Novel promoter for constitutive expression, target protein expression system comprising same, and method for preparing psicose using same

Abstract

One embodiment of the present invention provides a promoter which is characterized by comprising a base sequence represented by sequence 6 or a base sequence represented by sequence 27, and which regulates the expression of psicose epimerase in a Corynebacterium strain. The novel promoter provided by the invention can be used for constitutive high expression of a target protein, especially an enzyme, in a corynebacterium strain. For example, when using a recombinant Corynebacterium strain transformed with an expression vector comprising a novel promoter of the present invention, psicose epimerase can be economically mass-produced or psicose can be economically mass-produced from fructose.

Description

Technical Field

[0001] The present invention relates to a novel promoter and its use, and more particularly, to a novel promoter capable of constitutively overexpressing 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 discovered. Gene expression refers to a series of processes in which a protein is synthesized according to the code of an input gene through transcription and translation occurring within a cell. In particular, the transcription process is the initial stage of gene expression, which is initiated by RNA polymerase binding to a promoter sequence present upstream of the gene with the help of various cofactors. Transcription factors (TFs) are one of such cofactors and are known to bind directly to the promoter sequence. In particular, the regulation of gene expression in prokaryotes mainly occurs at the transcription stage, so researchers continuously discover new transcription factors and promoters.

[0003] Industrially, to produce exogenous proteins such as enzymes, transformants prepared by transforming prokaryotes such as Escherichia coli with a pET-type expression vector containing an exogenous protein gene are mainly used as an expression system. The prokaryotic expression system transformed with a pET-type expression vector usually requires an expensive expression inducer such as isopropyl-β-D-thiogalactopyranoside (IPTG), and has disadvantages such as the need to carefully adjust the inducer concentration, equipment, expression induction time, etc.

[0004] On the other hand, in order to mass-produce D-allulose epimerase (or allulose epimerase) having the activity of converting fructose into allulose (or D-allulose), an expression system using a Corynebacterium strain, which is generally recognized as safe (GRAS), as a host cell has been proposed. Regarding the D-allulose epimerase (or allulose epimerase) expression system based on Corynebacterium strains, Korean Patent No. 10-1695830 discloses a nucleic acid sequence encoding D-allulose epimerase, a gene expression cassette operably linked upstream thereof and containing a regulatory sequence that regulates the expression of the above D-allulose epimerase in Corynebacterium strains, and the above regulatory sequence contains an Escherichia coli (E. coli)-derived transcription promoter selected from the trc promoter, Tac1 promoter, and Tac2 promoter or the sod promoter as a transcription promoter derived from Corynebacterium glutamicum. Generally, a promoter contains a region that binds to RNA polymerase, and within the 5' UTR sequence and 3' UTR sequence centered on the core promoter region, there are binding regions of transcription factors that promote or inhibit the expression of various RNAs. The gene arrangement around the promoter sequence is important for improving the expression efficiency. Since the promoter sequence of the D-allulose epimerase expression system based on Corynebacterium strains disclosed in the above prior art is located in a bidirectional promoter region where the McrA gene and the sod gene are arranged to be expressed in opposite directions, a highly sophisticated regulatory mechanism for expressing bidirectional genes is required, and it is not suitable as a system for constitutively expressing a single target protein. In addition, the D-allulose epimerase expression system based on Corynebacterium strains disclosed in the above prior art has relatively weak expression intensity and is not suitable for mass-producing allulose due to the structural hindrance of the promoter and the complex regulation of the promoter. Summary of the Invention

[0005] The present invention is proposed under the background of the existing technology. The object of the present invention is to provide a novel promoter that can constitutively highly express a target protein. In addition, the object of the present invention is to provide various uses such as a target protein expression system and a method for preparing allulose as the above novel promoter.

[0006] The inventors of the present invention prepared a promoter excluding bidirectionality by deleting partial fragments in the bidirectional promoter region existing between the McaA gene and the sod gene in the genomic sequence of Corynebacterium glutamicum ATCC13032 strain, and confirmed that a specific promoter therein could constitutively highly express allulose epimerase in Corynebacterium strains, thereby completing the present invention. Moreover, the inventors of the present invention prepared a recombinant expression vector by operably linking the above-mentioned promoter eliminating bidirectionality with a polynucleotide encoding allulose epimerase, and as a result of introducing the above-mentioned recombinant expression vector into Corynebacterium glutamicum, a generally recognized as safe strain, for transformation, it was confirmed that random mutations occurred in some promoters, and a specific promoter variant among the promoter variants obtained by random mutation significantly increased the expression efficiency of allulose epimerase, thereby completing the present invention.

[0007] To solve the above problems, an example of the present invention provides a promoter, which is characterized by consisting of the base sequence shown in Sequence 6 or the base sequence shown in Sequence 27, and 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, which comprises a polynucleotide encoding allulose epimerase and a promoter operably linked thereto. The above-mentioned promoter consists of the base sequence shown in Sequence 6 or the base sequence shown in Sequence 27.

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

[0010] To solve the above problems, an example of the present invention provides a recombinant Corynebacterium strain, which transforms a Corynebacterium host strain by introducing an allulose epimerase expression cassette or a recombinant expression vector inserted with the above-mentioned expression cassette.

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

[0012] The novel promoter of the present invention can constitutively highly express a target protein, especially an enzyme, in Corynebacterium strains. For example, when using a recombinant Corynebacterium strain transformed with an expression vector containing the novel promoter of the present invention, allulose epimerase can be produced inexpensively in large quantities or allulose can be produced inexpensively in large quantities from fructose. Detailed Description

[0013] Hereinafter, the present invention will be specifically described.

[0014] The term "promoter" used in the present invention refers to the smallest nucleic acid sequence that is operably linked to a target nucleotide sequence to be transcribed and regulates the transcription of the above-mentioned target nucleotide sequence. Moreover, the above-mentioned promoter may include a promoter structure sufficient to express a regulatable promoter-dependent gene induced by cell type specificity or an external signal or agent, and such a structure may be located in the 5' or 3' portion of the gene. The above-mentioned promoter includes a constitutive promoter and an inducible promoter. The promoter sequence may be derived from prokaryotes, eukaryotes or viruses. A promoter in prokaryotes is generally defined as a binding site adjacent to the transcription start site where RNA polymerase binds.

[0015] The term "homology" used in the present invention refers to the identity of a nucleic acid sequence with a wild type or a variant having the same activity. The homology comparison can be visually inspected or the percentage (%) of homology between two or more sequences can be calculated using an easily purchasable comparison program. Moreover, "homology" is used to represent the identity of an amino acid sequence with a wild type or a variant having the same activity.

[0016] The term "target protein" used in the present invention refers to a protein that generally cannot be present in a transformed strain (or host cell) that expresses the above-mentioned protein as a foreign protein.

[0017] The term "polynucleotide" used in the present invention refers to all polyribonucleotides (RNA) or polydeoxyribonucleotides (DNA), whether unmodified or modified. The above-mentioned polynucleotide includes single-stranded or double-stranded DNA, DNA as a mixture of single-stranded regions and double-stranded regions, single-stranded and double-stranded RNA, RNA as a mixture of single-stranded regions and double-stranded regions, or hybrid molecules thereof, but is not limited thereto.

[0018] The term "operably linked" used in the present invention is defined as a state in which a promoter sequence and a nucleotide sequence encoding a target protein are functionally linked so 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 the coding sequence, or if a ribosome binding site is located at a position that can promote translation, the ribosome binding site is operably linked to the coding sequence. The coding sequence can be operably linked to the regulatory sequence in the sense or antisense direction.

[0019] 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.

[0020] The term "expression cassette" used in the present invention refers to a regulatory sequence functionally linked to a nucleotide sequence to be expressed, such as a polynucleotide sequence encoding allulose epimerase. Thus, different from an expression unit, an expression cassette not only contains nucleotide sequences that regulate transcription and translation, but also contains nucleotide sequences that are expressed as proteins as a result of transcription and translation.

[0021] The term "expression vector" used in the present invention is defined as a DNA sequence required for transcription and translation of cloned DNA in a suitable host, specifically referring to a gene construct containing essential gene regulatory elements operably linked to an insert, such that the insert is expressed when present in a cell. Expression vectors can be prepared and purified using standard recombinant DNA techniques. There is no particular limitation on the types of the above-mentioned expression vectors, as long as they have the function of expressing a desired gene and producing a desired protein in various host cells of prokaryotic and eukaryotic cells. An expression vector includes at least a promoter, a start codon, a gene encoding a desired protein, and a terminator codon. And, in addition to this, an expression vector may appropriately include DNA encoding a signal peptide, additional expression regulatory sequences, untranslated regions on the 5' side and 3' side of a desired gene, a selection marker region, or a replicable unit, etc. The above-mentioned selection marker region may be a selection marker gene for an antibiotic for screening a target vector.

[0022] The term "recombinant strain" used in the present invention refers to a cell transformed by introducing a polynucleotide encoding one or more target proteins or an expression vector having the polynucleotide into a host cell. Methods for introducing the above expression vector into a host cell to prepare a transformant include transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, 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. Also, the type of host cell of the recombinant strain is not particularly limited as long as the promoter present in the expression vector can operate smoothly, and prokaryotes are preferred.

[0023] The term "substrate" used in the present invention refers to any substance or compound that is converted or will be converted into other compounds by the action of an enzyme. The above term includes single compounds as well as combinations of compounds and their derivatives, such as solvents, mixtures, and other materials containing at least one substrate.

[0024] On the one hand, the present invention relates to a novel promoter capable of constitutively highly expressing a target protein. The novel promoter of an example of the present invention is composed of the base sequence shown in SEQ ID NO: 6 or the base sequence shown in SEQ ID NO: 27. The inventors of the present invention named the promoter composed of the base sequence shown in SEQ ID NO: 6 as "Pds2", and named the promoter composed of the base sequence shown in SEQ ID NO: 27 as "Pds4". The above promoter Pds2 is a promoter in which a partial fragment in the bidirectional promoter region between the McaA gene and the sod gene in the genomic sequence of the Corynebacterium glutamicum strain is deleted to eliminate bidirectionality and improve the expression efficiency of the target protein. And, the above promoter Pds4 is a promoter variant in which 6 bp bases at positions 195 to 200 of the promoter Pds2 are deleted and a new 3 bp base is inserted, which is caused by a mutation in the Ribosome-Binding Site (RBS) spacer. An expression system containing the promoter of an example of the present invention can constitutively highly express a target protein in a Corynebacterium strain. In particular, the promoter of an example of the present invention regulates the expression of allulose epimerase in a Corynebacterium strain. Therefore, the promoter Pds2 or the promoter Pds4 of an example of the present invention can be used as a promoter for constitutive expression in a Corynebacterium strain. The novel promoter of an example of the present invention is composed of the base sequence shown in SEQ ID NO: 6 or the base sequence shown in SEQ ID NO: 27, but the equivalent scope of the above promoter is not necessarily limited thereto. For example, the equivalent scope of the novel promoter of an example of the present invention includes sequences having substantial identity with the base sequence shown in SEQ ID NO: 6 or the base sequence shown in SEQ ID NO: 27. The above substantial identity means that by aligning the base sequence shown in SEQ ID NO: 6 or the base sequence shown in SEQ ID NO: 27 with any other sequence as much as possible and analyzing its sequence, any other sequence has a sequence homology of 70% or more, 90% or more, or 98% or more with the base sequence shown in SEQ ID NO: 6 or the base sequence shown in SEQ ID NO: 27. Those of ordinary skill in the art can easily understand that polynucleotides having the same or similar activities within the range of substantial homology can be prepared by substituting, adding, or deleting one or more bases in the base sequence of the above novel promoter by using gene recombination techniques well known in the art. Such homology comparison can be carried out by calculating the percentage (%) of homology between two or more sequences using a commercially available computer program. Therefore, the equivalent scope of the novel promoter of an example of the present invention can include base sequences having 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more homology with the base sequence shown in SEQ ID NO: 6 or the base sequence shown in SEQ ID NO: 27 within the range of maintaining the function of constitutively highly expressing a target protein.

[0025] One aspect of the present invention relates to a target protein expression system comprising a novel promoter. The above-mentioned novel promoter can be used to prepare a psicose epimerase expression cassette, a recombinant vector, and a recombinant strain.

[0026] An allulose epimerase expression cassette of an example of the present invention includes a polynucleotide encoding allulose epimerase and the above-mentioned promoter operably linked thereto. Preferably, the above-mentioned promoter is located upstream of the polynucleotide encoding allulose epimerase as the target protein. The promoter as a component of the above-mentioned allulose epimerase expression cassette consists of the base sequence shown in Sequence 6 or the base sequence shown in Sequence 27. There is no particular limitation on the type of polynucleotide encoding allulose epimerase as a component of the above-mentioned allulose epimerase expression cassette, as long as it is a polynucleotide encoding an enzyme having the activity of converting fructose into allulose. For example, the above-mentioned allulose epimerase may be derived from Flavonifractor plautii, Clostridiun scidens, Treponema primitia, Ensifer adhaerens, or Ruminococcus torques. Considering the activity of converting fructose into allulose, it is preferably derived from Flavonifractor plautii. Specifically, the above-mentioned allulose epimerase may consist of the amino acid sequence shown in Sequence 14, the amino acid sequence shown in Sequence 16, or the amino acid sequence shown in Sequence 18. The allulose epimerase consisting of the amino acid sequence shown in Sequence 14 above is a wild-type enzyme derived from Flavonifractor plautii. The allulose epimerase consisting of the amino acid sequence shown in Sequence 16 above is such that the tryptophan (Trp) at the 29th position, the glycine (Gly) at the 216th position, and the methionine (Met) at the 234th position in the amino acid sequence shown in Sequence 14 are replaced by lysine (Lys), serine (Ser), and isoleucine (Ile), respectively. The allulose epimerase shown by the amino acid sequence shown in Sequence 18 above is such that the tryptophan at the 29th position, the alanine (Ala) at the 77th position, the glycine at the 216th position, and the methionine at the 234th position in the amino acid sequence shown in Sequence 14 are replaced by lysine, serine, serine, and isoleucine, respectively. There is no particular limitation on the type of the above-mentioned polynucleotide encoding allulose epimerase. Preferably, it may include a base sequence consisting of the base sequence shown in Sequence 15 or a base sequence having 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more homology with the base sequence shown in Sequence 15. The base sequence shown in Sequence 15 is a polynucleotide encoding an allulose epimerase consisting of the amino acid sequence shown in Sequence 14.Furthermore, the polynucleotide encoding allose epimerase may consist of the nucleotide sequence shown in SEQ ID NO: 17 or the nucleotide sequence shown in SEQ ID NO: 19. The nucleotide sequence shown in SEQ ID NO: 17 is a polynucleotide encoding allose epimerase consisting of the amino acid sequence shown in SEQ ID NO: 16, and the nucleotide sequence shown in SEQ ID NO: 19 is a polynucleotide encoding allose epimerase consisting of the amino acid sequence shown in SEQ ID NO: 18. Regarding allose epimerase and the polynucleotide encoding the same, the present invention includes the contents disclosed in Korean Patent Publication No. 10-1919713, Korean Patent Publication No. 10-2187354, Korean Patent Publication No. 10-1656063, Korean Patent Publication No. 10-1695830, Korean Patent Publication No. 10-2189458, Korean Patent Publication No. 10-1539097, Korean Patent Publication No. 10-1539096, Korean Patent Publication No. 10-1455759, Korean Patent Publication No. 10-1318422, Korean Patent Publication No. 10-2023-0073739, etc.

[0027] The allose epimerase expression cassette of 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 above selection marker is used to screen cells transformed with a vector, and a marker that confers a selectable expression type such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins can be used. For example, the above selection marker may be an antibiotic resistance gene marker such as a kanamycin antibiotic resistance gene or an ampicillin antibiotic resistance gene. Preferably, the allose epimerase expression cassette of an example of the present invention may include a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 25 or a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 28. The polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 25 is an expression cassette fragment in which a promoter consisting of the nucleotide sequence shown in SEQ ID NO: 6 and an allose epimerase gene consisting of the nucleotide sequence shown in SEQ ID NO: 19 are sequentially linked. Furthermore, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 28 is an expression cassette fragment in which a promoter consisting of the nucleotide sequence shown in SEQ ID NO: 27 and an allose epimerase gene consisting of the nucleotide sequence shown in SEQ ID NO: 19 are sequentially linked.

[0028] A recombinant vector in one example of the present invention is a recombinant expression vector inserted with the above-mentioned allulose epimerase expression cassette. Preferably, the above-mentioned recombinant expression vector 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 connected in sequence.

[0029] A recombinant strain in one example of the present invention is a recombinant Corynebacterium strain transformed from a host cell by introducing the above-mentioned allulose epimerase expression cassette or a recombinant expression vector inserted with the above-mentioned expression cassette. There is no particular limitation on the type of the host strain used for preparing the above-mentioned recombinant Corynebacterium strain, 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.

[0030] One aspect of the present invention relates to a method for preparing allulose using a target protein expression system containing a novel promoter.

[0031] A method for preparing allulose in one example of the present invention includes the step of adding a recombinant Corynebacterium strain to a fructose-containing solution and reacting. The above-mentioned fructose serves as a substrate for allulose epimerase as the target protein expressed by the recombinant Corynebacterium strain. The above-mentioned fructose-containing solution may also contain metal ions such as Ca 2+ , Mn 2+ to promote the activity of allulose epimerase. And, in the method for preparing allulose from the above-mentioned 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 considering the smooth enzyme expression of the recombinant strain, the stability of the enzyme, and the maximum activity. The reaction pH is 6.5 to 8, preferably 6.5 to 7.5, and more preferably in the range of 6.5 to 7. And, in the method for preparing allulose from the above-mentioned fructose, the fructose concentration of the fructose-containing solution is not particularly limited, but considering productivity and economy, it is preferably 5 to 75% (w / w), more preferably 10 to 55% (w / w) based on the total weight of the fructose-containing solution.

[0032] Hereinafter, the present invention will be described more specifically by way of examples. However, the following examples are only used to clearly illustrate the technical features of the present invention and are not used to limit the protection scope of the present invention.

[0033] Example 1: Amplification and obtaining of a promoter sequence for expressing D-allulose 3-epimerase

[0034] Using the genomic DNA of Corynebacterium glutamicum ATCC 13032 strain as a template, PCR was performed using the Pctrl-F primer and the Pds-R primer set described in Table 1 to obtain a bidirectional promoter present in Corynebacterium sp. strain. As a result of cloning the obtained amplification product into a pGEM T-easy vector (Promega Co., USA) and analyzing the base sequence, it was confirmed that it was a polynucleotide fragment consisting of a base sequence shown in Sequence 1 with a length of 336 bp. The region corresponding to the promoter in the polynucleotide fragment consisting of the base sequence shown in Sequence 1 was named "Pctrl". Promoter Pctrl consists of the base sequence shown in Sequence 2. Also, using the genomic DNA of Corynebacterium glutamicum ATCC 13032 strain as a template, PCR for amplifying each variant promoter was performed using the 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 to obtain variant promoters of promoter Pctrl. The obtained amplification products were cloned into a pGEM T-easy vector (Promega Co., USA) and the base sequences were analyzed. As a result, the amplification product obtained using the Pds1-F primer and the Pds-R primer set was a polynucleotide fragment consisting of a base sequence shown in Sequence 3 with a length of 282 bp. The region corresponding to the promoter in the polynucleotide fragment consisting of the base sequence shown in Sequence 3 was named "Pds1". Promoter Pds1 consists of the base sequence shown in Sequence 4. Also, the amplification product obtained using the Pds2-F primer and the Pds-R primer set was a polynucleotide fragment consisting of a base sequence shown in Sequence 5 with a length of 236 bp. The region corresponding to the promoter in the polynucleotide fragment consisting of the base sequence shown in Sequence 5 was named "Pds2". Promoter Pds2 consists of the base sequence shown in Sequence 6. Also, the amplification product obtained using the Pds3-F primer and the Pds-R primer set was a polynucleotide fragment consisting of a base sequence shown in Sequence 7 with a length of 236 bp. The region corresponding to the promoter in the polynucleotide fragment consisting of the base sequence shown in Sequence 7 was named "Pds3". Promoter Pds3 consists of the base sequence shown in Sequence 8.The promoter Pctrl is a bidirectional promoter containing the intergenic region 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 variants with partial sequence deletions of the promoter Pctrl centered on the 282bp intergenic region.

[0035] Table 1

[0036]

[0037] Example 2: Amplification and Obtaining of the Polynucleotide Sequence Encoding D-psicose 3-epimerase

[0038] The applicant of the present invention disclosed a wild-type D-psicose epimerase derived from Flavonifractor plautii and the polynucleotide encoding the same through Korean Patent Publication No. 10-14739180. The above wild-type D-psicose epimerase is composed of the amino acid sequence shown in SEQ ID NO: 14, and the polynucleotide encoding the same is composed of the base sequence shown in SEQ ID NO: 15.

[0039] In addition, the applicant of the present invention disclosed a D-psicose epimerase variant W29K / G216S / M234I that improves the conversion rate of fructose to psicose and thermal stability and the polynucleotide encoding the same through Korean Patent Publication No. 10-2021-0132405. The above D-psicose epimerase variant W29K / G216S / M234I is formed by substituting the tryptophan at the 29th position in the amino acid sequence of the wild-type D-psicose epimerase derived from Flavonifractor plautii with lysine, substituting the glycine at the 216th position with serine, and substituting the methionine at the 234th position with isoleucine. The above D-psicose epimerase variant W29K / G216S / M234I is composed of the amino acid sequence shown in SEQ ID NO: 16, and the polynucleotide encoding the same is composed of the base sequence shown in SEQ ID NO: 17.

[0040] In addition, the applicant of the present invention disclosed a D-allulose epimerase variant W29K / A77S / G216S / M234I and a polynucleotide encoding the same, which improve the conversion rate of fructose to allulose and thermal stability, through Korean Patent Publication No. 10-2023-0073739. The above D-allulose epimerase variant W29K / A77S / G216S / M234I is formed by substituting lysine for tryptophan at the 29th position, serine for alanine at the 77th position, serine for glycine at the 216th position, and isoleucine for methionine at the 234th position in the amino acid sequence of the wild-type D-allulose epimerase derived from Flavonifractor plautii. The above D-allulose epimerase variant W29K / A77S / G216S / M234I is composed of the amino acid sequence shown in SEQ ID NO: 18, and the polynucleotide encoding the same is composed of the nucleotide sequence shown in SEQ ID NO: 19.

[0041] The inventors of the present invention named the D-allulose epimerase variant W29K / A77S / G216S / M234I disclosed in Korean Patent Publication No. 10-2023-0073739 as "FpDPE2". In the same manner as the content disclosed in Korean Patent Publication No. 10-2023-0073739, a recombinant vector pET28a::FpDPE2 was prepared by inserting a polynucleotide (SEQ ID NO: 19) fragment of the D-allulose epimerase variant W29K / A77S / G216S / M234I into pET28a (Novagen) as an expression vector. 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. As a result of cloning the obtained amplification product into the pGEM T-easy vector (Promega, USA) and analyzing the nucleotide sequence, it was confirmed that it was a polynucleotide fragment with a length of 921 bp and composed of the nucleotide sequence shown in SEQ ID NO: 20.

[0042] Table 2

[0043]

[0044] Example 3: Preparation of a ligation fragment of a promoter and an allulose epimerase variant gene

[0045] Using the polynucleotide fragment consisting of the base sequence shown in SEQ ID NO: 1 and the polynucleotide fragment consisting of the base sequence shown in SEQ ID NO: 20 as templates, and performing overlap extension PCR using the Pctrl-F primer described in Table 1 and the FpDPE2-R primer set described in Table 2, an expression cassette fragment Pctrl_FpDPE2 that links the promoter Pctrl-F and the allose epimerase gene FpDPE2 was prepared. As a result of cloning the expression cassette fragment Pctrl_FpDPE2 into the pGEM T-easy vector (Promega Corporation, USA) and analyzing the base sequence, it was confirmed that it was a fragment with a length of 1221 bp containing a polynucleotide consisting of the base sequence shown in SEQ ID NO: 23. Also, using the polynucleotide fragment consisting of the base sequence shown in SEQ ID NO: 3 and the polynucleotide fragment consisting of the base sequence shown in SEQ ID NO: 20 as templates, and performing overlap extension PCR using the Pds1-F primer described in Table 1 and the FpDPE2-R primer set described in Table 2, an expression cassette fragment Pds1_FpDPE2 that links the promoter Pds1 and the allose epimerase gene FpDPE2 was prepared. The expression cassette fragment Pds1_FpDPE2 is a fragment containing a polynucleotide consisting of the base sequence shown in SEQ ID NO: 24. Also, using the polynucleotide fragment consisting of the base sequence shown in SEQ ID NO: 5 and the polynucleotide fragment consisting of the base sequence shown in SEQ ID NO: 20 as templates, and performing overlap extension PCR using the Pds2-F primer described in Table 1 and the FpDPE2-R primer set described in Table 2, an expression cassette fragment Pds2_FpDPE2 that links the promoter Pds2 and the allose epimerase gene FpDPE2 was prepared. The expression cassette fragment Pds2_FpDPE2 is a fragment containing a polynucleotide consisting of the base sequence shown in SEQ ID NO: 25. Also, using the polynucleotide fragment consisting of the base sequence shown in SEQ ID NO: 7 and the polynucleotide fragment consisting of the base sequence shown in SEQ ID NO: 20 as templates, and performing overlap extension PCR using the Pds3-F primer described in Table 1 and the FpDPE2-R primer set described in Table 2, an expression cassette fragment Pds3_FpDPE2 that links the promoter Pds3 and the allose epimerase gene FpDPE2 was prepared. The expression cassette fragment Pds3_FpDPE2 is a fragment containing a polynucleotide consisting of the base sequence shown in SEQ ID NO: 26.Specifically, 1 pM of primer sets were added to a reaction solution supplemented with 100 μM of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP), and 100 ng each of a promoter and a D-psicose 3-epimerase variant DNA fragment used as a template were mixed. Then, a PCR reaction was carried out for 25 to 30 cycles in the presence of 1 unit of pfu-X DNA polymerase mixture (Bioneer) using a thermocycler (TP600, TAKARA BIO Inc., JAPAN).

[0046] Example 4: Preparation of D-psicose 3-epimerase variant expression vectors

[0047] Each of the expression cassette fragments Pctrl_FpDPE2, Pds1_FpDPE2, Pds2_FpDPE2, and Pds3_FpDPE2 prepared in Example 3 above was digested with restriction enzymes PstI and BamHI, and then ligated into a commercially available plasmid expression vector pVWEx1 having the same restriction enzyme sites to prepare D-psicose 3-epimerase variant expression vectors pPctrl_FpDPE2, pPds1_FpDPE2, pPds2_FpDPE2, and pPds3_FpDPE2. Then, by using the heat shock method (see Sambrook and Russell: Molecular Cloning), the D-psicose 3-epimerase variant expression vectors were transformed into Escherichia coli DH5α strain to obtain kanamycin-resistant colonies. The obtained colonies were inoculated into an LB liquid medium containing kanamycin and cultured overnight at 37°C. Then, recombinant plasmids were extracted and the base sequences were analyzed. As a result, it was confirmed that the cloned vector sequences were identical. By using the heat shock method (see Sambrook and Russell: Molecular Cloning), the extracted recombinant plasmids were transformed into Escherichia coli JM110 strain to obtain kanamycin-resistant colonies.

[0048] Example 5: Preparation of recombinant Corynebacterium strains expressing D-psicose 3-epimerase variant

[0049] The colonies of the recombinant Escherichia coli JM110 strain obtained in Example 4 above were inoculated into LB liquid medium containing kanamycin and cultured O / N at a temperature of 37°C. Then, 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 2YT solid medium containing kanamycin and cultured at a temperature of 30°C for 24 hours to obtain colonies with kanamycin antibiotic resistance. Then, the obtained colonies were inoculated into 2YT liquid medium containing kanamycin and cultured at a temperature of 30°C for 24 hours, and then the recombinant plasmid was extracted and the base sequence was analyzed. As a result, random mutations were confirmed in the promoter Pds2 or the allulose epimerase variant gene sequence in the partial recombinant plasmid containing the promoter Pds2. The content of the random mutations generated in the partial recombinant plasmid containing the promoter Pds2 is shown in Table 3 below.

[0050] Table 3

[0051]

[0052]

[0053] 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 variant gene sequence, so it was predicted that translation for preparing the required allulose epimerase variant would not be possible. 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 the enzyme gene sequences matched, so it was judged that the required enzyme might be expressed. 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 above promoter Pds4 consists of the base sequence shown in Sequence 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 above recombinant plasmid expression vector pPds4_FpDPE2 is a fragment containing a polynucleotide consisting of the base sequence shown in Sequence 28.

[0054] Example 6: Determination of the conversion rate of fructose to psicose according to different recombinant Corynebacterium strains and comparison of the enzyme expression intensity of promoters

[0055] The conversion rate of fructose to psicose is proportional to the expression level of D - psicose 3 - epimerase in Corynebacterium strains. The enzyme expression induction intensity of each promoter in recombinant Corynebacterium strains was compared by measuring the conversion rate of fructose to psicose according to different recombinant Corynebacterium strains.

[0056] To culture the recombinant Corynebacterium strains transformed with the 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 an orbital shaker incubator, and the recombinant Corynebacterium strain was cultured for 14 hours under the temperature condition of 30°C and the shaking condition of 140 rpm, and the cells were recovered by centrifuging the culture broth. Then, 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), and the reaction was carried out at 62°C for a specified time. Then, the temperature of the reaction product was lowered to 4°C to terminate the reaction, and the supernatant was recovered by centrifugation under the conditions of 16600×g and 4°C. Then, the concentrations of psicose and fructose in the supernatant were measured by high - performance liquid chromatography (HPLC), and the conversion rate of fructose to psicose was calculated from the measurement results, and the conversion rate was used as an index of enzyme activity. Table 4 below shows the conversion rate according to the reaction time when fructose was converted to psicose using the recombinant Corynebacterium strains prepared in the examples of the present invention.

[0057] Table 4

[0058]

[0059] As shown in Table 4 above, compared with the recombinant Corynebacterium strain introduced with the promoter Pctrl, the recombinant Corynebacterium strain introduced with the promoter Pds4 and the recombinant Corynebacterium strain introduced with the promoter Pds2 showed higher conversion rates of fructose to allulose. In particular, the highest conversion rate of fructose to allulose was shown in the recombinant Corynebacterium strain introduced with the promoter Pds4. From this result, it can be seen that the enzyme expression induction effects of the promoters Pds4 and Pds2 are stronger than that of the promoter Pctrl. Moreover, the promoters Pds4 and Pds2 are in the form of partial deletion or mutation of the promoter Pctrl in the Corynebacterium strain. Therefore, it is expected that they will not impose a burden on the recombinant Corynebacterium strain and are judged to be suitable as constitutive expression promoters.

[0060] As described above, the present invention has been illustrated by the above embodiments, but the protection scope of the present invention is not limited thereto. Naturally, various modifications can be made without departing from the scope and spirit of the present invention. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed as the best form, and should be construed to include all embodiments falling within the scope of the appended claims of the present invention.

Claims

1. A promoter, characterized in that The method comprises the base sequence shown in SEQ ID NO: 6 or the base sequence shown in SEQ ID NO: 27, and regulates the expression of psicose epimerase in a Corynebacterium strain.

2. A psicose epimerase expression cassette, characterized in that: The invention comprises a polynucleotide encoding a psicose epimerase and the promoter according to claim 1 operably linked thereto.

3. The expression cassette of psicose epimerase according to claim 2, characterized in that: The above-mentioned allulose epimerase is derived from Flavonoids perfringens, Clostridium fulgidus, Treponema bacteria, Sagittaria adhering or Ruminococcus contortus.

4. The expression cassette of psicose epimerase according to claim 2, characterized in that: The above-mentioned D-psicose isomerase consists of the amino acid sequence shown in SEQ ID NO: 14, the amino acid sequence shown in SEQ ID NO: 16 or the amino acid sequence shown in SEQ ID NO:

18.

5. The expression cassette of psicose epimerase according to claim 2, characterized in that: The polynucleotide encoding the psicose epimerase is composed of the base sequence shown in SEQ ID NO: 15, the base sequence shown in SEQ ID NO: 17, or the base sequence shown in SEQ ID NO:

19.

6. The expression cassette of psicose epimerase according to claim 2, characterized in that: It comprises a polynucleotide consisting of the base sequence shown in SEQ ID NO: 25 or a polynucleotide consisting of the base sequence shown in SEQ ID NO:

28.

7. A recombinant expression vector, characterized in that: An expression cassette according to any one of claims 2 to 6 is inserted.

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

9. The recombinant Corynebacterium strain according to claim 8, characterized in that The above-mentioned Corynebacterium host strain is selected from the group consisting of Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacetogenicum, Corynebacterium thermoammonigenicum, Corynebacterium molasses and Corynebacterium efficacious.

10. A method for preparing psicose, characterized in that: The method comprises the steps of adding the recombinant Corynebacterium strain according to claim 9 to a fructose-containing solution and carrying out a reaction.

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