Novel promoter for constitutive expression, target protein expression system including the same, and method for producing allulose using the same

Abstract

To provide a novel promoter that can consistently express a target protein at a high level, and to provide a target protein expression system and a method for producing allulose, as various uses of the promoter.SOLUTION: Provided is a promoter that is composed of a specific base sequence and that regulates the expression of allulose epimerization enzyme in Corynebacterium strains. The novel promoter according to the present invention is capable of constitutively and highly expressing a target protein, particularly an enzyme, in Corynebacterium strains. As an example, the use of a recombinant Corynebacterium strain transformed with an expression vector containing the novel promoter according to the present invention makes it possible to economically mass-produce allulose epimerization enzyme and to economically mass-produce allulose from fructose.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a novel promoter and its use, and more particularly to a novel promoter capable of constantly highly expressing a target protein, a target protein expression system containing the same, and a method for producing allulose using the same.

Background Art

[0002] With the development of molecular biology, various mechanisms for regulating gene expression have been revealed. Gene expression refers to a series of processes in which proteins are synthesized according to the codes input into genes through transcription and translation occurring in cells. In particular, the transcription process is the initial stage of gene expression, which is initiated by RNA polymerase binding to the promoter sequence existing upstream of the gene with the help of numerous cofactors. Transcription factors (TFs) are one of such cofactors and are known to directly bind to the promoter sequence. In particular, since the regulation of gene expression in prokaryotes mainly occurs at the transcription stage, new transcription factors and promoters have been continuously revealed by the present researchers.

[0003] In order to industrially produce foreign proteins such as enzymes, a transformant prepared by transforming a prokaryote such as Escherichia coli with a pET-type expression vector containing a foreign protein gene is mainly used as an expression system. In a prokaryotic expression system transformed with a pET-type expression vector, generally, an expensive expression inducer such as IPTG (isopropyl-β-D-thiogalactopyranoside) is required, and there are disadvantages that the inducer concentration, equipment, expression induction time, etc. must be finely adjusted.

[0004] On the one hand, in order to mass-produce psicose epimerase (or allulose epimerase) having an activity of converting fructose into allulose (or psicose), an expression system using a Corynebacterium strain, which is a GRAS (Generally Recognized As Safe) strain, as a host cell has been presented. In relation to the Corynebacterium strain-based psicose epimerase (or allulose epimerase) expression system, Korean Registered Patent No. 10-1695830 discloses a nucleic acid sequence encoding psicose epimerase and a regulatory sequence that is operably linked upstream thereof and regulates the expression of the psicose epimerase in a Corynebacterium strain, and the regulatory sequence includes a transcription promoter derived from Escherichia coli (E. coli) selected from a trc promoter, a Tac1 promoter, and a Tac2 promoter or a sod promoter that is a transcription promoter derived from Corynebacterium glutamicum. Generally, a binding site for RNA polymerase is conserved in a promoter, and binding sites for transcription factors that promote or suppress various RNA expressions are present in the 5'UTR sequence and the 3'UTR sequence centered on the core promoter site, and the gene sequence around the promoter sequence is important for improving the expression efficiency. In the Corynebacterium strain-based psicose epimerase expression system disclosed in the prior art, since the promoter sequence is located at a bidirectional promoter site where the McrA gene and the sod gene are arranged to be expressed in opposite directions to each other, a high-level regulatory mechanism for the expression of bidirectional genes is required, and it is not suitable as a system for the constitutive expression of a desired single target protein.In addition, the psicose epimerase expression system based on Corynebacterium strains disclosed in the prior art has relatively weak expression intensity due to the structural hindrance of the promoter or the complex regulation of the promoter, and is not suitable for the large-scale production of psicose.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention has been derived under the background of the prior art. The object of the present invention is to provide a novel promoter capable of constantly highly expressing a target protein. In addition, as various uses of the novel promoter, the object of the present invention is to provide a target protein expression system, a method for producing allulose, and the like.

Means for Solving the Problems

[0006] The inventors of the present invention deleted some fragments from the bidirectional promoter site existing between the McaA gene and the sod gene in the genomic sequence of Corynebacterium glutamicum ATCC 13032 strain to create a promoter with bidirectionality eliminated. Among them, it was confirmed that a specific promoter can constantly highly express allulose epimerase in Corynebacterium strains, and thus the present invention was completed. In addition, the inventors of the present invention operably linked the promoter with bidirectionality eliminated to a polynucleotide encoding allulose epimerase to create a recombinant expression vector, and introduced the recombinant expression vector into Corynebacterium glutamicum, which is a GRAS (Generally Recognized As Safe) strain, and transformed it. As a result, random mutations occurred in some promoters, and among the promoter mutants obtained through random mutations, it was confirmed that a specific promoter mutant significantly increased the expression efficiency of allulose epimerase, and thus the present invention was completed.

[0007] To solve the above problems, an example of the present invention provides a promoter composed of the nucleotide sequence of SEQ ID NO: 6 or the nucleotide sequence of SEQ ID NO: 27, which regulates the expression of allulose epimerase in Corynebacterium strains.

[0008] To solve the above problems, an example of the present invention provides an allulose epimerase expression cassette comprising a polynucleotide encoding allulose epimerase and a promoter operably linked thereto. The promoter is composed of the nucleotide sequence of SEQ ID NO: 6 or the nucleotide sequence of SEQ ID NO: 27.

[0009] To solve the above problems, an example of the present invention provides a recombinant expression vector into which the allulose epimerase expression cassette is inserted.

[0010] To solve the above problems, an example of the present invention provides a recombinant Corynebacterium strain, which is obtained by transforming a Corynebacterium host strain with an allulose epimerase expression cassette or a recombinant expression vector into which the expression cassette is inserted.

[0011] To solve the above problems, an example of the present invention provides a method for producing allulose, which includes a step of adding a recombinant Corynebacterium strain to a fructose-containing solution and reacting them.

Advantages of the Invention

[0012] The novel promoter according to the present invention can constantly and highly express a target protein, particularly an enzyme, in a Corynebacterium strain. As an example, when using a recombinant Corynebacterium strain transformed with an expression vector containing the novel promoter according to the present invention, allulose epimerase can be economically mass-produced, or allulose can be economically mass-produced from fructose.

Embodiments for Carrying Out the Invention

[0013] Hereinafter, the present invention will be specifically described. The term "promoter" used in the present invention means the minimum nucleic acid sequence that is operably linked to a target nucleotide sequence to be transcribed and regulates the transcription of the target nucleotide sequence. Further, the promoter may include a promoter configuration sufficient to express a regulatable promoter-dependent gene induced by a cell type-specific or external signal or agent, and such a configuration can be located in the 5' or 3' region of the gene. The promoter includes both a conserved promoter and an inducible promoter. The promoter sequence can be derived from a prokaryote, a eukaryote, or a virus. A promoter in a prokaryote is generally defined as a binding site in the vicinity of a transcription start site to which RNA polymerase binds.

[0014] As used herein, the term "homology" refers to identity with the nucleic acid sequence of a wild type or a mutant having the same activity. The homology comparison can be performed visually or using a readily available comparison program to calculate the homology between two or more sequences as a percentage (%). Also, "homology" is used to indicate identity with the amino acid sequence of a wild type or a mutant having the same activity.

[0015] As used herein, the term "target protein" refers to a foreign protein that cannot normally exist in a transformed strain (or host cell) expressing the protein.

[0016] As used herein, the term "polynucleotide" means all non-modified or modified polyribonucleotides (RNA) or polydeoxyribonucleotides (DNA). The polynucleotide includes, but is not limited to, single-stranded or double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, or hybrid molecules thereof.

[0017] As used herein, the term "operably linked" is defined as a state in which a promoter sequence and a nucleotide sequence encoding a target protein are functionally linked such that the promoter can regulate the expression of the target protein. For example, when a promoter can control the expression of a coding sequence (i.e., when the coding sequence is under the transcriptional regulation of the promoter), the promoter is operably linked to and operates with the coding sequence, or the ribosome binding site is located such that it can promote translation, then the ribosome binding site is operably linked to and operates with the coding sequence. The coding sequence is operably linked to the regulatory sequence in the sense or antisense direction.

[0018] The term "recombinant vector" used in the present invention is defined as recombinant DNA prepared by excising a promoter variant or a target gene using a restriction enzyme and inserting it into a vector.

[0019] The term "expression cassette" used in the present invention means a regulatory sequence functionally linked to a nucleotide sequence to be expressed, for example, a polynucleotide sequence encoding an allulose epimerase. Therefore, unlike an expression unit, an expression cassette includes not only nucleotide sequences that regulate transcription and translation, but also nucleotide sequences that are expressed as proteins as a result of transcription and translation.

[0020] The term "expression vector" used in the present invention is defined as a DNA sequence necessary for transcription and translation of DNA cloned in an appropriate host. Specifically, when present in the cells of an individual, it means a gene product containing essential regulatory elements operably linked to the insert so that the insert is expressed. Expression vectors can be produced and purified using standard recombinant DNA techniques. The type of the expression vector is not particularly limited as long as it functions to express a desired gene and produce a desired protein in various host cells of prokaryotic and eukaryotic cells. The expression vector includes at least a promoter, a start codon, a gene encoding a desired protein, and a stop codon terminator. In addition, the expression vector can appropriately include, among other things, DNA encoding a signal peptide, additional expression regulatory sequences, untranslated regions on the 5' side and 3' side of the desired gene, a selection marker region, or a replicable unit. The selection marker region can be a selection marker gene for an antibiotic for selecting a target vector.

[0021] As used herein, the term "recombinant strain" means a cell into which a polynucleotide encoding one or more target proteins or an expression vector having the same has been introduced and transformed into a host cell. As methods for introducing the expression vector into a host cell to produce a transformant, there are transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation method, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, electroinjection, chemical treatment methods such as PEG, methods using a gene gun, heat shock method, etc., but are not limited thereto. In addition, the host cell of the recombinant strain is not greatly limited in its type as long as the promoter present in the expression vector can operate smoothly, and it is preferably a prokaryote.

[0022] As used herein, the term "substrate" refers to any substance or compound that is or is to be converted into another compound by the action of an enzyme. The term includes not only a single compound, but also combinations of compounds such as solvents, mixtures, and other materials containing at least one substrate, as well as derivatives thereof.

[0023] One aspect of the present invention relates to a novel promoter capable of constantly highly expressing a target protein. The novel promoter according to an example of the present invention is composed of the nucleotide sequence of SEQ ID NO: 6 or the nucleotide sequence of SEQ ID NO: 27. The inventor of the present invention named the promoter composed of the nucleotide sequence of SEQ ID NO: 6 as "Pds2" and the promoter composed of the nucleotide sequence of SEQ ID NO: 27 as "Pds4". The promoter Pds2 is a promoter in which a part of the fragment is deleted from the bidirectional promoter site existing between the McaA gene and the sod gene in the genomic sequence of the Corynebacterium glutamicum strain, the bidirectionality is eliminated, and the expression efficiency of the target protein is improved. Further, the promoter Pds4 is a promoter mutant in which 6 bp of nucleotides located at positions 195 to 200 of the promoter Pds2 are deleted and 3 new bp of nucleotides are inserted, and is generated by a ribosome-binding site (RBS) spacer mutation. An expression system containing the promoter according to an example of the present invention can constantly highly express a target protein in a Corynebacterium strain. In particular, the promoter according to an example of the present invention regulates the expression of allose epimerase in a Corynebacterium strain. Therefore, the promoter Pds2 or the promoter Pds4 according to an example of the present invention can be used as a promoter for constitutive expression in a Corynebacterium strain. The novel promoter according to an example of the present invention is composed of the nucleotide sequence of SEQ ID NO: 6 or the nucleotide sequence of SEQ ID NO: 27, but the equivalent scope of the promoter is not necessarily limited thereto. For example, the equivalent scope of the novel promoter according to an example of the present invention includes sequences having substantial identity to the nucleotide sequence of SEQ ID NO: 6 or the nucleotide sequence of SEQ ID NO: 27.The above-mentioned substantial identity means aligning the nucleotide sequence of SEQ ID NO: 6 or the nucleotide sequence of SEQ ID NO: 27 with any other sequence to maximize correspondence, analyzing the sequence, and meaning that the any other sequence has a sequence homology of 70% or more, 90% or more, or 98% or more with the nucleotide sequence of SEQ ID NO: 6 or the nucleotide sequence of SEQ ID NO: 27. A person skilled in the art can easily understand that polynucleotides having the same or similar activities can be produced within the range having substantial homology by substituting, adding or deleting one or more bases in the nucleotide sequence of the novel promoter using gene recombination techniques known in the art. Such homology comparison can be performed by calculating the homology between two or more sequences as a percentage (%) using a commercially available computer program. Therefore, the equivalent range of the novel promoter according to an example of the present invention can include a nucleotide sequence having a homology of 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more with the nucleotide sequence of SEQ ID NO: 6 or the nucleotide sequence of SEQ ID NO: 27 within the range where the function of constantly highly expressing the target protein is maintained.

[0024] One aspect of the present invention relates to a target protein expression system containing a novel promoter. The novel promoter can be used for the production of an allulose epimerase expression cassette, a recombinant vector, and a recombinant strain.

[0025] An allulose epimerase expression cassette according to an example of the present invention includes a polynucleotide encoding an allulose epimerase and the aforementioned promoter operably linked thereto. The promoter is preferably located upstream of the polynucleotide encoding the allulose epimerase, which is the target protein. The promoter, which is a component of the allulose epimerase expression cassette, is composed of the nucleotide sequence of SEQ ID NO: 6 or the nucleotide sequence of SEQ ID NO: 27. The polynucleotide encoding the allulose epimerase, which is a component of the allulose epimerase expression cassette, is not greatly limited in type as long as it is a polynucleotide encoding an enzyme having an activity to convert fructose into allulose. For example, the allulose epimerase may be derived from Flavonifractor plautii, Clostridiun scidens, Treponema primitia, Ensifer adhaerens or Ruminococcus torques, and is preferably derived from Flavonifractor plautii when considering the conversion activity of fructose into allulose. Specifically, the allulose epimerase can be composed of the amino acid sequence of SEQ ID NO: 14, the amino acid sequence of SEQ ID NO: 16 or the amino acid sequence of SEQ ID NO: 18. The allulose epimerase composed of the amino acid sequence of SEQ ID NO: 14 is a wild-type enzyme derived from Flavonifractor plautii. The allulose epimerase composed of the amino acid sequence of SEQ ID NO: 16 has the tryptophan (Trp) at the 29th position in the amino acid sequence of SEQ ID NO: 14 replaced by lysine (Lys), the glycine (Gly) at the 216th position replaced by serine (Ser), and at the same time the methionine (Met) at the 234th position replaced by isoleucine (Ile).The allulose epimerase composed of the amino acid sequence of SEQ ID NO: 18 is such that tryptophan (Trp) at the 29th position in the amino acid sequence of SEQ ID NO: 14 is substituted with lysine (Lys), alanine (Ala) at the 77th position is substituted with serine (Ser), glycine (Gly) at the 216th position is substituted with serine (Ser), and simultaneously methionine (Met) at the 234th position is substituted with isoleucine (Ile). The polynucleotide encoding the allulose epimerase is not highly restricted in its type, and preferably, it can be composed of the nucleotide sequence of SEQ ID NO: 15 or include a nucleotide sequence having 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more homology with the nucleotide sequence of SEQ ID NO: 15. The nucleotide sequence of SEQ ID NO: 15 is a polynucleotide encoding the allulose epimerase composed of the amino acid sequence of SEQ ID NO: 14. Further, the polynucleotide encoding the allulose epimerase can be composed of the nucleotide sequence of SEQ ID NO: 17 or the nucleotide sequence of SEQ ID NO: 19. The nucleotide sequence of SEQ ID NO: 17 is a polynucleotide encoding the allulose epimerase composed of the amino acid sequence of SEQ ID NO: 16, and the nucleotide sequence of SEQ ID NO: 19 is a polynucleotide encoding the allulose epimerase composed of the amino acid sequence of SEQ ID NO: 18. The present invention relates to an allulose epimerase and a polynucleotide encoding the same, and includes the contents disclosed in Korean Patent Registration Publication No. 10-1919713, Korean Patent Registration Publication No. 10-2187354, Korean Patent Registration Publication No. 10-1656063, Korean Patent Registration Publication No. 10-1695830, Korean Patent Registration Publication No. 10-2189458, Korean Patent Registration Publication No. 10-1539097, Korean Patent Registration Publication No. 10-1539096, Korean Patent Registration Publication No. 10-1455759, Korean Patent Registration Publication No. 10-1318422, Korean Patent Publication No. 10-2023-0073739, etc.

[0026] An allulose epimerase expression cassette according to an example of the present invention may further include one or more sequences selected from the group consisting of a replication origin, a multi-cloning site (MCS) for cloning a target protein gene, a transcription termination sequence, and a selection marker. The selection marker is for screening cells transformed with the vector, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins can be used. For example, the selection marker may be an antibiotic resistance gene marker such as a kanamycin antibiotic resistance gene or an ampicillin antibiotic resistance gene. An allulose epimerase expression cassette according to an example of the present invention preferably can include a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 25 or a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 28. The polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 25 is an expression cassette fragment in which a promoter consisting of the nucleotide sequence of SEQ ID NO: 6 and an allulose epimerase gene consisting of the nucleotide sequence of SEQ ID NO: 19 are sequentially linked. Also, the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 28 is a fragment of an expression cassette in which a promoter consisting of the nucleotide sequence of SEQ ID NO: 27 and an allulose epimerase gene consisting of the nucleotide sequence of SEQ ID NO: 19 are sequentially linked.

[0027] A recombinant vector according to an example of the present invention is a recombinant expression vector into which the above-described allulose epimerase expression cassette is inserted. The recombinant expression vector preferably has a structure in which a replication origin, a promoter, a polynucleotide encoding allulose epimerase, a transcription terminator, a kanamycin resistance gene marker, etc. are sequentially linked.

[0028] A recombinant strain according to an example of the present invention is obtained by transforming a host cell by introducing the aforementioned allulose epimerase expression cassette or a recombinant expression vector into which the expression cassette has been inserted, and is a recombinant Corynebacterium strain. The host strain used for producing the recombinant Corynebacterium strain is not greatly limited as long as it is a Corynebacterium strain. For example, it can be selected from the group consisting of Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium thermoaminogenes, Corynebacterium melassecola, and Corynebacterium efficiens.

[0029] One aspect of the present invention relates to a method for producing allulose using a target protein expression system containing a novel promoter.

[0030] An allulose production method according to an example of the present invention includes a step of adding a recombinant Corynebacterium strain to a fructose-containing solution and reacting them. The fructose acts as a mechanism of the allulose epimerase, which is the target protein expressed by the recombinant Corynebacterium strain. The fructose-containing solution contains Ca 2+ , Mn 2+It can further contain metal ions such as [metal ions]. Also, in the method for producing allose from the fructose, the reaction temperature is 50 to 70 °C, preferably 55 to 65 °C, and more preferably in the range of 60 to 65 °C when considering the smooth enzyme expression of the recombinant strain, the stability and maximum activity of the enzyme. The reaction pH is in the range of 6.5 to 8, preferably 6.5 to 7.5, and more preferably 6.5 to 7. Further, in the method for producing allose from the fructose, the fructose concentration of the fructose-containing solution is not particularly limited, but when considering productivity or economy, it is preferably 5 to 75% (w / w) based on the total weight of the fructose-containing solution, and more preferably 10 to 55% (w / w).

[0031] Hereinafter, the present invention will be described more specifically through examples. However, the following examples are only for clearly exemplifying the technical features of the present invention and do not limit the protection scope of the present invention.

Examples

[0032] Example 1: Amplification and acquisition of a promoter sequence for the expression of D-allose 3-epimerase To obtain the bidirectional promoter present in the Corynebacterium sp. strain, genomic DNA of Corynebacterium glutamicum ATCC 13032 strain was used as a template, and PCR was performed using the Pctrl-F primer and Pds-R primer set described in Table 1 below. The resulting amplification product was cloned into the pGEM T-easy vector (Promega Co., USA), and the nucleotide sequence was analyzed. As a result, it was confirmed that it was a polynucleotide fragment having a length of 336 bp and composed of the nucleotide sequence of SEQ ID NO: 1. In the polynucleotide fragment composed of the nucleotide sequence of SEQ ID NO: 1, the site corresponding to the promoter was named "Pctrl". The promoter Pctrl is composed of the nucleotide sequence of SEQ ID NO: 2. Also, to obtain a mutant promoter of promoter Pctrl, genomic DNA of Corynebacterium glutamicum ATCC 13032 strain was used as a template, and PCR for amplifying each mutant promoter was performed using the following primer sets: Pds1-F and Pds-R primer set; Pds2-F and Pds-R primer set; Pds3-F and Pds-R primer set described in Table 1 below. The resulting amplification products were cloned into the pGEM T-easy vector (Promega Co., USA), and the nucleotide sequences were analyzed. As a result, the amplification product obtained using the Pds1-F primer and Pds-R primer set had a length of 282 bp and was a polynucleotide fragment composed of the nucleotide sequence of SEQ ID NO: 3. In the polynucleotide fragment composed of the nucleotide sequence of SEQ ID NO: 3, the site corresponding to the promoter was named "Pds1". The promoter Pds1 is composed of the nucleotide sequence of SEQ ID NO: 4. Also, the amplification product obtained using the Pds2-F primer and Pds-R primer set had a length of 236 bp and was a polynucleotide fragment composed of the nucleotide sequence of SEQ ID NO: 5.Among the polynucleotide fragments composed of the nucleotide sequence of SEQ ID NO: 5, the site corresponding to the promoter was named "Pds2". The promoter Pds2 is composed of the nucleotide sequence of SEQ ID NO: 6. Also, the amplification product obtained using the Pds3-F primer and Pds-R primer set had a length of 236 bp and was a polynucleotide fragment composed of the nucleotide sequence of SEQ ID NO: 7. Among the polynucleotide fragments composed of the nucleotide sequence of SEQ ID NO: 7, the site corresponding to the promoter was named "Pds3". The promoter Pds3 is composed of the nucleotide sequence of SEQ ID NO: 8. The promoter Pctrl is a bidirectional promoter containing an intergenic region existing between the McaA gene and the sod gene in the genomic sequence of Corynebacterium glutamicum ATCC 13032 strain. The promoters Pds1, Pds2, and Pds3 are promoter mutants in which a partial sequence of the promoter Pctrl is deleted centering on an intergenic region having a length of 282 bp.

Table 1

[0033] Example 2: Amplification and acquisition of a polynucleotide sequence encoding D-allose 3-epimerase The applicant of the present invention disclosed a wild-type D-allose epimerase derived from Flavonifractor plautii and a polynucleotide encoding the same through Korean Patent Registration Publication No. 10-14739180. The wild-type D-allose epimerase is composed of the amino acid sequence of SEQ ID NO: 14, and the polynucleotide encoding the same is composed of the nucleotide sequence of SEQ ID NO: 15.

[0034] In addition, the applicant of the present invention disclosed a D-allose epimerase mutant W29K / G216S / M234I with improved conversion rate of fructose to allose and thermal stability, and a polynucleotide encoding the same, through Korean Patent Publication No. 10-2021-0132405. The D-allose epimerase mutant W29K / G216S / M234I has the tryptophan (Trp) at the 29th position in the amino acid sequence of the wild-type D-allose epimerase derived from Flavonifractor plautii replaced by lysine (Lys), the glycine (Gly) at the 216th position replaced by serine (Ser), and at the same time the methionine (Met) at the 234th position replaced by isoleucine (Ile). The D-allose epimerase mutant W29K / G216S / M234I is composed of the amino acid sequence of SEQ ID NO: 16, and the polynucleotide encoding the same is composed of the nucleotide sequence of SEQ ID NO: 17.

[0035] In addition, the applicant of the present invention disclosed a D-allose epimerase mutant W29K / A77S / G216S / M234I with improved conversion rate of fructose to allose and thermal stability, and a polynucleotide encoding the same, through Korean Patent Publication No. 10-2023-0073739. The D-allose epimerase mutant W29K / A77S / G216S / M234I has the tryptophan (Trp) at the 29th position in the amino acid sequence of the wild-type D-allose epimerase derived from Flavonifractor plautii replaced by lysine (Lys), the alanine (Als) at the 77th position replaced by serine (Ser), the glycine (Gly) at the 216th position replaced by serine (Ser), and at the same time the methionine (Met) at the 234th position replaced by isoleucine (Ile). The D-allose epimerase mutant W29K / A77S / G216S / M234I is composed of the amino acid sequence of SEQ ID NO: 18, and the polynucleotide encoding the same is composed of the nucleotide sequence of SEQ ID NO: 19.

[0036] The inventor of the present invention named the D-allose epimerase mutant W29K / A77S / G216S / M234I disclosed in Korean Patent Publication No. 10-2023-0073739 as "FpDPE2". Similar to the content disclosed in Korean Patent Publication No. 10-2023-0073739, a polynucleotide (SEQ ID NO: 19) fragment of the D-allose epimerase mutant W29K / A77S / G216S / M234I was inserted into the expression vector pET28a (Novagen) to prepare a recombinant vector pET28a::FpDPE2. Then, using the recombinant vector pET28a::FpDPE2 as a template, PCR was performed using the FpDPE2-F primer and the FpDPE2-R primer set described in Table 2 below. The resulting amplification product was cloned into a pGEM T-easy vector (Promega Co., USA), and the nucleotide sequence was analyzed. As a result, it was confirmed that it was a polynucleotide fragment having a length of 921 bp and composed of the nucleotide sequence of SEQ ID NO: 20.

Table 2

[0037] Example 3: Preparation of a Linked Fragment of a Promoter and an Allose Epimerase Mutant Gene Using the polynucleotide fragment composed of the nucleotide sequence of SEQ ID NO: 1 and the polynucleotide fragment composed of the nucleotide sequence of SEQ ID NO: 20 as templates, overlap extension PCR was performed using the Pctrl-F primer described in Table 1 and the FpDPE2-R primer set described in Table 2 to prepare an expression cassette fragment Pctrl_FpDPE2 in which the promoter Pctrl-F and the allose epimerase gene FpDPE2 were ligated. The expression cassette fragment Pctrl_FpDPE2 was cloned into the pGEM T-easy vector (Promega Co., USA), and as a result of analyzing the nucleotide sequence, it was confirmed to be a fragment having a length of 1,221 bp and containing a polynucleotide composed of the nucleotide sequence of SEQ ID NO: 23. Also, using the polynucleotide fragment composed of the nucleotide sequence of SEQ ID NO: 3 and the polynucleotide fragment composed of the nucleotide sequence of SEQ ID NO: 20 as templates, overlap extension PCR was performed using the Pds1-F primer described in Table 1 and the FpDPE2-R primer set described in Table 2 to prepare an expression cassette fragment Pds1_FpDPE2 in which the promoter Pds1 and the allose epimerase gene FpDPE2 were ligated. The expression cassette fragment Pds1_FpDPE2 is a fragment containing a polynucleotide composed of the nucleotide sequence of SEQ ID NO: 24. Also, using the polynucleotide fragment composed of the nucleotide sequence of SEQ ID NO: 5 and the polynucleotide fragment composed of the nucleotide sequence of SEQ ID NO: 20 as templates, overlap extension PCR was performed using the Pds2-F primer described in Table 1 and the FpDPE2-R primer set described in Table 2 to prepare an expression cassette fragment Pds2_FpDPE2 in which the promoter Pds2 and the allose epimerase gene FpDPE2 were ligated. The expression cassette fragment Pds2_FpDPE2 is a fragment containing a polynucleotide composed of the nucleotide sequence of SEQ ID NO: 25.Also, using the polynucleotide fragment composed of the nucleotide sequence of SEQ ID NO: 7 and the polynucleotide fragment composed of the nucleotide sequence of SEQ ID NO: 20 as templates, overlap extension PCR was performed using the Pds3-F primer described in Table 1 and the FpDPE2-R primer set described in Table 2 to prepare an expression cassette fragment Pds3_FpDPE2 in which the promoter Pds3 and the allose epimerase gene FpDPE2 were ligated. The expression cassette fragment Pds3_FpDPE2 is a fragment containing a polynucleotide composed of the nucleotide sequence of SEQ ID NO: 26. Specifically, 1 pM of the primer set was added to a reaction solution supplemented with 100 μM of deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP), and 100 ng each of the promoter and the D-allose epimerase mutant DNA fragment used as templates were mixed. Using a Thermocycler (TP600, TAKARA BIO Inc., JAPAN), PCR reaction was performed in the presence of 1 unit of pfu-X DNA polymerase mixture (Bioneer) for 25 to 30 cycles.

[0038] Example 4: Preparation of D-allose epimerase mutant expression vector After cutting each of the expression cassette fragments Pctrl_FpDPE2, Pds1_FpDPE2, Pds2_FpDPE2, and Pds3_FpDPE2 prepared in Example 3 above with restriction enzymes PstI and BamHI, this was ligated with the commercial plasmid expression vector pVWEx1 having the same restriction enzyme sites to prepare D-allose epimerase mutant expression vectors pPctrl_FpDPE2, pPds1_FpDPE2, pPds2_FpDPE2, and pPds3_FpDPE2. Then, the D-allose epimerase mutant expression vectors were transformed into Escherichia coli DH5α strain by the heat shock method (see Sambrook and Russell: Molecular Cloning), and colonies having kanamycin resistance were secured. The secured colonies were inoculated into LB liquid medium containing kanamycin and cultured overnight at 37°C. After that, the recombinant plasmid was extracted and the nucleotide sequence was analyzed. As a result, it was confirmed that the vector sequences of the clones were identical. The extracted recombinant plasmid was transformed into Escherichia coli JM110 strain by the heat shock method (see Sambrook and Russell: Molecular Cloning), and colonies having kanamycin resistance were secured.

[0039] Example 5: Preparation of a recombinant Corynebacterium strain expressing a D-allose epimerase mutant The colonies of the recombinant Escherichia coli JM110 strain obtained in Example 4 were inoculated into an LB liquid medium containing kanamycin and cultured overnight at 37°C. After that, the recombinant plasmid was extracted again, and the extracted recombinant plasmid was transformed into a Corynebacterium glutamicum strain by the heat shock method (see Sambrook and Russell: Molecular Cloning). Then, the transformed recombinant Corynebacterium glutamicum strain was inoculated into a 2YT solid medium containing kanamycin and cultured at 30°C for 24 hours to obtain colonies having kanamycin resistance. Then, the obtained colonies were inoculated into a 2YT liquid medium containing kanamycin and cultured at 30°C for 24 hours. After that, the recombinant plasmid was extracted and the nucleotide sequence was analyzed. As a result, in some of the recombinant plasmids containing the promoter Pds2, random mutations were confirmed in the promoter Pds2 or the allose epimerase mutant gene sequence, respectively. Table 3 below summarizes the details of the random mutations that occurred in some of the recombinant plasmids containing the promoter Pds2.

Table 3

[0040] As shown in Table 3 above, in the case of the recombinant plasmid expression vectors recovered from colonies 1, 3, 4, 5, and 8, mutations occurred in the allulose epimerase mutant gene sequence, and it is predicted that translation to create the target allulose epimerase mutant cannot be performed. On the contrary, in the case of the recombinant plasmid expression vectors recovered from colonies 2, 6, and 7, mutations occurred in the promoter sequence, and since the enzyme gene sequences were identical, it was determined that there was a possibility of expressing the target enzyme. The mutant promoter in the recombinant plasmid expression vector recovered from colony 2 was named "Pds4", the mutant promoter in the recombinant plasmid expression vector recovered from colony 6 was named "Pds4-1", and the mutant promoter in the recombinant plasmid expression vector recovered from colony 7 was named "Pds4-2". The promoter Pds4 is composed of the nucleotide sequence of SEQ ID NO: 27. Also, the recombinant plasmid expression vector recovered from colony 2 was renamed pPds4_FpDPE2, the recombinant plasmid expression vector recovered from colony 6 was renamed pPds4-1_FpDPE2, and the recombinant plasmid expression vector recovered from colony 7 was renamed pPds4-2_FpDPE2. The expression cassette fragment Pds4_FpDPE2 present in the recombinant plasmid expression vector pPds4_FpDPE2 is a fragment containing a polynucleotide composed of the nucleotide sequence of SEQ ID NO: 28.

[0041] Example 6: Measurement of the conversion rate of fructose to allulose and comparison of the intensity of enzyme expression of promoters for each recombinant Corynebacterium strain The conversion rate of fructose to allulose is proportional to the expression level of D-allulose epimerase in Corynebacterium strains. By measuring the conversion rate of fructose to allulose for each recombinant Corynebacterium strain, the intensity of enzyme expression induction of each promoter in the recombinant Corynebacterium strain was compared.

[0042] To culture a recombinant Corynebacterium strain transformed with a recombinant expression vector, 100 ml of LB medium with a kanamycin concentration of 50 μg / ml was placed in a 1 L flask, and 1 ml of the recombinant Corynebacterium strain prepared in Example 5 was inoculated therein. Then, the flask was transferred to a shaking incubator, and the recombinant Corynebacterium strain was cultured for 14 hours (hr) while maintaining the temperature condition of 30°C and the shaking condition of 140 rpm. The culture solution was centrifuged to recover the cells. Thereafter, the recovered cells were added at a concentration of 1 mg / ml to a 50 mM PIPES buffer solution (pH 7.0) containing 30% (w / w) fructose and 1 mM metal ion of manganese sulfate (MnSO4). After allowing the reaction to proceed for a predetermined time at 62°C, the temperature of the reaction product solution was lowered to 4°C to stop the reaction, and the solution was centrifuged under the conditions of 16,600×g and 4°C to recover the supernatant. Thereafter, using high performance liquid chromatography (HPLC), the concentrations of allose and fructose in the supernatant were measured, and after calculating the conversion rate of fructose to allose from the measured results, the conversion rate was used as an index of enzyme activity.

[0043] The following Table 4 summarizes the conversion rates depending on the reaction time when fructose was converted to allose using the recombinant Corynebacterium strain prepared in the examples of the present invention.

Table 4

[0044] As shown in Table 4 above, the recombinant Corynebacterium strain into which promoter Pds4 was introduced and the recombinant Corynebacterium strain into which promoter Pds2 was introduced showed a higher conversion rate of fructose to allose than the recombinant Corynebacterium strain into which promoter Pctrl was introduced. In particular, the recombinant Corynebacterium strain into which promoter Pds4 was introduced showed the highest conversion rate of fructose to allose. From such results, it can be understood that the enzyme expression induction effects of promoters Pds4 and Pds2 are stronger than that of promoter Pctrl. In addition, since promoters Pds4 and Pds2 are in a form in which a part of Pctrl, which is a promoter in the Corynebacterium strain, is deleted or mutated, it is expected that they will not impose a burden on the recombinant Corynebacterium strain and are judged to be suitable as a constitutive expression promoter.

[0045] As described above, the present invention has been described through the above embodiments. However, the protection scope of the present invention is not necessarily limited thereto, and it goes without saying that various modifications can be made without departing from the scope and spirit of the present invention. Therefore, the protection scope of the present invention should not be limited to the specific embodiments disclosed as the best mode, but should be construed as including all embodiments belonging to the scope of the claims attached to the present invention.

Claims

1. A promoter characterized in that it is composed of the base sequence of SEQ ID NO: 6 or the base sequence of SEQ ID NO: 27 and regulates the expression of allulose epimerization enzyme in a Corynebacterium strain.

2. An allulose epimerization enzyme expression cassette comprising a polynucleotide encoding an allulose epimerization enzyme and the promoter of claim 1 operably linked thereto.

3. The allulose epimerization enzyme expression cassette according to claim 2, wherein the allulose epimerization enzyme is derived from Flavonifractor plautii, Clostridium scidens, Treponema primitia, Ensifer adhaerens or Ruminococcus torques.

4. The allulose epimerization enzyme expression cassette of claim 2, wherein the allulose epimerization enzyme is composed of the amino acid sequence of SEQ ID NO: 14, the amino acid sequence of SEQ ID NO: 16, or the amino acid sequence of SEQ ID NO:

18.

5. The allulose epimerization enzyme expression cassette of claim 2, wherein the polynucleotide encoding the allulose epimerization enzyme is composed of the base sequence of SEQ ID NO: 15, the base sequence of SEQ ID NO: 17, or the base sequence of SEQ ID NO:

19.

6. The allulose epimerization enzyme expression cassette of claim 2, comprising a polynucleotide consisting of the base sequence of SEQ ID NO: 25 or a polynucleotide consisting of the base sequence of SEQ ID NO:

28.

7. A recombinant expression vector into which the expression cassette according to any one of claims 2 to 6 has been inserted.

8. A recombinant Corynebacterium strain, which is obtained by transforming a Corynebacterium host strain by introducing an expression cassette according to any one of claims 2 to 6 or a recombinant expression vector into which the expression cassette is inserted.

9. 9. The recombinant Corynebacterium strain of claim 8, wherein the Corynebacterium host strain is selected from the group consisting of Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium thermoaminogenes, Corynebacterium melassecola, and Corynebacterium efficiens.

10. A method for producing allulose, comprising the step of adding the recombinant Corynebacterium strain described in claim 9 to a fructose-containing solution and reacting it.

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