KR20220162390A - a method for providing determination gene group for rare sugar metabolizing ability for rare sugar non-metabolizable strain - Google Patents

Abstract

The present invention relates to a method for providing a rare sugar assimilation ability-determining gene group for rare sugar non-metabolizable strains. A new metabolic pathway can be established through an expression cassette and a vector containing the same according to the present invention. In addition, a mutant strain transformed through the vector or a mutant strain containing a mutated gene has the new sugar metabolic pathway established therein.

Description

A method for providing determination gene group for rare sugar metabolizing ability for rare sugar non-metabolizable strain}

The present invention relates to a method for providing rare saccharide magnetization ability-determining gene groups of non-metabolizing strains of rare saccharides.

D-tagatose is an isomer of D-galactose and is a natural sugar present in fruits, milk, and cheese. Because D-tagatose has various health functional properties and a sweet taste very similar to sugar, it is used as an alternative sweetener that can satisfy both health and taste when applied to various products.

On the other hand, in general, directed evolution technology is used as an improvement technology for changing the characteristics of an enzyme to meet a desired purpose, such as enhancing the activity and structural stability of the enzyme or imparting activity to a new substrate. The most commonly used method to prepare a mutant library for performing this technology is an error-prone PCR method, which randomly introduces mutations by controlling the error rate of DNA polymerase during PCR. After expressing proteins using these mutant strains, improved enzymes with excellent activity are obtained by selecting mutant strains with good activity. can do.

Korean Patent Publication No. 10-2018-0074550 discloses a recombinant strain having a modified sugar metabolism pathway by introducing enzymes derived from other strains into the strain and the possibility of obtaining D-tagatose through this, but recently As the yield decreases due to the increase, the need to develop a strain capable of obtaining D-tagatose through a new metabolic pathway is increasing.

Thus, the present inventors completed the present invention by confirming a mutant strain having a modified sugar metabolism pathway and its use through research.

1. KR 10-2018-0074550 A

One aspect of the present invention provides an expression cassette comprising a gene encoding a novel fructose-1-phosphate kinase in which the 39th alanine (A) is substituted with serine (S) in the amino acid sequence of SEQ ID NO: 1 aims to

Another aspect of the present invention aims to provide a recombinant vector comprising the expression cassette.

Another aspect of the present invention aims to provide a mutant strain transformed with the recombinant vector, thereby providing a strain having D-tagatose metabolic ability in a D-tagatose non-metabolizing strain.

Another aspect of the present invention is 1) a gene sequence encoding the novel fructose-1-phosphate kinase of SEQ ID NO: 2; 2) deletion of the gene sequence of SEQ ID NO: 3, which is a cra binding site; and 3) to provide a mutant strain comprising any one or more of the gene sequences of SEQ ID NO: 4.

Another aspect of the present invention comprises culturing the mutant strain in a medium containing D-tagatose; It is an object to provide a method for producing a strain having a D- tagatose metabolic ability comprising a.

One aspect of the present invention provides an expression cassette comprising a gene encoding a novel fructose-1-phosphate kinase in which the 39th alanine (A) is substituted with serine (S) in the amino acid sequence of SEQ ID NO: 1. .

The amino acid sequence of SEQ ID NO: 1 refers to the amino acid sequence of wild-type fructose-1-phosphate kinase (fruK), and in the present invention, the 39th alanine (A) of the amino acid sequence of SEQ ID NO: 1 is converted to serine (S). Through the new substituted fructose-1-phosphate kinase, D-tagatose can be metabolized instead of D-fructose.

The fructose-1-phosphate kinase (fruK) is an in vivo enzyme, also called 1-phosphofructokinase, which converts fructose-1-phosphate and ATP into fructose-1,6-phosphate in vivo. It functions to convert to diphosphate and ADP.

In one embodiment of the present invention, the gene encoding the fructose-1-phosphate kinase may be SEQ ID NO: 2.

sequence name Sequence E.coli BL21(DE3) FruK WT amino acid sequence
(SEQ ID NO: 1) MSRRVATITLNPAYDLVGFCPEIERGEVNLVKTTGLHA A GKGINVAKVLKDLGIDVTVGGFLGKDNQDGFQQLFSELGIANRFQVVQGRTRINVKLTEKDGEVTDFNFSGFEVTPADWERFVTDSLSWLGQFDMVCVSGSLPSGVSPEAFTDWMTRLRSQCPCIIFDSSREALVAGLKAAPWLVKPNRRELEIWAGRKLPEMKDVIEAAHALREQGIAHVVISLGAEGALWVNASGEWIAKPPSVDVVSTVGAGDSMVGGLIYGLLMRESSEHTLRLATAVAALAVSQSNVGITDRPQLAAMMARVDLQPFN E.coli BL21(DE3) FruK A39S mutant amino acid sequence MSRRVATITLNPAYDLVGFCPEIERGEVNLVKTTGLHA S GKGINVAKVLKDLGIDVTVGGFLGKDNQDGFQQLFSELGIANRFQVVQGRTRINVKLTEKDGEVTDFNFSGFEVTPADWERFVTDSLSWLGQFDMVCVSGSLPSGVSPEAFTDWMTRLRSQCPCIIFDSSREALVAGLKAAPWLVKPNRRELEIWAGRKLPEMKDVIEAAHALREQGIAHVVISLGAEGALWVNASGEWIAKPPSVDVVSTVGAGDSMVGGLIYGLLMRESSEHTLRLATAVAALAVSQSNVGITDRPQLAAMMARVDLQPFN FruK wt nucleotide sequence ATGAGCAGAC GTGTTGCTACTATCACCCTTAATCCGGCTTATGACCTTGTTGGTTTCTGC CCGGAAATTGAACGCGGCGAAGTGAACCTGGTGAAAACCACCGGTCTGCATGCG G TGGCCGCAATGATGGCGCGCGTCGACTTACAACCTTTTAACTGA mutant FruK A39S nucleotide sequence (SEQ ID NO: 2) ATGAGCAGAC GTGTTGCTACTATCACCCTTAATCCGGCTTATGACCTTGTTGGTTTCTGC CCGGAAATTGAACGCGGCGAAGTGAACCTGGTGAAAACCACCGGTCTGCATGCG T TGGCCGCAATGATGGCGCGCGTCGACTTACAACCTTTTAACTGA

In one embodiment of the present invention, the expression cassette may be deficient in the cra binding site, and the deficient cra binding site may be formed by not including the sequence of SEQ ID NO: 3 in the expression cassette.

sequence name Sequence Cra binding site deleted nucleotide sequence (SEQ ID NO: 3) Tgaaacgatt cagcctctat gagaaaaaaa gcgccaacct ggcttagggt taaagacaag atcgcgc

The cra (catabolite repressor/activator) is induced by the fructose-1-phosphate and fructose-1,6-biphosphate, and when there is a cra binding site in the expression cassette, the novel fructose- Although the expression of the 1-phosphate kinase gene may be restricted, the expression level of the novel fructose-1-phosphate kinase gene is increased by deleting this binding site.

In one embodiment of the present invention, the expression cassette may further include a mutant sequence between the lacI-encoding sequence and the T7 RNAP-encoding sequence, and more specifically, the lacI-encoding sequence and the T7 RNAP The mutant sequence between the coding sequences may be a T7 RNAP core promoter region, and more specifically, the mutant sequence between the lacI coding sequence and the T7 RNAP coding sequence is SEQ ID NO: 4 can

sequence name Sequence Wt T7RNAP core promoter region gcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtaagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtat aa tgtgtg g aattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaactt Mutated T7RNAP core promoter region (SEQ ID NO: 4) gcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtaagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtat gt tgtgtg a aattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaactt

The lacI refers to a gene encoding a lac repressor (LacI), a DNA-binding protein that inhibits the expression of a gene encoding a protein participating in lactose metabolism in microorganisms.

The T7 RNAP refers to an RNA polymerase derived from T7 bacteriophage that catalyzes the formation of RNA from DNA in the 5'->3' direction (EC:2.7.7.).

One aspect of the present invention provides a recombinant vector comprising the expression cassette.

As used herein, the term "recombinant vector" refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences essential for expressing the operably linked coding sequence in a specific host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

As used herein, the term "operably linked" means a functional linkage between a gene expression control sequence and another nucleotide sequence. The gene expression control sequence may be one or more selected from the group consisting of a replication origin, a promoter, and a transcription termination sequence. The transcription termination sequence may be a polyadenylation sequence (pA), and the origin of replication may be the f1 origin of replication, the SV40 origin of replication, the pMB1 origin of replication, the adeno origin of replication, the AAV origin of replication, or the BBV origin of replication, but is not limited thereto. .

The recombinant vector according to one embodiment of the present invention may be selected from the group consisting of plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retroviral vectors and adeno-associated virus vectors. Vectors that can be used as recombinant expression vectors include plasmids used in the art (eg, pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1 , pHV14, pGEX series, pET series, pUC19, etc.), phage (eg, λgt4λB, λ-Charon, λΔz1, M13, etc.) or viral vectors (eg, adeno-associated virus (AAV) vectors, etc.) It may be manufactured based on the like, but is not limited thereto.

The recombinant vector of the present invention may further include one or more selectable markers. The marker is a nucleic acid sequence having characteristics that can be selected by conventional chemical methods, and includes all genes capable of distinguishing transfected cells from non-transfected cells. For example, herbicide resistance genes such as glyphosate, glufosinate ammonium or phosphinothricin, ampicillin, kanamycin, G418, bleomycin ), hygromycin, and antibiotic resistance genes such as chloramphenicol, but are not limited thereto.

The construction of the recombinant vector of the present invention can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cutting and linking can be performed using enzymes generally known in the art. .

Another aspect of the present invention provides a mutant strain transformed with the recombinant vector.

As a method of transformation with the recombinant vector, an insertion method widely known in the art may be used. As the delivery method, for example, when the host cell is a prokaryotic cell, a CaCl2 method or an electroporation method may be used, and when the host cell is a eukaryotic cell, a microinjection method, a calcium phosphate precipitation method, an electroporation method, or a liposome method. -mediated transfection, heat shock and gene bombardment, etc. may be used, but are not limited thereto.

In addition, another aspect of the present invention is 1) a gene sequence encoding the novel fructose-1-phosphate kinase of SEQ ID NO: 2; 2) deletion of the gene sequence of SEQ ID NO: 3, which is a cra binding site; and 3) providing a mutant strain comprising any one or more gene sequences of the gene mutant sequences of SEQ ID NO: 4.

The mutant strain, in addition to the mutant strain transformed with the above-described recombinant vector, is mutated through various methods: 1) the gene mutant sequence encoding the novel fructose-1-phosphate kinase of SEQ ID NO: 2; 2) deletion of the gene sequence of SEQ ID NO: 3, which is a cra binding site; and 3) any one or more of the gene sequences of SEQ ID NO: 4 may be included.

Gene sequences included in the mutant strain include: 1) a gene mutant sequence encoding the novel fructose-1-phosphate kinase of SEQ ID NO: 2; 2) deletion of the gene sequence of SEQ ID NO: 3, which is a cra binding site; Or 3) may include any one of the genetic sequence of the gene mutation sequence of SEQ ID NO: 4,

1) a gene mutant sequence encoding the novel fructose-1-phosphate kinase of SEQ ID NO: 2; and 2) a deletion of the gene sequence of SEQ ID NO: 3, which is a cra binding site, or 1) a gene mutation sequence encoding the novel fructose-1-phosphate kinase of SEQ ID NO: 2; and 3) a gene mutation sequence of SEQ ID NO: 4, or 2) a deletion of the gene sequence of SEQ ID NO: 3, which is a cra binding site; and 3) may include the genetic mutation sequence of SEQ ID NO: 4,

1) a gene sequence encoding the novel fructose-1-phosphate kinase of SEQ ID NO: 2; 2) a gene sequence in which the sequence of SEQ ID NO: 4 is deleted at the cra binding site of SEQ ID NO: 3; and 3) a gene mutation sequence of SEQ ID NO: 4.

Any host known in the art may be used as the host of the mutant strain, and prokaryotic cells include, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli strains of the genus Bacillus, such as B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and enterobacteriaceae and strains such as Salmonella typhimurium, Serratia marcessons, and various Pseudomonas species. , In the case of transformation into a eukaryotic cell, as a host cell, yeast (Saccharomyce cerevisiae), insect cells, plant cells and animal cells, such as SP2/0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6 , W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN and MDCK cell lines may be used, and specifically, the host of the mutant strain may be Escherichia coli.

In one embodiment of the present invention, the mutant strain may have D-tagatose metabolic ability. Specifically, the recombinant strain may have D-tagatose metabolic ability because it contains the aforementioned expression cassette or mutation. The D-tagatose metabolic ability means to utilize D-tagatose as an energy source.

Another aspect of the present invention comprises culturing the mutant strain in a medium containing D-tagatose; It provides a method for producing a strain having D- tagatose metabolic ability comprising a.

The culturing step is a step of culturing the mutant strain in a medium containing D-tagatose, and the medium may contain known components for culturing the mutant strain.

A new sugar metabolism pathway can be constructed through the expression cassette according to the present invention and a vector containing the same, and a mutant strain transformed through this or a mutant strain including a mutated gene has a new sugar metabolism pathway.

1 is a view showing a process for preparing a mutant strain of the present invention. Specifically, FIG. 1A compares three strains capable of using tagatose (S.enterica, K. pneumociae, K.oxytoca, and B.licheniformis), a gene cluster, and E. coli , and FIG. 1B shows a comparison of the parent strain ( E. coli) . BL12 (DE3)) and the mutant strain (pET28a-gatY/ E. coli BL21 (DE3)) were compared to confirm the mutation site, and FIG. 1c is a graph confirming the growth rate according to subculture of the mutant strain. .
Figure 2 confirms the activity of the mutant strain, Figure 2a is the result of comparing the activity of the kinase WT and mutant strain, Figure 2b is the three glycotrophic medium of the mutant strain (Glc (glucose), Fru (fructose), Tag (tagatose)) is the result of mRNA level analysis through qRT-PCR, and FIG. 2c is a picture showing the metabolic activity of tagatose introduced by mutation.
Figure 3 is the result of confirming the growth of the mutant strain of the present invention, Figure 3a is the result of confirming the growth curve of the mutant strain in which fruK A39S and Cra gene binding sites were deleted (two sites were mutated), Figure 3b is This is the result of confirming the growth curve of the mutant strain in the fruK A39S and Cra gene binding sites and the T7RNAP core promoter region (strain with three sites mutated).

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of mutant strains having tagatose metabolism

Example 1-1. Preparation of mutant strains

Through gene cluster comparison with three strains capable of using tagatose ( S.enterica, K. pneumociae, K.oxytoca, B.licheniformis ), E.coli , a tagatose-incompatible strain, has tagatose 1, It was confirmed that the gatY gene, a 6bp aldolase, was missing (Fig. 1a).

B. licheniformis- derived gatY gene was transformed into E. coli and adapted for evolution in tagatose medium. After confirming growth after about 500 hours, whole-length genomic analysis was performed on strains that were continuously subcultured and no longer increased in growth rate, and mutant sites (fruK, Cra binding site, T7RNAP promoter) were identified (Fig. 1b, Fig. 1c) Mutant strains were prepared.

Experimental Example 1: Confirmation of activity of mutant strains

The results of comparing the activities of kinase WT and mutant strains to identify the factors affecting the acquisition of tagatose availability. In the case of the mutant strain, it was confirmed that the activity was decreased for Fru-6p and increased for Tag-6p (FIG. 2a).

The degree of gene expression in three glycotrophic media (Glc (glucose), Fru (fructose), Tag (tagatose)) of strains that have undergone whole-length genome analysis inducing adaptive evolution (three sites mutated strains) mRNA level analysis was performed through qRT-PCR. As a result, it was confirmed that the expression level of FruK A39S was increased in tagatose than in other media (FIG. 2b), and through this, activity in the tagatose metabolic cycle was predicted (FIG. 2c).

Experimental Example 2: Confirmation of growth of mutant strains

As a result of checking the growth curve of the mutant strain lacking the binding site of fruK A39S and Cra gene (strain with two sites mutated), it was confirmed that the growth was excellent when tagatose was used as a nutrient source (Fig. 3a).

In addition, as a result of confirming the growth curve of a mutant strain of fruK A39S and Cra gene binding sites and T7RNAP promoter site mutation (strain with three sites mutated), it was confirmed that growth was excellent when tagatose was used as a nutrient source ( Fig. 3b).

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> a method for providing determination gene group for rare sugar metabolizing ability for rare sugar non-metabolizable strain <130> PN200407 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 312 <212> PRT <213> artificial sequence <220> <223> E. coli BL21(DE3) FruK WT amino acid sequence <400> 1 Met Ser Arg Arg Val Ala Thr Ile Thr Leu Asn Pro Ala Tyr Asp Leu 1 5 10 15 Val Gly Phe Cys Pro Glu Ile Glu Arg Gly Glu Val Asn Leu Val Lys 20 25 30 Thr Thr Gly Leu His Ala Ala Gly Lys Gly Ile Asn Val Ala Lys Val 35 40 45 Leu Lys Asp Leu Gly Ile Asp Val Thr Val Gly Gly Gly Phe Leu Gly Lys 50 55 60 Asp Asn Gln Asp Gly Phe Gln Gln Leu Phe Ser Glu Leu Gly Ile Ala 65 70 75 80 Asn Arg Phe Gln Val Val Gln Gly Arg Thr Arg Ile Asn Val Lys Leu 85 90 95 Thr Glu Lys Asp Gly Glu Val Thr Asp Phe Asn Phe Ser Gly Phe Glu 100 105 110 Val Thr Pro Ala Asp Trp Glu Arg Phe Val Thr Asp Ser Leu Ser Trp 115 120 125 Leu Gly Gln Phe Asp Met Val Cys Val Ser Gly Ser Leu Pro Ser Gly 130 135 140 Val Ser Pro Glu Ala Phe Thr Asp Trp Met Thr Arg Leu Arg Ser Gln 145 150 155 160 Cys Pro Cys Ile Ile Phe Asp Ser Ser Arg Glu Ala Leu Val Ala Gly 165 170 175 Leu Lys Ala Ala Pro Trp Leu Val Lys Pro Asn Arg Arg Glu Leu Glu 180 185 190 Ile Trp Ala Gly Arg Lys Leu Pro Glu Met Lys Asp Val Ile Glu Ala 195 200 205 Ala His Ala Leu Arg Glu Gln Gly Ile Ala His Val Val Ile Ser Leu 210 215 220 Gly Ala Glu Gly Ala Leu Trp Val Asn Ala Ser Gly Glu Trp Ile Ala 225 230 235 240 Lys Pro Pro Ser Val Asp Val Val Ser Thr Val Gly Ala Gly Asp Ser 245 250 255 Met Val Gly Gly Leu Ile Tyr Gly Leu Leu Met Arg Glu Ser Ser Glu 260 265 270 His Thr Leu Arg Leu Ala Thr Ala Val Ala Ala Leu Ala Val Ser Gln 275 280 285 Ser Asn Val Gly Ile Thr Asp Arg Pro Gln Leu Ala Ala Met Met Ala 290 295 300 Arg Val Asp Leu Gln Pro Phe Asn 305 310 <210> 2 <211> 939 <212> DNA <213> artificial sequence <220> <223> mutant FruK A39S nucleotide sequence <400> 2 atgagcagac gtgttgctac tatcaccctt aatccggctt atgaccttgt tggtttctgc 60 ccggaaattg aacgcggcga agtgaacctg gtgaaaacca ccggtctgca tgcgtcgggt 120 aaaggcatca acgtggccaa agtattaaaa gacctgggaa ttgatgtcac cgttggcggc 180 ttcctcggta aagacaatca ggatggtttt cagcaactgt tcagcgagct gggcattgcc 240 aaccgtttcc aggttgtaca ggggcgcact cgaattaacg ttaagctgac ggaaaaagac 300 ggcgaagtga ccgacttcaa cttctcgggt tttgaagtca cccccgccga ctgggaacgc 360 tttgtgactg attctctgag ctggctcggt cagttcgata tggtctgtgt cagcggaagc 420 ttaccgtcag gcgtcagccc ggaagcgttc accgactgga tgactcgcct gcgtagtcag 480 tgtccttgca ttatctttga tagtagccgt gaagcgttag tagcaggttt gaaagcggca 540 ccgtggctgg tgaaacctaa ccgccgcgag ctggaaatct gggcaggccg taaactgcct 600 gaaatgaaag atgtgattga agctgcgcat gcgctgcgtg aacaaggtat cgcgcatgtt 660 gttatttcac tgggtgccga aggcgcgctt tgggttaatg cctccggcga atggatcgcc 720 aaaccaccgt cagtcgatgt cgtaagcacc gttggcgcag gggattctat ggttggtggc 780 ctgattatg gcttgctgat gcgtgaatcc agtgaacaca cactgcgtct ggcgacagct 840 gttgcagccc tggcggtaag tcaaagcaat gtgggtatta ccgatcgtcc gcagttggcc 900 gcaatgatgg cgcgcgtcga cttacaacct tttaactga 939 <210> 3 <211> 67 <212> DNA <213> artificial sequence <220> <223> Cra binding site deleted nucleotide sequence <400> 3 tgaaacgatt cagcctctat gagaaaaaaa gcgccaacct ggcttagggt taaagacaag 60 atcgcgc 67 <210> 4 <211> 279 <212> DNA <213> artificial sequence <220> <223> Mutated T7RNAP core promoter region <400> 4 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc 60 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg 120 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtgaaatt gtgagcggat 180 aacaatttca cacaggaaac agctatgacc atgattacgg attcactggc cgtcgtttta 240 caacgtcgtg actgggaaaa ccctggcgtt acccaactt 279

Claims (13)

An expression cassette comprising a gene encoding a novel fructose-1-phosphate kinase in which alanine (A) at position 39 in the amino acid sequence of SEQ ID NO: 1 is substituted with serine (S).
According to claim 1,
The gene encoding the novel fructose-1-phosphate kinase expression cassette of SEQ ID NO: 2.
According to claim 1,
The expression cassette is an expression cassette in which the cra binding site is deleted.
According to claim 3,
The expression cassette wherein the deletion of the cra binding site is made by not including the sequence of SEQ ID NO: 3 in the expression cassette.
According to claim 1,
The expression cassette further comprises a mutant sequence between the sequence encoding lacI and the sequence encoding T7 RNAP.
According to claim 5,
The mutant sequence between the sequence encoding the lacI and the sequence encoding the T7 RNAP is a mutant of the T7 RNAP core promoter region.
According to claim 5,
The expression cassette of SEQ ID NO: 4, wherein the mutant sequence between the sequence encoding lacI and the sequence encoding T7 RNAP is SEQ ID NO: 4.
A recombinant vector comprising the expression cassette of claim 1.
A mutant strain transformed with the recombinant vector of claim 8.
1) a gene mutant sequence encoding the novel fructose-1-phosphate kinase of SEQ ID NO: 2;
2) deletion of the gene sequence of SEQ ID NO: 3, which is a cra binding site; and
3) A mutant strain containing any one or more mutations in the gene mutation sequence of SEQ ID NO: 4.
According to claim 9 or 10,
The recombinant strain is a mutant strain of Escherichia coli.
The method of claim 9 or 10,
The recombinant strain is a mutant strain having D-tagatose metabolism.
Culturing the mutant strain of claim 9 or claim 10 in a medium containing D-tagatose; Method for producing a strain having D- tagatose metabolic ability comprising a.

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