[Technical Field]
The present application relates to an O-phosphoserine (OPS) export protein variant, and
a method for producing O-phosphoserine, cysteine, and cysteine derivatives using the same.
5
[Background Art]
L-Cysteine, an amino acid having an important role in sulfur metabolism in all living
organisms, is used not only in the synthesis of biological proteins such as hair keratin,
glutathione, biotin, methionine, and other sulfur-containing metabolites, but also as a precursor
10 for biosynthesis of coenzyme A.
Methods of producing L-cysteine using microorganisms known in the art include: 1) a
method of biologically converting D,L-2-aminothiazoline-4-carboxylic acid (D,L-ATC) into
L-cysteine using microorganisms, 2) a method of producing L-cysteine by direct fermentation
using E. coli (US 5972663 A; Wada M and Takagi H, Appl. Microbiol. Biochem., 73:48–54,
15 2006), and 3) a method of producing O-phosphoserine (hereinafter, “OPS”) by fermentation
using microorganisms, and then converting O-phosphoserine into L-cysteine by reacting
O-phosphoserine with a sulfide under the catalytic action of O-phosphoserine sulfhydrylase
(hereinafter, “OPSS”) (US 8557549 B2).
In particular, in order to produce cysteine by way of method 3) in high yield, OPS, the
20 precursor, should be produced in an excess amount.
[Disclosure]
[Technical Problem]
The present inventors completed the present application by identifying a variant with
25 increased activity in the export factor capable of smoothly exporting the OPS produced in the
OPS-producing strain out of the cell, and confirming that OPS export is improved due to the
variant.
[Technical Solution]
30 It is one object of the present application to provide a polypeptide having
O-phosphoserine (OPS) exporting activity.
It is another object of the present application to provide a polynucleotide encoding the
polypeptide of the present application.
It is still another object of the present application to provide a vector containing the
3
polynucleotide of the present application.
It is yet another object of the present application to provide an
O-phosphoserine-producing microorganism, including any one or more of the polypeptide, the
polynucleotide, and the vector of the present application.
5 It is even another object of the present application to provide a method for producing
O-phosphoserine, including culturing the O-phosphoserine-producing microorganism of the
present application in a medium.
It is further another object of the present application to provide a method for producing
cysteine or a derivative thereof, including:
10 a) producing O-phosphoserine (OPS) or a medium containing the same by culturing an
O-phosphoserine-producing microorganism, which includes any one or more of the polypeptide,
the polynucleotide encoding the polypeptide of the present application, and the vector containing
the polynucleotide of the present application, in a medium; and
b) reacting the O-phosphoserine or a medium containing the same produced in step a)
15 with a sulfide in the presence of O-phosphoserine sulfhydrylase (OPSS) or a microorganism
expressing the same.
[Advantageous Effects]
When the microorganism having an O-phosphoserine producing capability is cultured
20 using the polypeptide having O-phosphoserine exporting activity of the present application, it
can lead to high-yield production of OPS compared to using an existing non-modified or variant
protein.
[Best Mode]
25 The present application will be described in detail. 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.
30
In one aspect of the present application to achieve the objects above, the present
application provides a polypeptide having O-phosphoserine (OPS) exporting activity, including a)
a substitution of isoleucine (I) at a position corresponding to 241 in the amino acid sequence of
SEQ ID NO: 11 with threonine (T), a substitution of aspartic acid (D) at a position corresponding
4
to 246 in the amino acid sequence of SEQ ID NO: 11 with valine (V), and a substitution of
valine (V) at a position corresponding to 330 in the amino acid sequence of SEQ ID NO: 11 with
isoleucine (I), and having an amino acid sequence, wherein b) the amino acid at a position
corresponding to 88 is phenylalanine and c) the amino acid at a position corresponding to 207 is
5 lysine (K).
As used herein, the term “O-phosphoserine” (hereinafter, “OPS”) refers to a phosphoric
acid ester of serine which serves as a constituting component for many proteins. In particular,
the OPS is a precursor of L-cysteine and can be converted to cysteine by reacting with a sulfide
under the catalytic action of OPS sulfhydrylase (hereinafter, “OPSS”), but is not limited thereto
10 (US 8557549 B2).
As used herein, the term “a polypeptide having OPS exporting activity” refers to a
membrane protein which has the activity of exporting OPS to the outside of the cell, and the
membrane protein may be derived from E. coli. In the present application, the polypeptide
having OPS exporting activity may be a YhhS major facilitator superfamily (MFS) transporter or
15 a variant thereof. Specifically, the polypeptide of the present application may be a variant of
the YhhS MFS transporter exhibiting improved activity compared to that of a wild-type YhhS
MFS transporter, which has been identified as a protein having OPS exporting activity in E. coli,
in which growth inhibition is released in a condition where excess OPS is present.
As used herein, the term “variant” refers to a protein having at least one amino acid
20 sequence different from the recited sequence due to conservative substitution and/or
modification such that functions and properties of the protein are retained. Variants differ from
an identified sequence due to substitution, deletion, or addition of several amino acids. Such
variants may generally be identified by modifying one of the above amino acid sequences of the
protein and evaluating the properties of the modified protein. That is, the ability of the variants
25 may be enhanced, unchanged, or diminished relative to a native protein. Further, some variants
may include those in which one or more portions, such as an N-terminal leader sequence or
transmembrane domain, have been removed. Other variants may include those in which a
portion has been removed from the N- and/or C-terminus of a mature protein. The term
“variant” may be used interchangeably with terms such as modified, modification, modified
30 protein, modified polypeptide, mutant, mutein, divergent, variant, etc. without limitation, as long
as the terms are used to indicate variation. For the purpose of the present application, the
variant may be those having an increased activity of a modified protein compared to a natural
wild-type or non-modified protein, but is not limited thereto.
As used herein, the term “conservative substitution” refers to substitution of an amino
5
acid with another amino acid having similar structural and/or chemical properties. The variant
may have, for example, at least one conservative substitution while retaining at least one
biological activity. Such amino acid substitution may generally occur based on similarity of
polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of a
5 residue. For example, among electrically charged amino acids, positively charged (basic)
amino acids include arginine, lysine, and histidine, and negatively charged (acidic) amino acids
include glutamic acid and aspartic acid; among uncharged amino acids, non-polar amino acids
include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and
proline; polar or hydrophilic amino acids include serine, threonine, cysteine, tyrosine, asparagine,
10 and glutamine; and aromatic amino acids among the amino acids include phenylalanine,
tryptophan, and tyrosine.
Additionally, variants may also include deletion or addition of amino acids that have
minimal influence on the properties and secondary structure of the polypeptide. For example,
the polypeptide may be conjugated to a signal (or leader) sequence at the N-terminus of a protein
15 involved in the transfer of proteins co-translationally or post-translationally. Further, the
polypeptide may also be conjugated with another sequence or linker to identify, purify, or
synthesize the polypeptide.
Specifically, the polypeptide having OPS exporting activity of the present application
20 may be a polypeptide having OPS exporting activity, including a) a substitution of isoleucine (I)
at a position corresponding to 241 in the amino acid sequence of SEQ ID NO: 11 with threonine
(T), a substitution of aspartic acid (D) at a position corresponding to 246 in the amino acid
sequence of SEQ ID NO: 11 with valine (V), and a substitution of valine (V) at a position
corresponding to 330 in the amino acid sequence of SEQ ID NO: 11 with isoleucine (I), and
25 having an amino acid sequence, wherein b) the amino acid at a position corresponding to 88 is
phenylalanine and c) the amino acid at a position corresponding to 207 is lysine (K), or a
polypeptide having OPS exporting activity, including a) a substitution of isoleucine (I) at a
position corresponding to 241 in the amino acid sequence of SEQ ID NO: 11 with threonine (T),
a substitution of aspartic acid (D) at a position corresponding to 246 in the amino acid sequence
30 of SEQ ID NO: 11 with valine (V), and a substitution of valine (V) at a position corresponding to
330 in the amino acid sequence of SEQ ID NO: 11 with isoleucine (I), and having an amino acid
sequence, wherein b) the amino acid at a position corresponding to 88 is phenylalanine and c) the
amino acid at a position corresponding to 207 is lysine (K).
The polypeptide having OPS exporting activity of the present application may be a
6
polypeptide having OPS exporting activity, including a) a substitution of isoleucine (I) at a
position corresponding to 241 in the amino acid sequence of SEQ ID NO: 11 with threonine (T),
a substitution of aspartic acid (D) at a position corresponding to 246 in the amino acid sequence
of SEQ ID NO: 11 with valine (V), and a substitution of valine (V) at a position corresponding to
5 330 in the amino acid sequence of SEQ ID NO: 11 with isoleucine (I), and having an amino acid
sequence, wherein b) the amino acid at a position corresponding to 88 is phenylalanine and c) the
amino acid at a position corresponding to 207 is lysine (K), or a polypeptide having OPS
exporting activity, including a) a substitution of isoleucine (I) at a position corresponding to 241
in the amino acid sequence of SEQ ID NO: 11 with threonine (T), a substitution of aspartic acid
10 (D) at a position corresponding to 246 in the amino acid sequence of SEQ ID NO: 11 with valine
(V), and a substitution of valine (V) at a position corresponding to 330 in the amino acid
sequence of SEQ ID NO: 11 with isoleucine (I), and consisting of or essentially consisting of an
amino acid sequence, wherein b) the amino acid at a position corresponding to 88 is
phenylalanine and c) the amino acid at a position corresponding to 207 is lysine (K).
15 Additionally, the polypeptide having OPS exporting activity of the present application
may include without limitation any polypeptide having an amino acid sequence showing an
identity of at least 70%, 80%, 90%, 95%, or 99% or higher, and less than 100% with the amino
acid sequence of SEQ ID NO: 11 or SEQ ID NO: 1 and exhibiting an OPS exporting capability
substantially identical or corresponding to that of the polypeptide, while being a polypeptide,
20 which includes a) a substitution of isoleucine (I) at a position corresponding to 241 in the amino
acid sequence of SEQ ID NO: 11 with threonine (T), a substitution of aspartic acid (D) at a
position corresponding to 246 in the amino acid sequence of SEQ ID NO: 11 with valine (V),
and a substitution of valine (V) at a position corresponding to 330 in the amino acid sequence of
SEQ ID NO: 11 with isoleucine (I), and has an amino acid sequence, wherein b) the amino acid
25 at a position corresponding to 88 is phenylalanine and c) the amino acid at a position
corresponding to 207 is lysine (K). Further, it is apparent that any polypeptide variant having
an amino acid sequence, in which a part of the amino acid sequence is deleted, modified,
substituted, or added at amino acid positions corresponding to 88, 207, 241, 246, and 330 of the
amino acid sequence of SEQ ID NO: 11, may also fall within the scope of the present application,
30 as long as it is a polypeptide having an amino acid sequence substantially having OPS exporting
activity as a sequence having such identity.
Specifically, the polypeptide having OPS exporting activity of the present application
may be a polypeptide having an amino acid sequence of SEQ ID NO: 1.
In the present application, the polypeptide having O-phosphoserine exporting activity
7
having the amino acid sequence of SEQ ID NO: 1 may be a polypeptide having O-phosphoserine
exporting activity including the amino acid sequence of SEQ ID NO: 1, a polypeptide having
O-phosphoserine exporting activity represented by the amino acid sequence of SEQ ID NO: 1, a
polypeptide having O-phosphoserine exporting activity consisting essentially of the amino acid
5 sequence of SEQ ID NO: 1, or a polypeptide having O-phosphoserine exporting activity
consisting of the amino acid sequence of SEQ ID NO: 1. Furthermore, the polypeptide having
O-phosphoserine exporting activity of the present application does not exclude a meaningless
sequence addition upstream or downstream of the amino acid sequence of SEQ ID NO: 1.
In the present application, the SEQ ID NO: 1 may mean an amino acid sequence having
10 OPS exporting activity. Specifically, the SEQ ID NO: 1 may be an amino acid sequence
constituting a variant of the YhhS MFS transporter, a protein that exhibits OPS exporting activity
encoded by the yhhS gene.
The amino acid sequence of the YhhS MFS transporter, a protein that exhibits OPS
exporting activity encoded by the yhhS gene, can be obtained from GenBank of NCBI, a known
15 database. The amino acid sequence of the YhhS MFS transporter may be, for example, SEQ ID
NO: 11. Additionally, the amino acid sequence of the YhhS MFS transporter may be an amino
acid sequence derived from Escherichia coli (E. coli), but is not limited thereto.
In another aspect of the present application, the present application provides a
20 polynucleotide encoding the polypeptide having O-phosphoserine exporting activity, including a)
a substitution of isoleucine (I) at a position corresponding to 241 in the amino acid sequence of
SEQ ID NO: 11 with threonine (T), a substitution of aspartic acid (D) at a position corresponding
to 246 in the amino acid sequence of SEQ ID NO: 11 with valine (V), and a substitution of
valine (V) at a position corresponding to 330 in the amino acid sequence of SEQ ID NO: 11 with
25 isoleucine (I), and having an amino acid sequence, wherein b) the amino acid at a position
corresponding to 88 is phenylalanine and c) the amino acid at a position corresponding to 207 is
lysine (K), or the polypeptide having O-phosphoserine exporting activity having the amino acid
sequence of SEQ ID NO: 1.
The SEQ ID NO: 11, SEQ ID NO: 1, O-phosphoserine, and polypeptide having
30 O-phosphoserine exporting activity are the same as described above.
As used herein, the “polynucleotide”, which is a polymer of nucleotides composed of
nucleotide monomers connected in a lengthy chain by a covalently bond, is a DNA or RNA
strand having at least a certain length.
The polynucleotide may include any polynucleotide encoding a polypeptide having OPS
8
exporting activity of the present application without limitation. In the present application, the
gene encoding the amino acid sequence of the OPS exporting protein may be the yhhS gene.
Additionally, the gene may be derived from Escherichia coli (E. coli), but is not limited thereto.
Specifically, the polynucleotide encoding the polypeptide having OPS exporting activity
5 of the present application may have or include a nucleotide sequence encoding the amino acid
sequence represented by SEQ ID NO: 1. Additionally, the polynucleotide encoding the
polypeptide having OPS exporting activity of the present application may consist of or consist
essentially of a nucleotide sequence encoding the amino acid sequence represented by SEQ ID
NO: 1. The polynucleotide of the present application may undergo various modifications in the
10 coding region within the scope that does not change the amino acid sequence of the polypeptide,
due to codon degeneracy or in consideration of the codons preferred in an organism in which the
polypeptide is to be expressed. The polynucleotide of the present application may include or
have, for example, a nucleotide sequence having homology or identity of at least 80%, 90%,
95%, or 99% or higher with the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.
15 In one embodiment, the polynucleotide of the present application may consist of or consist
essentially of a nucleotide sequence having homology or identity of at least 80%, 90%, 95%, or
99% or higher with the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.
Additionally, the polynucleotide of the present application may include a probe that may
be prepared from a known gene sequence, for example, any sequence which can hybridize with a
20 sequence complementary to all or part of the nucleotide sequence under stringent conditions to
encode the amino acid sequence of SEQ ID NO: 1 without limitation. The “stringent
conditions” refer to conditions under which specific hybridization between polynucleotides is
allowed. Such conditions are specifically described in the literature (see J. Sambrook et al.,
Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press,
25 Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular
Biology, John Wiley & Sons, Inc., New York). For example, the stringent conditions may
include conditions under which genes having a high homology or identity of 40% or higher,
specifically 90% or higher, more specifically 95% or higher, much more specifically 97% or
higher, and still much more specifically 99% or higher are hybridized with each other, and genes
30 having homology or identity lower than the above homologies or identities are not hybridized
with each other, or washing conditions of Southern hybridization, that is, washing once,
specifically twice or three times at a salt concentration and a temperature corresponding to 60°C,
1× SSC, 0.1% SDS, specifically 60°C, 0.1× SSC, 0.1% SDS, and more specifically 68°C, 0.1×
SSC, 0.1% SDS.
9
Hybridization requires that two nucleic acids contain complementary sequences,
although mismatches between bases are possible depending on the stringency of the
hybridization. The term “complementary” is used to describe the relationship between
nucleotide bases that can hybridize with each other. For example, with respect to DNA,
5 adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore,
the polynucleotide of the present application may include isolated nucleotide fragments
complementary to the entire sequence as well as nucleic acid sequences substantially similar
thereto.
As used herein, the term “homology” or “identity” refers to a degree of relevance
10 between two given amino acid sequences or nucleotide sequences, and may be expressed as a
percentage. The terms homology and identity may often be used interchangeably with each
other.
The sequence homology or identity of conserved polypeptide or polynucleotide
sequences may be determined using standard alignment algorithms and can be used with a
15 default gap penalty established by the program being used. Substantially, homologous or
identical sequences are generally expected to hybridize to all or at least about 50%, 50%, 60%,
70%, 80%, or 90% of the entire length of the sequences under moderate or highly stringent
conditions. Polynucleotides that contain degenerate codons instead of codons in hybridizing
polynucleotides are also considered.
20 Whether any two polynucleotide sequences have homology, similarity, or identity may
be, for example, determined by a known computer algorithm such as the “FASTA” program
using default parameters (Pearson et al., (1988) Proc. Natl. Acad. Sci. USA 85:2444).
Alternatively, it may be determined by the Needleman–Wunsch algorithm (Needleman and
Wunsch, 1970, J. Mol. Biol. 48:443–453), which is performed using the Needleman program of
25 the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice
et al., 2000, Trends Genet. 16:276–277) (version 5.0.0 or later) (GCG program package
(Devereux, J., et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA
(Atschul, S. F., et al., J MOLEC BIOL 215:403 (1990); Guide to Huge Computers, Martin J.
Bishop, ed., Academic Press, San Diego, 1994, and CARILLO et al. (1988) SIAM J Applied
30 Math 48:1073). For example, the homology, similarity, or identity may be determined using
BLAST or ClustalW of the National Center for Biotechnology Information (NCBI).
The homology, similarity, or identity of polypeptides or polynucleotides may be
determined by comparing sequence information using, for example, the GAP computer program,
such as Needleman et al. (1970), J Mol Biol. 48:443 as disclosed in Smith and Waterman, Adv.
10
Appl. Math (1981) 2:482. In summary, the GAP program defines the homology, similarity, or
identity as the value obtained by dividing the number of similarly aligned symbols (i.e.,
nucleotides or amino acids) by the total number of the symbols in the shorter of the two
sequences. Default parameters for the GAP program may include (1) a unary comparison
5 matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted
comparison matrix of Gribskov et al. (1986), Nucl. Acids Res. 14:6745, as disclosed in Schwartz
and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research
Foundation, pp. 353–358 (1979) (or EDNAFULL substitution matrix (EMBOSS version of
NCBI NUC4.4)); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each
10 symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3)
no penalty for end gaps.
Further, whether any two polynucleotide or polypeptide sequences have homology,
similarity, or identity with each other may be identified by comparing the sequences in a
Southern hybridization experiment under stringent conditions as defined, and appropriate
15 hybridization conditions defined are within the skill of the art, and may be determined by a
method well known to those skilled in the art (for example, J. Sambrook et al.).
Specifically, the polynucleotides having homology or identity may be detected using the
hybridization conditions including a hybridization step at a Tm value of 55°C under the
above-described conditions. Further, the Tm value may be 60°C, 63°C, or 65°C, but is not
20 limited thereto, and may be appropriately adjusted by those skilled in the art depending on the
purpose thereof.
The appropriate stringency for hybridizing polynucleotides depends on the length of the
polynucleotides and the degree of complementation, and these variables are well known in the
art (see Sambrook et al., supra, 9.50–9.51, 11.7–11.8).
25
In still another aspect of the present application, the present application provides a
vector containing the polynucleotide encoding the polypeptide having O-phosphoserine
exporting activity, including a) a substitution of isoleucine (I) at a position corresponding to 241
in the amino acid sequence of SEQ ID NO: 11 with threonine (T), a substitution of aspartic acid
30 (D) at a position corresponding to 246 in the amino acid sequence of SEQ ID NO: 11 with valine
(V), and a substitution of valine (V) at a position corresponding to 330 in the amino acid
sequence of SEQ ID NO: 11 with isoleucine (I), and having an amino acid sequence, wherein b)
the amino acid at a position corresponding to 88 is phenylalanine and c) the amino acid at a
position corresponding to 207 is lysine (K), or the polynucleotide encoding the polypeptide
11
having O-phosphoserine exporting activity having the amino acid sequence of SEQ ID NO: 1.
The SEQ ID NO: 11, SEQ ID NO: 1, O-phosphoserine, polypeptide having
O-phosphoserine exporting activity, and polynucleotide are the same as described above.
As used herein, the term “vector” refers to a DNA construct containing the nucleotide
5 sequence of a polynucleotide encoding the target polypeptide or protein operably linked to a
suitable regulatory sequence so as to be able to express the target polypeptide or protein in a
suitable host cell. The regulatory sequence may include a promoter capable of initiating
transcription, any operator sequence for regulating the transcription, a sequence encoding a
suitable mRNA ribosome binding site, and a sequence for regulating termination of transcription
10 and translation. Once transformed into a suitable host cell, the vector may replicate or function
independently from the host genome, or may integrate into the genome thereof.
The vector used in the present application is not particularly limited as long as it is able
to replicate in the host cell, and any vector known in the art may be used. Examples of the
vector conventionally used may include natural or recombinant plasmids, cosmids, viruses, and
15 bacteriophages. For example, as a phage vector or cosmid vector, pWE15, M13, MBL3, MBL4,
IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used; and as a plasmid vector,
those based on pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pSK, pSKH, and pET may be used.
Specifically, pCL, pSK, pSKH130, pDZ, pACYC177, pACYC184, pECCG117, pUC19,
pBR322, pMW118, and pCC1BAC vectors may be used.
20 The insertion of the polynucleotide into the chromosome may be performed by any
method known in the art, for example, by homologous recombination, but the method is not
limited thereto.
The vector may further include a selection marker to confirm the insertion into the
chromosome. The selection marker is for selecting the cells transformed with the vector, that is,
25 for confirming whether the target nucleic acid molecule has been inserted, and markers that
provide selectable phenotypes, such as drug resistance, auxotrophy, resistance to cell toxic
agents, or expression of surface-modified proteins, may be used. Only cells expressing the
selection marker are able to survive or to show different phenotypes under the environment
treated with the selective agent, and thus the transformed cells may be selected.
30 As used herein, the term “transformation” refers to the introduction of a vector including
a polynucleotide encoding a target polypeptide or protein into a host cell so that the polypeptide
or protein encoded by the polynucleotide can be expressed in a host cell. As long as the
transformed polynucleotide can be expressed in the host cell, it does not matter whether the
transformed polynucleotide is integrated into the chromosome of the host cell and located therein
12
or located extrachromosomally, and both cases can be included. Further, the polynucleotide
may include DNA and RNA encoding the target polypeptide or protein.
The polynucleotide may be introduced in any form, as long as it can be introduced into
the host cell and expressed therein. For example, the polynucleotide may be introduced into the
5 host cell in the form of an expression cassette, which is a gene construct including all elements
required for its autonomous expression. The expression cassette may commonly include a
promoter operably linked to the polynucleotide, a transcription terminator, a ribosome binding
site, or a translation terminator. The expression cassette may be in the form of a self-replicable
expression vector. Additionally, the polynucleotide may be introduced into the host cell as it is
10 and operably linked to sequences required for expression in the host cell, but is not limited
thereto.
Additionally, as used herein, the term “operably linked” means that the gene sequence is
functionally linked to a promoter sequence that initiates and mediates transcription of the
polynucleotide encoding the target polypeptide or protein of the present application.
15
In yet another aspect of the present application, the present application provides an
O-phosphoserine-producing microorganism, including any one or more of the polypeptide
having O-phosphoserine (OPS) exporting activity, including a) a substitution of isoleucine (I) at
a position corresponding to 241 in the amino acid sequence of SEQ ID NO: 11 with threonine
20 (T), a substitution of aspartic acid (D) at a position corresponding to 246 in the amino acid
sequence of SEQ ID NO: 11 with valine (V), and a substitution of valine (V) at a position
corresponding to 330 in the amino acid sequence of SEQ ID NO: 11 with isoleucine (I), and
having an amino acid sequence, wherein b) the amino acid at a position corresponding to 88 is
phenylalanine and c) the amino acid at a position corresponding to 207 is lysine (K), or the
25 polypeptide having O-phosphoserine exporting activity having the amino acid sequence of SEQ
ID NO: 1, the polynucleotide encoding the polypeptide of the present application, and the vector
containing the polynucleotide encoding the polypeptide of the present application.
The SEQ ID NO: 11, SEQ ID NO: 1, O-phosphoserine, polypeptide having
O-phosphoserine exporting activity, polynucleotide, and vector are the same as described above.
30 As used herein, the term “an OPS-producing microorganism” refers to a microorganism
that has a naturally weak OPS producing capability or a microorganism that has been given an
OPS producing capability by natural or artificial genetic modification of a parent strain that does
not have an OPS producing capability. Specifically, the microorganism may be a
microorganism expressing a polypeptide having the amino acid sequence of SEQ ID NO: 1, but
13
is not limited thereto. In the present application, the “OPS-producing microorganism” may be
used interchangeably with “a microorganism having OPS producing capability”,
“OPS-producing strain”, and “OPS-producing strain line”.
For the purpose of the present application, in the case of the OPS-producing
5 microorganism, the microorganism may include any one or more of the polypeptide having
O-phosphoserine exporting activity of the present application, the polynucleotide encoding the
polypeptide of the present application, and the vector containing the polynucleotide of the
present application, thereby enhancing the activity of the polypeptide expressed therefrom, and
thus, the production amount of OPS may be increased compared to that of a wild type or the
10 microorganism before modification. This is significant in that the production of OPS can be
increased by introducing the polypeptide having OPS exporting activity of the present
application and enhancing its activity, while wild-type microorganisms cannot produce OPS or
can only produce trace amounts even if they are able to produce OPS. That is, the
OPS-producing microorganism of the present application may be one in which the activity of the
15 polypeptide having OPS exporting activity of the present application is enhanced compared to its
endogenous activity, but is not limited thereto.
As used herein, the term “enhancement compared to its endogenous activity” refers to an
increased activity of a protein when compared to the activity of the protein possessed by a
microorganism in its natural state.
20 As used herein, the term “to be expressed/being expressed” refers to a state in which a
target polypeptide or protein is introduced into a microorganism or in which a target polypeptide
or protein is modified to be expressed in the microorganism. When the target polypeptide or
protein is a polypeptide or protein present in a microorganism, it may mean a state in which its
activity is enhanced compared to the endogenous activity or activity before modification.
25 As used herein, the term “enhancement of activity” of a polypeptide or protein means
that the activity of a polypeptide or protein is enhanced compared to its endogenous activity.
As used herein, the term “endogenous activity” refers to the activity of a particular polypeptide
or protein originally possessed by a parent strain before transformation or a non-modified
microorganism, when a trait of a microorganism is altered due to genetic modification caused by
30 a natural or artificial factor, and may be used interchangeably with “activity before modification”.
The “enhancement” or “increase” of the activity of a polypeptide or protein compared to its
endogenous activity means that the activity is enhanced compared to the activity of a particular
polypeptide or protein originally possessed by a parent strain before transformation or a
non-modified microorganism.
14
The “increase of activity” may be achieved by introducing a foreign polypeptide or
protein, or by enhancing the activity of an endogenous polypeptide or protein, but specifically, it
may be achieved by enhancing the activity of an endogenous polypeptide or protein. Whether
or not the activity of the polypeptide or protein is enhanced may be confirmed from an increase
5 in the activity level, the expression level of the target polypeptide or protein, or the amount of the
product exported from the target protein.
In the present application, the polypeptide or protein targeted for the enhancement of
activity, that is, the target polypeptide or protein, may be a variant of the YhhS MFS transporter,
and specifically, it may be a variant of the YhhS MFS transporter having OPS exporting activity
10 which is enhanced compared to that of the wild-type YhhS MFS transporter, but is not limited
thereto.
Additionally, in the present application, the product exported from the target polypeptide
or protein may be O-phosphoserine, but is not limited thereto.
15 The enhancement of the activity of the polypeptide or protein may be achieved by
various methods well known in the art, and may not be limited as long as the activity of the
target polypeptide or protein can be enhanced compared to that of the microorganism before
modification. The method may include genetic engineering or protein engineering, but is not
limited thereto.
20 The method of enhancing the activity of a polypeptide or protein using the genetic
engineering may be achieved, for example, by way of:
1) a method of increasing the intracellular copy number of a gene or polynucleotide
encoding the polypeptide or protein;
2) a method of replacing the expression regulatory sequence of a gene encoding the
25 polypeptide or protein on a chromosome with a sequence having a strong activity;
3) a method of modifying the nucleotide sequence of the initiation codon or 5′-UTR of
the polypeptide or protein;
4) a method of modifying a polynucleotide sequence on a chromosome such that the
activity of the polypeptide or protein is enhanced;
30 5) a method of introducing a foreign polynucleotide having the activity of the
polypeptide or protein or a codon-optimized modified polynucleotide of the polynucleotide; or
6) a combination thereof, but is not limited thereto.
The method of enhancing the activity of a polypeptide or protein using the protein
engineering may be achieved, for example, by analyzing the tertiary structure of the polypeptide
15
or protein and selecting and modifying the exposed site, or chemically modifying the same, but
is not limited thereto.
The 1) method of increasing the intracellular copy number of a gene encoding the
5 polypeptide or protein may be performed by way of a method known in the art, for example, by
introducing a vector, which is operably linked to the gene or polynucleotide encoding the
polypeptide or protein and is able to replicate and function regardless of a host cell, into the host
cell. Alternatively, the method may be performed by introducing a vector, which is able to
insert the gene or polynucleotide into the chromosome of a host cell, to which the gene is
10 operably linked, into the host cell, but is not limited thereto. The vector is the same as
described above.
The 2) method of replacing the expression regulatory sequence of a gene encoding the
polypeptide or protein on a chromosome with a sequence having a strong activity may be
15 performed by way of a method known in the art, for example, by inducing a modification on the
sequence through deletion, insertion, non-conservative or conservative substitution of the nucleic
acid sequence, or a combination thereof to further enhance the activity of the expression
regulatory sequence, or by replacing the polynucleotide sequence with a nucleic acid sequence
having a stronger activity. The expression regulatory sequence may include, but is not
20 particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome
binding site, and a sequence regulating the termination of transcription and translation.
Specifically, the method may include linking a strong heterologous promoter, instead of the
original promoter, but is not limited thereto.
Examples of the strong promoter may include CJ7 promoter (US 7662943 B2), CJ1
25 promoter (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda
phage PR promoter, PL promoter, and tet promoter, but is not limited thereto.
The 3) method of modifying the nucleotide sequence of the initiation codon or 5′-UTR
of the polypeptide or protein may be performed by way of a method known in the art, for
30 example, by substituting the endogenous initiation codon of the polypeptide or protein with
another initiation codon having a higher expression rate of the polypeptide or protein compared
to the endogenous initiation codon, but is not limited thereto.
The 4) method of modifying a polynucleotide sequence on a chromosome such that the
16
activity of the polypeptide or protein is enhanced may be performed by way of a method known
in the art, for example, by inducing a modification on the expression regulatory sequence
through deletion, insertion, non-conservative or conservative substitution of the nucleotide
sequence, or a combination thereof to further enhance the activity of the polynucleotide sequence,
5 or by replacing the polynucleotide sequence with a polynucleotide sequence modified to have a
stronger activity. The replacement may specifically be achieved by inserting the gene into the
chromosome by homologous recombination, but is not limited thereto.
The vector used herein may further include a selection marker to confirm the insertion
into the chromosome. The selection marker is the same as described above.
10
The 5) method of introducing a foreign polynucleotide having the activity of the
polypeptide or protein may be performed by way of a method known in the art, for example, by
introducing into a host cell a foreign polynucleotide encoding a polypeptide or protein that
exhibits the same or similar activity to the polypeptide or protein or a codon-optimized modified
15 polynucleotide thereof. The foreign polynucleotide may be used without limitation regardless
of its origin or sequence as long as it exhibits the same or similar activity to the polypeptide or
protein. Additionally, for the optimized transcription and translation of the foreign
polynucleotide in a host cell, its codon may be optimized and introduced into the host cell. The
introduction may be performed by one of ordinary skill in the art by selecting a suitable
20 transformation method known in the art, and the expression of the introduced polynucleotide in
the host cell enables production of the polypeptide or protein, thereby increasing its activity.
Lastly, the 6) combination of the methods above may be performed by applying any one
or more of methods of 1) to 5) in combination.
25 Such enhancement of the activity of the polypeptide or protein activity may be an
increase in the activity or concentration of the target polypeptide or protein based on the activity
or concentration of the polypeptide or protein expressed in a wild-type or microbial strain before
modification, or may be an increase in the amount of product produced from the target
polypeptide or protein, but is not limited thereto. As used herein, the term “strain before
30 modification” or “microorganism before modification” does not exclude strains containing
mutations that may occur naturally in microorganisms, and it may refer to a natural strain itself
or a strain before modification in which a trait is altered due to a genetic mutation caused by
natural or artificial factors. The “strain before modification” or “microorganism before
modification” may be used interchangeably with “non-mutated strain”, “non-modified strain”,
17
“non-mutated microorganism”, “non-modified microorganism”, or “platform microorganism”.
In the present application, the platform microorganism may be CA07-0012, a known
microorganism producing OPS, CA07-0022/pCL_Prmf-serA*(G336V)-serC (KCCM11103P
(US 8557549 B2), a strain in which the activities of the endogenous SerA
5 (D-3-phosphoglycerate dehydrogenase) and SerC (3-phosphoserine aminotransferase) are
enhanced, and CA07-0012 (KCCM11121P, US 8557549 B2), or a strain in which the activity of
endogenous phosphoserine phosphatase (SerB) is weakened, but is not limited thereto.
The microorganism of the present application may be a recombinant microorganism
10 produced by transforming with a vector containing a polynucleotide encoding the polypeptide,
but is not limited thereto.
The microorganism of the present application is not limited by its type as long as it can
produce OPS, and may be any prokaryotic or eukaryotic microorganism, specifically a
prokaryotic microorganism. The prokaryotic microorganism may include microbial strains
15 belonging to the genus Escherichia, the genus Erwinia, the genus Serratia, the genus
Providencia, the genus Corynebacterium, and the genus Brevibacterium, specifically a
microorganism belonging to the genus Escherichia, and more specifically Escherichia coli, but
is not limited thereto. In particular, in the case of the microorganism belonging to the genus
Escherichia, OPS and L-serine can be produced through SerA, SerC, and SerB, which are
20 enzymes of the biosynthetic pathway of L-serine (Ahmed Zahoor, Computational and structural
biotechnology journal, Vol. 3, 2012 October; Wendisch V. F. et al., Curr Opin Microbiol. 2006
Jun;9(3):268–74; Peters-Wendisch P. et al., Appl Environ Microbiol. 2005 Nov;71(11):7139–
44.).
25 The OPS-producing microorganism of the present application may be one in which the
activity of phosphoserine phosphatase (SerB) may be further weakened compared to its
endogenous activity.
The SerB of the present application has an activity of converting OPS to L-serine, and
thus the microorganism modified to weaken the SerB activity has the property of accumulating
30 OPS therein, and is thus useful for the production of OPS. The SerB of the present application
may be a protein having or including an amino acid sequence represented by SEQ ID NO: 3, or
may be a protein consisting of or consisting essentially of an amino acid sequence represented by
SEQ ID NO: 3, but is not limited thereto. Additionally, the SerB may have or include an amino
acid sequence having a sequence homology or identity of 80%, 90%, 95%, or 99% or higher to
18
the amino acid sequence represented by SEQ ID NO: 3, as long as it shows the SerB activity.
Moreover, the SerB of the present application may consist of or consist essentially of an amino
acid sequence having homology or identity of 80%, 90%, 95%, or 99% or higher to the amino
acid sequence represented by SEQ ID NO: 3, but is not limited thereto. In addition, the
5 polynucleotide encoding the SerB may have or include a nucleotide sequence encoding the
amino acid sequence represented by SEQ ID NO: 3. Further, the polynucleotide encoding the
SerB may consist of or consist essentially of a nucleotide sequence encoding the amino acid
sequence represented by SEQ ID NO: 3. The polynucleotide encoding SerB of the present
application may undergo various modifications in the coding region within the scope that does
10 not change the amino acid sequence of the SerB protein, due to codon degeneracy or in
consideration of the codons preferred in an organism in which the SerB protein is to be
expressed. The polynucleotide encoding SerB of the present application may have or include a
nucleotide sequence having homology or identity of 80%, 90%, 95%, or 99% or higher, and less
than 100% to the nucleotide sequence of SEQ ID NO: 4. Additionally, the polynucleotide
15 encoding SerB of the present application may consist of or consist essentially of a nucleotide
sequence having homology or identity of 80%, 90%, 95%, or 99% or higher, and less than 100%
to the nucleotide sequence of SEQ ID NO: 4, but is not limited thereto.
As used herein, the term “weakening of activity compared to its endogenous activity”
means that a natural wild-type strain, a parent strain, or the target protein have no expression of
20 the enzyme or protein, or have no activity or decreased activity even when expressed, as
compared to a non-modified strain. In particular, the decrease is a comprehensive concept
including the case where the protein activity is decreased compared to the activity of the protein
originally possessed by a microorganism due to a mutation of the gene encoding the protein,
modification of the expression regulatory sequence, or deletion in a part or all of genes, etc.; the
25 case where the overall level of intracellular protein activity is decreased compared to that of a
natural strain or a strain before modification due to the inhibition of expression of the gene
encoding the protein or the inhibition of translation; and a combination thereof.
The weakening of the protein activity may be achieved by way of various methods well
known in the art. Examples of the methods may include: a method for modifying the gene
30 sequence encoding the protein such that the protein activity is removed or weakened; a method
for modifying the expression regulatory sequence such that the expression of the gene is
decreased; a method for deleting a part or all of the gene encoding the protein; a method of
introducing an antisense oligonucleotide (e.g., antisense RNA), which inhibits the translation
from the mRNA into a protein via a complementary binding to the transcript of the gene on the
19
chromosome; a method of making the attachment of a ribosome impossible by forming a
secondary structure by artificially adding a complementary sequence to the Shine–Dalgarno (SD)
sequence on the front end of the SD sequence of the gene encoding the protein; and a reverse
transcription engineering (RTE) method, which adds a promoter so as to be reversely transcribed
5 on the 3′ terminus of the open reading frame (ORF) of the polynucleotide sequence of the gene
encoding the protein; and a combination thereof, but are not particularly limited thereto.
Specifically, the method of modifying the gene sequence on the chromosome may be
performed by inducing a modification in the sequence via deletion, insertion, non-conservative
substitution, conservative substitution, or a combination thereof so as to further weaken the
10 activity of the protein; or by replacing the sequence with a gene sequence modified to have a
weaker activity or a gene sequence modified to have no activity at all.
The method of modifying the expression regulatory sequence may be performed by
inducing a modification in the expression regulatory sequence via deletion, insertion,
conservative substitution, non-conservative substitution, or a combination thereof so as to further
15 weaken the activity of the expression regulatory sequence; or by replacing the sequence with a
nucleic acid sequence having a weaker activity. The expression regulatory sequence may
include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a
sequence for regulating transcription and translation.
The method of deleting a part or the entirety of a gene encoding the protein may be
20 performed by replacing the polynucleotide encoding the endogenous target protein within the
chromosome with a polynucleotide or a marker gene having a partially deleted nucleic acid
sequence using a vector for chromosomal insertion into bacteria. For example, a method of
deleting a gene by way of homologous recombination may be used. Additionally, as used
herein, the term “part”, although it may vary depending on the kinds of polynucleotide, may
25 specifically refer to 1 to 300 nucleotides, more specifically 1 to 100 nucleotides, and even more
specifically 1 to 50 nucleotides, but is not particularly limited thereto.
In addition, the method of modifying the expression regulatory sequence may be
performed by inducing a modification in the expression regulatory sequence via deletion,
insertion, conservative substitution, non-conservative substitution, or a combination thereof so as
30 to further weaken the activity of the expression regulatory sequence; or by replacing the
sequence with a nucleic acid sequence having a weaker activity. The expression regulatory
sequence may include a promoter, an operator sequence, a sequence encoding a ribosome
binding site, and a sequence for regulating transcription and translation.
Further, the method of modifying the gene sequence on the chromosome may be
20
performed by inducing a modification in the sequence via deletion, insertion, conservative
substitution, non-conservative substitution, or a combination thereof so as to further weaken the
activity of the protein; or by replacing the sequence with a gene sequence modified to have a
weaker activity or a gene sequence modified to have no activity at all.
5 Additionally, the OPS-producing microorganism of the present application may be one
in which the activity of phosphoglycerate dehydrogenase (SerA) or phosphoserine
aminotransferase (SerC) is further enhanced compared to its endogenous activity.
The SerA is a protein capable of converting 3-phosphoglycerate into
3-phospho-hydroxypyruvate. The SerC is a protein capable of converting
10 3-phospho-hydroxypyruvate into OPS. Accordingly, any microorganism with enhanced SerA
and/or SerC activities may be effectively used as an OPS-producing microorganism.
The SerA may be a protein having or including an amino acid sequence represented by
SEQ ID NO: 5 or 6, or a protein consisting of or consisting essentially of an amino acid sequence
represented by SEQ ID NO: 5 or 6, although it is not limited thereto. The amino acid sequence
15 represented by SEQ ID NO: 5 is a sequence of the wild-type SerA, and the amino acid sequence
represented by SEQ ID NO: 6 is a sequence of a SerA variant where the feedback inhibition on
serine is released. Additionally, the SerA of the present application may have or include an
amino acid sequence having homology or identity of at least 80%, 90%, 95%, or 99% or higher,
and less than 100% with the amino acid sequence represented by SEQ ID NO: 5 or 6, as long as
20 it shows the activity of the wild-type SerA or the activity of the SerA variant in which the
feedback inhibition on serine is released, but is not limited thereto. Moreover, the SerA of the
present application may consist of or consist essentially of an amino acid sequence having
homology or identity of at least 80%, 90%, 95%, or 99% or higher, and less than 100% with the
amino acid sequence represented by SEQ ID NO: 5 or 6, as long as it shows the activity of the
25 wild-type SerA or the activity of the SerA variant in which the feedback inhibition on serine is
released. The SerA variants in which the feedback inhibition on serine is released refer to those
proteins in which a modification is introduced on the SerA-encoding gene by inserting a
nucleotide of the gene encoding SerA or substituting the gene encoding the wild-type SerA, etc.,
thereby maintaining the activity from the feedback inhibition by serine or glycine, or having
30 enhanced activities thereof, and those variants where the feedback inhibition on serine is released
are already well known (Grant G. A. et al., J. Biol. Chem., 39:5357–5361, 1999; Grant G. A. et
al., Biochem., 39:7316–7319, 2000; Grant G. A. et al., J. Biol. Chem., 276:17844–17850, 2001;
Peters-Wendisch P. et al., Appl. Microbiol. Biotechnol., 60:37–441, 2002; US 6258573 B1).
21
Additionally, the polynucleotide sequence encoding the wild-type SerA or the SerA
variant where the feedback inhibition on serine is released may have or include a nucleotide
sequence encoding any one amino acid sequence represented by SEQ ID NO: 5 or 6. Further,
the polynucleotide sequence encoding the wild-type SerA or the SerA variant where the
5 feedback inhibition on serine is released may consist of or consist essentially of a nucleotide
sequence encoding any one amino acid sequence represented by SEQ ID NO: 5 or SEQ ID
NO: 6, but is not limited thereto. The polynucleotide sequence encoding the wild-type SerA or
the SerA variant where the feedback inhibition on serine is released may undergo various
modifications in the coding region within the scope that does not change the amino acid
10 sequence of the polypeptide encoding the wild-type SerA or the SerA variant where the feedback
inhibition on serine is released, due to codon degeneracy or in consideration of the codons
preferred in an organism in which the polypeptide is to be expressed. For example, the
nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 5 may be a
nucleotide sequence having homology or identity of at least 80%, 90%, 95%, or 99% or higher
15 with the nucleotide sequence of SEQ ID NO: 7. Additionally, the nucleotide sequence
encoding the amino acid sequence represented by SEQ ID NO: 6 may be a nucleotide sequence
having homology or identity of at least 80%, 90%, 95%, or 99% or higher with the nucleotide
sequence of SEQ ID NO: 8, but is not limited thereto.
The SerC may be, for example, a protein having or including an amino acid sequence
20 represented by SEQ ID NO: 9, or a protein consisting of or consisting essentially of an amino
acid sequence represented by SEQ ID NO: 9, but is not limited thereto. Additionally, the SerC
may have or include an amino acid sequence having homology or identity of at least 80%, 90%,
95%, or 99% or higher, and less than 100% with the amino acid sequence represented by SEQ
ID NO: 9, as long as it shows the activity of SerC. Further, the SerC may consist or consist
25 essentially of an amino acid sequence having homology or identity of at least 80%, 90%, 95%, or
99% or higher, and less than 100% with the amino acid sequence represented by SEQ ID NO: 9,
as long as it shows the activity of SerC.
In addition, the polynucleotide encoding the SerC may have a nucleotide sequence
encoding the amino acid sequence represented by SEQ ID NO: 9. The polynucleotide may
30 undergo various modifications in the coding region within the scope that does not change the
amino acid sequence of the polypeptide, due to codon degeneracy or in consideration of the
codons preferred in an organism in which the polypeptide is to be expressed. The
polynucleotide encoding the SerC may have or include, for example, a nucleotide sequence
having homology or identity of 80%, 90%, 95%, or 99% or higher with the nucleotide sequence
22
of SEQ ID NO: 10, but is not limited thereto. Moreover, the polynucleotide encoding the SerC
may consist or consist essentially of a nucleotide sequence having homology or identity of 80%,
90%, 95%, or 99% or higher with the nucleotide sequence of SEQ ID NO: 10, but is not limited
thereto.
5 As used herein, the term “enhancement compared to its endogenous activity” and the
enhancement method are the same as described above.
Additionally, the microorganism may be a microorganism in which its capability to
introduce OPS into a cell or decompose OPS is further weakened.
10 Regarding the contents of the OPS-producing microorganism, the disclosures in
US 8557549 B2 may be used as references of the present application, in addition to those
described above.