwards, polymerization reaction was performed at 72°C for 5 minutes. In addition, to use the CJ7 promoter derived from Corynebacterium ammoniagenes (Korean Patent No. 10-0620092) as a promoter to induce the expression of panC in six species derived from Corynebacterium glutamicum and foreign species, Corynebacterium Using the chromosomal DNA sequence of Rhium ammoniagenes ATCC6872 as a template, SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 14 and SEQ ID NO: 15, SEQ ID NO: 18 and SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: PCR was performed using primers of SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, and SEQ ID NO: 31. PCR was performed using PfuUltraTM high-reliability DNA polymerase (Stratagene), and the PCR conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds, repeated 30 times. Afterwards, polymerization reaction was performed at 72°C for 5 minutes. As a result, it was possible to obtain panC gene fragments from one Corynebacterium glutamicum species and six foreign species, as well as CJ7 promoters for each of the seven panC genes.

pDCM2-△NCgl2284 vector treated with restriction enzyme SmaI The amplified CJ7 promoter and panC gene fragments were each grown at a molar concentration (M) of 2:1:1 (pDCM2-△NCgl2284 vector: CJ7 promoter: panC) and incubated at TaKaRa. After reaction according to the manual provided using the Infusion Cloning Kit, it was transformed into E. coli DH5α and plated on LB solid medium containing kanamycin (25 mg/l). PCR was performed using primers of SEQ ID NO: 34 and SEQ ID NO: 35, and colonies transformed with the vector into which the target gene was inserted were selected. A total of 7 plasmids were extracted using the plasmid extraction method and named as follows:

pDCM2_△NCgl2284::Pcj7-panC(Cgl) (panC introduction vector derived from Corynebacterium glutamicum)

pDCM2_△NCgl2284::Pcj7-panC(Eco) (panC introduction vector from Escherichia coli)

pDCM2_△NCgl2284::Pcj7-panC(Cak) (panC introduction vector from Coralliomargarita acajimensis)

pDCM2_△NCgl2284::Pcj7-panC(Aba) (panC introduction vector from Actinobacterium bacterium)

pDCM2_△NCgl2284::Pcj7-panC(Nla) (panC introduction vector from Nitrosospira lacus),

pDCM2_△NCgl2284::Pcj7-panC(Nwa) (panC introduction vector from Nitrosococcus Watsoni)

pDCM2_△NCgl2284::Pcj7-panC(Gha) (panC introduction vector from Glutamicibacter halopytocola).

The names, introduced gene information, and sequence information of the seven types of plasmids obtained above are shown in Table 1 below.

Gene and sequence information for construction of Corynebacterium glutamicum and 6 foreign panC gene enhancement vectors PanC origin plasmid Primer SEQ ID NO: Corynebacterium glutamicum
* Gene sequence: SEQ ID NO: 36 pDCM2_△NCgl2284::Pcj7-panC(Cgl) SEQ ID NO: 6, 7 / 8, 9 Escherichia coli
* Gene sequence: SEQ ID NO: 37 pDCM2_△NCgl2284::Pcj7-panC(Eco) SEQ ID NO: 10, 11 / 12, 13 Coraliomargarita akajimensis
* Gene sequence: SEQ ID NO: 38 pDCM2_△NCgl2284::Pcj7-panC(Cak) SEQ ID NO: 14, 15 / 16, 17 Actinobacteria bacterium
* Gene sequence: SEQ ID NO: 39 pDCM2_△NCgl2284::Pcj7-panC(Aba) SEQ ID NO: 18, 19 / 20, 21 Nitrosospira lacus
* Gene sequence: SEQ ID NO: 40 pDCM2_△NCgl2284::Pcj7-panC(Nla) SEQ ID NO: 22, 23 / 24, 25 Nitrosococcus watsonii
* Gene sequence: SEQ ID NO: 41 pDCM2_△NCgl2284::Pcj7-panC(Nwa) SEQ ID NO: 26, 27 / 28, 29 Glutamicibacter halophytocola
* Gene sequence: SEQ ID NO: 42 pDCM2_△NCgl2284::Pcj7-panC(Gha) SEQ ID NO: 30, 31 / 32, 33

Example 2. Evaluation of panthenol production ability of pantoate-beta-alanine-ligase enhanced strain

pDCM2-△NCgl2284, pDCM2-△NCgl2284::Pcj7-panC(Cgl), pDCM2-△NCgl2284::Pcj7-panC(Eco), pDCM2-△NCgl2284::Pcj7-panC(Cak) produced in Example 1, pDCM2-△NCgl2284::Pcj7-panC(Aba), pDCM2_△NCgl2284::Pcj7-panC(Nla), pDCM2_△NCgl2284::Pcj7-panC(Nwa) and pDCM2-△NCgl2284::Pcj7-panC(Gha) vectors. were transformed into the wild-type Corynebacterium glutamicum ATCC13032 strain by electroporation by homologous recombination on the chromosome. (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Afterwards, a second recombination was performed on a solid medium containing sucrose. Using the primers of SEQ ID NO: 34 and SEQ ID NO: 35, the Corynebacterium glutamicum transformant for which secondary recombination was completed was subjected to PCR to produce a strain with NCgl2284 deleted and a strain with NCgl2284 deleted and the panC gene inserted. The strains were confirmed, and each recombinant strain was 13032_△NCgl2284, CJPTN-01 (13032_△NCgl2284::Pcj7-panC(Cgl)), CJPTN-02 (13032_△NCgl2284::Pcj7-panC(Eco)), CJPTN- 03 (13032_△NCgl2284::Pcj7-panC(Cak)), CJPTN-04 (13032_△NCgl2284::Pcj7-panC(Aba)), CJPTN-05 (13032_△NCgl2284::Pcj7-panC(Nla)), CJPTN They were named -06 (13032_△NCgl2284::Pcj7-panC(Nwa)) and CJPTN-07 (13032_△NCgl2284::Pcj7-panC(Gha)).

To analyze the panthenol production ability of the above strains, they were cultured in the following manner.

Inoculate 13032_△NCgl2284, CJPTN-01, CJPTN-02, CJPTN-03, CJPTN-04, CJPTN-05, CJPTN-06 and CJPTN-07 in a 250 ml corner-baffle flask containing 25 ml of the following production medium. , the culture obtained by shaking at 200 rpm for 40 hours at 33°C was centrifuged at 20,000 rcf for 10 minutes, the supernatant was diluted 1/5 with TDW (triple distilled water), and then HPLC analysis was performed to measure the panthenol concentration. , the results are shown in Table 2 below.

<Panthenol production medium>

Glucose 10% (w/v), yeast extract 0.4% (w/v), ammonium sulfate 1.5% (w/w), monobasic potassium phosphate 0.1% (w/v), magnesium sulfate heptahydrate 0.05% (w/ v), iron sulfate heptahydrate 10 mg/l, manganese sulfate monohydrate 6.7 mg/l, biotin 50 μg/l, thiamine·HCl 100 μg/l, 3-aminopropanol 0.3%, pantoate 0.3%, pH 7.0

Panthenol production ability of strains with increased panC expression Panthenol concentration (g/L) 13032_ΔNCgl2284 0.0 CJPTN-01 0.0 CJPTN-02 0.2 CJPTN-03 0.0 CJPTN-04 0.0 CJPTN-05 0.0 CJPTN-06 0.0 CJPTN-07 0.9

As can be seen in Table 2, strain CJPTN-01 overexpressing the endogenous pantoate-beta-alanine-ligase did not produce panthenol, and pantoate-beta-alanine-derived from Escherichia coli. Ligase overexpression strain CJPTN-02 produced 0.2 g/L of panthenol.

Pantoate-beta-alanine-ligase from Coraliomargarita akajimensis , Actinobacteria bacterium, Nitrosospira lacus, and Nitrosococcus watsonii The enzyme overexpression strains CJPTN-03, CJPTN-04, CJPTN-05 and CJPTN-06 failed to produce panthenol, and the pantoate-beta-alanine-ligase overexpression strain from Glutamicibacter halophytocola CJPTN-07 produced 0.9 g/L panthenol, which is 4.5 times higher than the strain into which PanC from Escherichia coli was introduced.

Through the above results, it was confirmed that panthenol production was significantly increased by pantoate-beta-alanine-ligase derived from Glutamicibacter halophytocola.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be interpreted as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Sequence list electronic file attached

Claims (12)

A microorganism producing panthenol into which pantoate-beta-alanine ligase derived from Glutamicibacter halophytocola or a polynucleotide encoding the same has been introduced. The microorganism according to claim 1, wherein the pantoate-beta-alanine ligase is a PanC protein. The microorganism according to claim 1, wherein the pantoate-beta-alanine ligase comprises the amino acid sequence of SEQ ID NO: 43 or an amino acid sequence having at least 90% sequence identity thereto. The microorganism according to claim 1, wherein the pantoate-beta-alanine ligase is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 42. The microorganism according to claim 1, wherein the microorganism is a microorganism of the genus Corynebacterium. The microorganism according to claim 5, wherein the microorganism of the Corynebacterium genus is Corynebacterium glutamicum. The microorganism according to any one of claims 1 to 6, wherein the microorganism has an increased ability to produce panthenol compared to the parent strain in which the pantoate-beta-alanine ligase or the polynucleotide encoding it is not introduced. A method for producing panthenol, comprising culturing a microorganism into which pantoate-beta-alanine ligase derived from Glutamicibacter halophytocola or a polynucleotide encoding the same has been introduced in a medium. The method of claim 8, wherein the microorganism is a microorganism of the genus Corynebacterium. The method of claim 8, further comprising the step of recovering panthenol from the culture medium or microorganism. A composition for producing panthenol comprising a microorganism into which pantoate-beta-alanine ligase derived from Glutamicibacter halopytocola or a polynucleotide encoding the same has been introduced. The composition for producing panthenol according to claim 11, wherein the microorganism is a microorganism of the genus Corynebacterium.