TECHNICAL FIELD
The present application relates to a novel promoter and a method for producing target
materials using the same. More specifically, the present application relates to a novel
polynucleotide having promoter activity, a vector and a host cell including the same, and a
method for producing target materials using the microorganism.
BACKGROUND ART
The process of producing target materials (e.g., amino acids) from microorganisms has
been studied in various ways as an eco-friendly and safe production method. Among these,
research has been continuously conducted to produce a large amount of target materials in
microorganisms of the genus Corynebacterium. Microorganisms of the genus
Corynebacterium, particularly Corynebacterium glutamicum, are gram-positive microorganisms
that are widely used to produce L-amino acids and other useful materials. In order to produce
the L-amino acids and other useful materials, various studies are being conducted to develop
microorganisms with high-efficiency production and technologies for fermentation processes.
L-Lysine, a representative material produced by the microorganisms of the genus
Corynebacterium, is used in animal feeds, pharmaceuticals for human use, and the cosmetics
industry, and is produced by fermentation using strains of Corynebacterium. A microorganism
in which the L-lysine biosynthesis-related genes are enhanced and a method for producing Llysine
using the same, etc., are known in the art (KR 10-0924065 B1).
Additionally, L-threonine is an essential amino acid and is widely used in feed and food
additives, and is also used in infusions for medicine and as raw materials for pharmaceuticals.
Since L-threonine is low in vegetable protein, it is effectively used as an additive for animal feed.
L-Threonine is mainly produced by fermentation using E. coli or microorganisms of the genus
Corynebacterium developed using artificial mutation or genetic recombination. Typically, a
method using a gene recombinant strain to produce L-threonine by introducing an E. coli-derived
threonine operon into Brevibacterium flavum, a threonine-producing strain, etc., is known in the
art (TURBA E, et al., Agric. Biol. Chem. 53:2269–2271, 1989).
O-Acetyl homoserine is a substance used as a precursor of methionine production and is
an intermediate in the methionine biosynthesis pathway (WO 2008/013432). O-Acetyl-Lhomoserine
is synthesized from L-homoserine and acetyl-CoA as substrates by way of
homoserine O-acetyl transferase.
L-Isoleucine shares a major biosynthetic pathway with other branched-chain amino
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acids, L-valine and L-leucine. In regard to the biosynthetic pathway of L-isoleucine, 2-
ketobutyrate produced from L-threonine, an amino acid derived from pyruvate and aspartic acid
which are produced in glycolysis, is used as a precursor. From the two precursors, 2-aceto-2-
hydroxyacetate is synthesized through the action of an enzyme acetohydroxyacid synthase
(AHAS), and then 2,3-dihydroxy-3-methylvalerate is produced by way of acetohydroxyacid
isomeroreductase. Subsequently, 2-keto-3-methylvalerate is produced by the action of
dihydroxyacid dehydratase, and L-isoleucine is ultimately produced through the
aminotransferase reaction. In addition, the acetohydroxyacid synthase catalyzes
decarboxylation of pyruvate and a condensation reaction with other pyruvate molecules to
produce acetolactate, a precursor of valine and leucine.
L-Tryptophan, one of the essential amino acids, has been widely used in feed additives,
or as a raw material for pharmaceuticals such as infusions, and as a material for health food.
Currently, L-tryptophan is mainly produced by direct fermentation using microorganisms.
L-Tyrosine is an amino acid and is used as an important material for pharmaceutical raw
materials, food additives, animal feed, nutritional supplements, etc. In order to produce the Ltyrosine
and other useful materials, various studies are being conducted to develop
microorganisms with high-efficiency production and technologies for fermentation processes.
The production process of L-tyrosine by microorganisms starts with 3-deoxy-D-arobinoheptulosonate-
7-phosphate (DAHP) produced by the polymerization reaction of
phosphoenolpyruvate (PEP), which is an intermediate of glycolysis, with erythrose-4-phosphate
(E4P), which is an intermediate of the pentose phosphate pathway. Then, DAHP is
biosynthesized from chorismate to prephenate through the common aromatic biosynthetic
pathway and is ultimately converted to L-tyrosine through the L-tyrosine biosynthetic pathway.
During this process, chorismate can be shunted into L-tryptophan, and prephenate can be shunted
into L-tyrosine or L-phenylalanine. Thus, when the common aromatic biosynthetic pathway is
strengthened so as to increase the amount of L-tyrosine produced, it can be expected that the
production of L-tryptophan and L-phenylalanine will also increase at the same time. That is, in
order to produce L-tyrosine, phenylalanine and tryptophan are produced together as by-products,
and accordingly, various studies such as gene recombination, purification, etc. must be carried
out. Meanwhile, it is known that the production of L-tryptophan is regulated by repressors and
attenuators according to the concentration of L-tryptophan produced by microorganisms (Korean
Patent No. 10-0792095).
L-Phenylalanine is the starting material for L-aspartyl-L-phenylalanine methyl ester,
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which is a sweetener.
If a universal promoter that is not limited to specific useful materials is developed, it is
expected that it can be utilized in the production of various useful materials.
Under such circumstances, the present inventors have developed a polynucleotide
having novel promoter activity capable of producing target materials with high productivity in
microorganisms of the genus Corynebacterium, and have confirmed that the productivity of the
target materials can be increased by introducing the polynucleotide into the microorganisms,
thereby completing the present application.
DISCLOSURE
TECHNICAL PROBLEM
The present inventors have made extensive efforts to prepare a novel polynucleotide
having promoter activity, and as a result, they have confirmed that the target gene promoter can
be enhanced by nucleotide substitution, and the enhanced promoter can regulate the expression
of genes operably linked to the promoter, thereby completing the present application.
TECHNICAL SOLUTION
One object of the present application is to provide a polynucleotide having promoter
activity, in which the nucleotides at positions 218, 219, 220, 221, 222, 225, and 227 in the
polynucleotide sequence of SEQ ID NO: 1 are substituted with other nucleotides.
Another object of the present application is to provide a gene expression cassette,
including: the polynucleotide; and a gene encoding a target protein which is operably linked to
the polynucleotide.
Still another object of the present application is to provide a host cell, including: the
polynucleotide; and a gene encoding a target protein which is operably linked to the
polynucleotide.
Yet another object of the present application is to provide a method for producing a
target material, including: culturing the host cell in a medium; and recovering the target material
from the medium.
Even another object of the present application is to provide the use of a polynucleotide,
in which the nucleotides at positions 218, 219, 220, 221, 222, 225, and 227 in the polynucleotide
sequence of SEQ ID NO: 1 are substituted with other nucleotides, as a promoter.
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ADVANTAGEOUS EFFECTS
The novel promoter of the present application may be introduced into a microorganism
for producing target materials to increase the production of target materials of the microorganism.
Due to the improved production yield, effects such as production cost reduction along with the
convenience of production may be expected in the industrial aspect.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present application will be described in detail as follows. Meanwhile, each
description and embodiment disclosed herein can be applied to other descriptions and
embodiments, respectively. That is, all combinations of various elements disclosed herein fall
within the scope of the present application. Further, the scope of the present application is not
limited by the specific description described below.
Additionally, those of ordinary skill in the art may be able to recognize or confirm, using
only conventional experimentation, many equivalents to the particular aspects of the invention
described herein. Furthermore, it is also intended that these equivalents be included in the
present application.
One aspect of the present application provides a polynucleotide having promoter activity.
Specifically, the polynucleotide having promoter activity of the present application may
be a polynucleotide having promoter activity, including at least one nucleotide substitution in the
polynucleotide sequence of SEQ ID NO: 1.
As used herein, the term “polynucleotide” is a DNA strand having more than a certain
length as a nucleotide polymer, which is a long chain of nucleotide monomers connected by
covalent bonds.
As used herein, the term “promoter” refers to an untranslated nucleotide sequence
located upstream of a coding region, which includes a binding site for a polymerase and has the
activity of initiating transcription of a promoter-target gene into mRNA, i.e., a DNA region that
leads to the initiation of transcription of a particular gene when a polymerase binds thereto.
The promoter may be located at the 5′ region of an mRNA transcription initiation site.
As used herein, the term “polynucleotide having promoter activity” refers to a DNA
region present in the vicinity of a region, which is involved in the transcription of a target gene
including a binding site of an RNA polymerase or an enhancer, for the expression of a gene to be
operably connected downstream thereof, i.e., the target gene. For the purpose of the present
6
application, the polynucleotide may be used as a promoter for general use. Additionally, the
polynucleotide may regulate (e.g., increase or decrease), in a cell, as compared to an existing
promoter or a cell-internal promoter, the expression of a target gene operably linked thereto and
the production and/or activity of a protein encoded by the target gene, and may regulate (e.g.,
increase or decrease) the production and/or activity of target products (as biologically active
material, for example, at least one selected from the group consisting of amino acids, nucleic
acids, vitamins, proteins, fatty acids and organic acids, etc.) involved in the production of
proteins, but is not limited thereto.
For example, the polynucleotide having promoter activity of the present application may
be used as a promoter capable of enhancing the expression of a polypeptide having transketolase
(tkt) activity. Additionally, the polynucleotide may be a polynucleotide involved in increasing
the production or production amount of lysine, threonine, O-acetyl homoserine, isoleucine,
tryptophan, tyrosine or phenylalanine.
The polynucleotide of the present application may include any polynucleotide sequences
having promoter activity without limitation.
In one example, the polynucleotide having promoter activity of the present application
may include a polynucleotide having promoter activity, in which at least one nucleotide in the
nucleotide sequence of SEQ ID NO: 1 is substituted with another nucleotide. Specifically, in
the present application, the polynucleotide having promoter activity may be a polynucleotide
having promoter activity, including one or more, two or more, three or more, four or more, five
or more, six or more or seven or more nucleotide substitutions in the polynucleotide sequence of
SEQ ID NO: 1. In the present application, the polynucleotide having promoter activity may be
used interchangeably with “modified promoter”, and all of the above-described terms may be
used herein.
In the present application, the polynucleotide sequence of SEQ ID NO: 1 may be, for
example, a polynucleotide having promoter activity of transketolase. Additionally, a
polynucleotide in which specific nucleotides are substituted in the polynucleotide sequence of
SEQ ID NO: 1 may also be a polynucleotide having promoter activity of transketolase, as long as
it has promoter activity. The polynucleotide sequence of SEQ ID NO: 1 may be a
representative polynucleotide for indicating a mutation position, and other polynucleotide
sequences having promoter activity, which correspond thereto, are also included in the sequence
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that can introduce a mutation. For example, any polynucleotide sequences capable of serving
as a promoter for transketolase (tkt) or a polypeptide having a corresponding activity may be
included in the range of sequences that can introduce a mutation of the present application,
without limitation.
The nucleotide sequence of SEQ ID NO: 1 can be confirmed from NCBI GenBank, a
known database, and the sequence corresponding to SEQ ID NO: 1, as a sequence that can serve
as a promoter for transketolase, may be derived from Corynebacterium sp., and specifically, it
may be a sequence of Corynebacterium glutamicum, but a sequence having an activity equal to
or higher than that of the polynucleotide may be included in the promoter of the present
application, without limitation.
The polynucleotide having promoter activity of the present application may be one in
which the promoter activity is enhanced by substitution of nucleotides at specific positions in the
polynucleotide sequence having existing promoter activity.
In one embodiment, the polynucleotide having promoter activity of the present
application may include a polynucleotide having promoter activity in which at least one
nucleotide in the nucleotide sequence of SEQ ID NO: 1 is substituted with another nucleotide.
Specifically, it may be composed of a polynucleotide having promoter activity, in which at least
one nucleotide in the nucleotide sequence of SEQ ID NO: 1 is substituted with another
nucleotide. In the present application, the polynucleotide having promoter activity may be used
interchangeably with “modified promoter”.
In one embodiment, the modified promoter may be a polynucleotide having promoter
activity, including substitutions of at least one nucleotide selected from the group consisting of
nucleotides at positions 218, 219, 220, 221, 222, 225, and 227 with other nucleotides.
Specifically, the modified promoter may be one in which any one or more, two or more, three or
more, four or more, five or more, six or more or seven of the above positions or their
corresponding positions are substituted with other nucleotides.
As used herein, the term “another/other nucleotide” is not limited as long as it is
different from the nucleotide before substitution. For example, when it is described that “the
nucleotide at position 218 in SEQ ID NO: 1 is substituted with another nucleotide”, it means that
it is substituted with adenine (A), thymine (T), or guanine (G), except for cytosine (C). In
addition, unless otherwise indicated, when it is described that a specific nucleotide is
“substituted”, it means that the nucleotide is substituted with another nucleotide which is
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different from the nucleotide before substitution.
Meanwhile, those skilled in the art can identify a nucleotide at a position corresponding
to the nucleotides at positions 218, 219, 220, 221, 222, 225, and 227 of SEQ ID NO: 1 of the
present application in any polynucleotide sequences through sequence alignment known in the
art. Further, unless otherwise indicated herein, when it is described that “a nucleotide at a
specific position in a specific SEQ ID NO, it is obvious that it is meant to include the
“nucleotides at the corresponding position” in any polynucleotide sequences. Therefore, the
polynucleotide sequences in which any one or more nucleotides selected from the group
consisting of nucleotides at positions corresponding to 218, 219, 220, 221, 222, 225, and 227 of
SEQ ID NO: 1 in any polynucleotide sequence having promoter activity is substituted with other
nucleotides also fall within the scope of the present application.
In one example, when one or more, two or more, three or more, four or more, five or
more, six or more or seven nucleotides at positions corresponding to 218, 219, 220, 221, 222,
225, and 227 in the polynucleotide sequence of SEQ ID NO: 1 is substituted with other
nucleotides, promoters with higher activity than unsubstituted (unmodified) promoter sequences
can be obtained.
Specifically, the polynucleotide having promoter activity of the present application may
be one in which the nucleotides at positions 218, 219, 220, 221, 222, 225, and 227 in the
polynucleotide sequence of SEQ ID NO: 1 are substituted with other nucleotides, but is not
limited thereto.

WE CLAIM:
1. A polynucleotide having promoter activity, in which the nucleotides at positions 218, 219,
220, 221, 222, 225, and 227 in the polynucleotide sequence of SEQ ID NO: 1 are
substituted with other nucleotides.
2. The polynucleotide of claim 1, wherein the polynucleotide comprises a substitution of
cytosine (C), the nucleotide at position 218, with thymine (T), a substitution of cytosine
(C), the nucleotide at position 219, with guanine (G), a substitution of adenine (A), the
nucleotide at position 220, with thymine (T), a substitution of adenine (A), the nucleotide
at position 221, with guanine (G), a substitution of thymine (T), the nucleotide at position
222, with guanine (G), a substitution of adenine (A), the nucleotide at position 225, with
thymine (T), and a substitution of cytosine (C), the nucleotide at position 227, with
adenine (A), in the polynucleotide sequence of SEQ ID NO: 1.
3. The polynucleotide of claim 1, wherein the polynucleotide is composed of a nucleotide
sequence of SEQ ID NO: 2.
4. The polynucleotide of any one of claims 1 to 3, wherein the polynucleotide is operably
linked to a gene encoding a target protein.
5. A gene expression cassette, comprising: the polynucleotide of any one of claims 1 to 3;
and a gene encoding a target protein which is operably linked to the polynucleotide.
6. The gene expression cassette of claim 5, wherein the target protein is transketolase.
7. A host cell, comprising: the polynucleotide of any one of claims 1 to 3; and a gene
encoding a target protein which is operably linked to the polynucleotide.
8. The host cell of claim 7, wherein the target protein is transketolase.
9. The host cell of claim 7, wherein the host cell is a microorganism of the genus
Corynebacterium.
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10. The host cell of claim 9, wherein the microorganism of the genus Corynebacterium is
Corynebacterium glutamicum.
11. A method for producing a target material, comprising: culturing the host cell of claim 7 in
a medium; and recovering the target material from the medium.
12. The method of claim 11, wherein the target material is an amino acid.
13. The method of claim 11, wherein the target material is at least one selected from the
group consisting of lysine, threonine, O-acetyl homoserine, isoleucine, tryptophan,
tyrosine, and phenylalanine.