DESCRIPTION
Invention Title
MICROORGANISM PRODUCING O-ACETYL-HOMOSERINE AND METHOD
FOR PRODUCING O-ACETYLHOMOSERINE USING THE SAME
Technical Field
The present disclosure relates to a microorganism producing O-acetylhomoserine in high
yield and a method for producing O-acetylhomoserine using the microorganism.
Background Art
O-acetylhomoserine acts as a precursor of methionine, which is a kind of essential amino
acid in the body. Methionine is used not only as an additive for feeds and foods but also as a
raw material for infusion solutions and pharmaceutical drugs.
Methionine is produced by chemical and biological syntheses. Recently, a two-step
process for producing L-methionine from an L-methionine precursor, which was produced via
fermentation, by enzymatic conversion was published (International Publication No. WO
2008/013432).
The two-step process employs O-succinylhomoserine and O-acetylhomoserine as
precursors, and it is very important to produce O-acetylhomoserine in high yield for the purpose
of economical mass production.
Disclosure
Technical Problem
Under the circumstances, the present inventors have made efforts to improve the
production of O-acetylhomoserine, and as a result, they have discovered a protein capable of
exporting O-acetylhomoserine, thereby completing the present disclosure.
Technical Solution
An object of the present disclosure is to provide a microorganism having enhanced
O-acetylhomoserine productivity.
WO/2015/186990 PCT/KR2015/005659
3
Another object of the present disclosure is to provide a method for efficient production
of O-acetylhomoserine using the microorganism having enhanced O-acetylhomoserine
productivity.
Advantageous Effects of the Invention
The microorganism of the present disclosure with an enhanced activity of YjeH, i.e., an
inner membrane protein, has an improved ability of exporting O-acetylhomoserine and thus the
efficiency of O-acetylhomoserine production can be increased. Therefore, the microorganism
of the present disclosure can be widely used for the production of O-acetylhomoserine.
Description of Drawings
Fig. 1 shows a cleavage map of the yjeH vector (pBAC-yjeH vector) according to the
present disclosure.
Best Mode
An aspect of the present disclosure provides a microorganism having
O-acetylhomoserine productivity, in which the activity of YjeH, i.e., an inner membrane protein,
is enhanced compared to a non-modified microorganism.
As used herein, the term “O-acetylhomoserine” refers to an acetyl derivative of
L-homoserine, which is a specific intermediate material in the biosynthetic pathway of
methionine in microorganisms. O-Acetylhomoserine is known to be produced by a reaction
between homoserine and acetyl-CoA catalyzed by homoserine acetyltransferase. It has a
chemical formula of C6H11NO4.
As used herein, the term “a microorganism having O-acetylhomoserine productivity”
refers to a microorganism which, when cultured in a culture medium, can produce
O-acetylhomoserine within a bioorganism and secrete it into the culture medium. The
O-acetylhomoserine productivity may be provided or enhanced by improvement of species.
Specifically, the microorganism having O-acetylhomoserine productivity may be a
microorganism of the genus Escherichia having O-acetylhomoserine productivity, and more
specifically, E. coli. For example, the microorganism may be E. coli having lysine, threonine,
WO/2015/186990 PCT/KR2015/005659
4
isoleucine, or methionine productivity, but is not limited thereto. As used herein, the term
“YjeH” is known as a protein which, being a member of the Amino
Acid-Polyamine-Organocation (APC) family for amino acid transporters, is present in an inner
membrane. YjeH is expected to act as an amino acid transporter but its exact function is not
known yet. As such, the present inventors have confirmed first that YjeH specifically exports
O-acetylhomoserine.
Specifically, YjeH may be derived from a microorganism of the genus Escherichia, and
more specifically, from E. coli. In particular, YjeH may be a protein having an amino acid
sequence of SEQ ID NO: 1, or any amino acid sequence having a homology of 70% or higher,
specifically 80% or higher, or more specifically 90% or higher, to the amino acid sequence of
SEQ ID NO: 1. Additionally, it is obvious that any amino acid sequence which has the same
amino acid sequence as that of SEQ ID NO: 1, or any amino acid sequence having the activity of
exporting O-acetylhomoserine, can belong to the scope of the present disclosure, although the
sequence may have a partial deletion, modification, substitution, or addition therein.
Additionally, based on genetic codon degeneracy, nucleotide sequences encoding the same
amino acid and variants thereof belong to the scope of the present disclosure, for example, a
nucleotide sequence represented by SEQ ID NO: 2, but are not limited thereto.
As used herein, the term “homology” refers to a degree of identity between two different
sequences of nucleotides or amino acid residues in a particular comparison region of the
nucleotide or amino acid sequences of a gene encoding a protein, after aligning both sequences
to a maximal match. When the homology is sufficiently high, the expression products of the
corresponding gene may have the same or similar activity. The homology may be determined
using a sequence comparison program known in the art, e.g., BLAST (NCBI), CLC Main
Workbench (CLC bio), MegAlignTM (DNASTAR Inc.), etc.
As used herein, the term “a non-modified microorganism” refers to a microorganism
which is not introduced with a modification in the activity of the corresponding protein, and the
microorganism refers to a base strain to be introduced with the modification in the activity of the
corresponding protein, which may be a native or modified microorganism.
As used herein, the term “enhancement” of a protein activity refers to improving the
WO/2015/186990 PCT/KR2015/005659
5
activity state of proteins possessed by a microorganism. The enhancement of a protein activity
is not limited as long as the activity of each protein can be enhanced compared to that of a
non-modified microorganism, as is the case with the enhancement of the activity of a target
protein. For example, the enhancement may be performed by methods selected from the group
consisting of i) increasing the copy number of a polynucleotide encoding each protein, ii)
modifying the expression control sequence for increasing the expression of the polynucleotide,
iii) modifying the polynucleotide sequence on the chromosome for enhancing the activity of each
protein; and iv) a combination thereof. Specifically, the enhancement of a protein activity may
be performed by a method selected from the group consisting of a method of introducing a
nucleotide sequence encoding each protein into the chromosome, a method of introducing the
nucleotide sequence into a microorganism after introducing it into a vector system, a method of
introducing a promoter exhibiting an improved activity into the upstream of the nucleotide
sequence encoding each protein or introducing each protein with a modification on the promoter,
a method of modifying the nucleotide sequence of the 5’-UTR region, and a method of
introducing a variant of the nucleotide sequence encoding each protein, but the method is not
limited thereto.
In a specific embodiment of the present disclosure, the YjeH activity may be enhanced
compared to that of a non-modified microorganism, by increasing the copy number or enhancing
the activity of a given promoter. Specifically, the YjeH activity may be enhanced by
introducing a promoter exhibiting an improved activity to YjeH, which is an inner membrane.
In a specific embodiment of the present disclosure, the promoter with an improved activity may
include without limitation any promoter having an improved activity compared to that of the
self-promoter of yjeH. Examples of such promoter may include without limitation any
promoter of a gene having a higher activity of gene expression compared to that of the
self-promoter of yjeH, or a modified promoter with an improved activity due to modification of
the gene in the self-promoter of yjeH, etc. Specifically, the promoter of the present disclosure
exhibiting an improved activity may be selected from the group consisting of icd promoter, pro
promoter, and cysk promoter. Specifically, the icd promoter may consist of the nucleotide
sequence of SEQ ID NO: 51; the pro promoter may consist of the nucleotide sequence of SEQ
ID NO: 52; and the cysk promoter may consist of the nucleotide sequence of SEQ ID NO: 53,
but each of the promoters may consist of a nucleotide sequence having a homology of 70% or
WO/2015/186990 PCT/KR2015/005659
6
higher, specifically 80% or higher, and more specifically 90% or higher, to each of the above
nucleotide sequences.
In a specific aspect of the present disclosure, the microorganism of the genus
Escherichia having O-acetylhomoserine productivity may be one in which the activity of
cystathionine synthase is further weakened or inactivated. Specifically, the microorganism may
be one in which the activity of cystathionine synthase is weakened or inactivated compared to
that of a non-modified microorganism, and specifically the gene, metB which encodes
cystathionine synthase, is deleted, but is not limited thereto. The amino acid sequence of metB
may be obtained from a known database, and any amino acid sequence having the activity of
cystathionine synthase may be included without limitation, for example, it may refer to a protein
having the amino acid sequence of SEQ ID NO: 3. The protein having the amino acid sequence
of SEQ ID NO: 3 may be a protein encoded by the nucleotide sequence of SEQ ID NO: 4, but is
not limited thereto. Additionally, in another aspect of the present disclosure, the microorganism
of the genus Escherichia may be one in which the activity of homoserine kinase is further
weakened or inactivated. Specifically, the microorganism may be one in which the activity of
homoserine kinase is weakened or inactivated compared to the endogenous activity of a
non-modified microorganism, and specifically one in which the gene, thrB which encodes
homoserine kinase, is deleted, but is not limited thereto. The amino acid sequence of thrB may
be obtained from a known database, and any amino acid sequence having the activity of
homoserine kinase may be included without limitation, for example, it may refer to a protein
having the amino acid sequence of SEQ ID NO: 5. The protein having the amino acid sequence
of SEQ ID NO: 5 may be a protein encoded by the nucleotide sequence of SEQ ID NO: 6, but is
not limited thereto.
In the present disclosure, “weakening” of the activity of the protein may be performed
by a method selected from the group consisting of i) deleting a part or entirety of the gene
encoding each protein, ii) modifying the expression control sequence for decreasing the
expression of the gene, iii) modifying the gene sequence on the chromosome for weakening the
activity of the protein; and iv) a combination thereof, but is not limited thereto.
Specifically, the term “weakening of a protein activity” refers to a decrease in the
activity of an enzyme compared to the endogenous activity of the enzyme possessed by a
microorganism in its natural or base strain state. The weakening is a concept including a case
WO/2015/186990 PCT/KR2015/005659
7
when there is a decrease in the activity of an enzyme in a microorganism compared to that
originally possessed by the enzyme itself due to a modification of the enzyme-encoding gene,
etc., a case when the level of overall enzyme activity in a cell is lower than that of the wild-type
strain due to inhibition of expression or inhibition of translation of the enzyme-encoding gene, or
a combined case thereof, but is not limited thereto.
The “inactivation” refers to a case when the enzyme-encoding gene in a microorganism
is not expressed at all and a case when the gene is expressed but exhibits no activity, compared
to that of the wild-type strain.
The inactivation of an enzyme may be achieved by applying various methods known in
the art. Examples of the methods may include a method of substituting the enzyme-encoding
gene on the chromosome with a gene mutated to reduce the activity of the enzyme, including the
case when the enzyme activity is eliminated; a method of introducing a modification in the
expression control sequence of the enzyme-encoding gene on the chromosome; a method of
substituting the expression control sequence of the enzyme-encoding gene with a sequence
having weak or no activity; a method of deleting the entirety or a part of the enzyme-encoding
gene on the chromosome; a method of introducing an antisense oligonucleotide (e.g., antisense
RNA) which binds complementary to a transcript of the gene on the chromosome, thereby
inhibiting the translation from the mRNA into the enzyme; a method of artificially incorporating
a complementary sequence to the SD sequence into the upstream of the SD sequence of the
enzyme-encoding gene, forming a secondary structure, thereby making the attachment of
ribosome thereto impossible; a method of incorporating a promoter to the 3’ terminus of the open
reading frame (ORF) of the corresponding sequence to induce a reverse transcription (reverse
transcription engineering (RTE)), etc., and also a combination thereof, but are not limited
thereto.
Specifically, the method of deleting the entirety or a part of a gene encoding an enzyme
may be performed by substituting the polynucleotide encoding the endogenous target protein
within the chromosome with a polynucleotide or marker gene having a partial deletion in the
nucleic acid sequence using a vector for chromosomal insertion within a strain. In an
exemplary embodiment of the method of deleting a part or the entirety of a gene, a method for
deleting a gene by homologous recombination may be used, but is not limited thereto.
As used herein, the term “a part” may vary depending on the kinds of polynucleotides,
and it may specifically refer to 1 to 300, more specifically 1 to 100, and even more specifically 1
WO/2015/186990 PCT/KR2015/005659
8
to 50, but is not particularly limited thereto.
As used herein, the term “homologous recombination” refers to genetic recombination
that occurs via crossover at a locus of a gene chain having a mutual homology.
Specifically, the expression regulatory sequence may be modified by inducing a
modification of the expression control sequence by deletion, insertion, non-conservative or
conservative substitution, or a combination thereof to further weaken the activity of the
expression control sequence; or by substituting with a promoter having much weaker activity.
The expression control sequence may include a promoter, an operator sequence, a sequence
encoding a ribosome-binding region, and sequences controlling the termination of transcription
and translation, but is not limited thereto.
Furthermore, the gene sequence on the chromosome may be modified by inducing a
modification in the sequence by deletion, insertion, non-conservative or conservative substitution,
or a combination thereof in the gene sequence for further weakening the enzyme activity; or by
substituting with a gene sequence which was improved to have a weaker activity or a gene
sequence which was improved to have no activity, but is not limited thereto.
In a specific embodiment of the present disclosure, the activity of each protein was
weakened by deleting the metB gene encoding cystathionine synthase and/or the thrB gene by
homologous recombination.
Additionally, in a specific embodiment of the present disclosure, the microorganism of
the genus Escherichia may be one in which, additionally, the activity of homoserine
acetyltransferase is enhanced compared to that of a non-modified microorganism. Specifically,
the microorganism may be one in which the activity of homoserine acetyltransferase is increased
compared to that of a non-modified microorganism, and in particular, the microorganism may be
one in which a modified metA gene encoding homoserine acetyltransferase having an enhanced
activity is introduced. The modified metA gene may be a gene encoding homoserine
acetyltransferase, in which the 111th amino acid of homoserine acetyltransferase is substituted
with glutamic acid and the 112th amino acid is substituted with histidine, and in particular, a gene
which consists of the nucleotide sequence of SEQ ID NO: 8, but is not limited thereto. The
modified metA gene may include without limitation any amino acid in which the activity of
homoserine acetyltransferase is enhanced compared to that of wild-type, e.g., a protein having
the amino acid sequence of SEQ ID NO: 7. An exemplary embodiment with respect to the
WO/2015/186990 PCT/KR2015/005659
9
preparation, utilization of the modified metA gene, a strain with an enhanced activity of
homoserine acetyltransferase, etc., is disclosed in Korean Patent No. 10-1335841, and the
entirety of the Korean Patent may be included as a reference to the present disclosure.
Additionally, in a specific aspect of the present disclosure, the microorganism of the
genus Escherichia may be one in which the activity of aspartate kinase (EC 2.7.2.4) is enhanced
compared to that of a non-modified microorganism. Specifically, the microorganism may be
one in which the activity of aspartate kinase is increased compared to that of the endogenous
activity of the microorganism, but is not limited thereto.
In a specific embodiment of the present disclosure, the biosynthetic pathway was further
enhanced for the purpose of maximizing the O-acetylhomoserine productivity. Specifically,
aspartate kinase and homoserine O-acetyltransferase were introduced using a plasmid, and the
change in O-acetylhomoserine productivity was measured after further enhancing YjeH in the
strain with an enhanced biosynthetic pathway for homoserine. As a result of the simultaneous
enhancement of both biosynthetic pathway and yjeH, it was confirmed that the
O-acetylhomoserine productivity was further improved. In particular, it was confirmed that
when the YjeH activity was enhanced using the cysk promoter, the YjeH productivity was
increased by about 93% (2.8 g/L -> 5.4 g/L) (Table 5).
Additionally, in a specific embodiment of the present disclosure, it was examined
whether the enhancement of YjeH activity in an existing strain having high-yield
O-acetylhomoserine productivity can further increase its O-acetylhomoserine productivity.
More specifically, the amount of O-acetylhomoserine production was examined using a strain
KCCM11146P (International Publication No. WO2012/087039) producing O-acetylhomoserine
prepared from the strain having threonine productivity by NTG mutation, which a wild-type
W3110-derived strainafter substituting the promoter of the endogenous yjeH gene with a
promoter with high expression-inducing activity. As a result, it was confirmed that the strain,
which already has high-yield O-acetylhomoserine productivity, can further increase the activity
of producing O-acetylhomoserine by the enhancement of YjeH activity by about 14% (14.2 g/L
-> 18.2 g/L), and in particular, by the enhancement of YjeH activity using the cysk promoter
(Table 7).
Information on the genes used in the present disclosure, the sequences of the proteins
WO/2015/186990 PCT/KR2015/005659
10
encoded by the genes, and promoter sequences can be obtained from a known database, e.g.,
NCBI GenBank, but is not limited thereto.
The genes encoding each of the proteins and promoters of the present disclosure include
not only the nucleotide sequences represented by each of the SEQ ID NOS. but also include
without limitation any gene sequence having a homology of 80% or higher, specifically 90% or
higher, more specifically 95% or higher, even more specifically 98% or higher, and most
specifically 99% or higher, to these sequences, as long as the gene sequence encodes an enzyme
which exhibits an effect substantially the same as or corresponding to that of each of the
enzymes described above. Additionally, it is obvious that any nucleotide sequence having the
above sequence homology must also belong to the scope of the present disclosure, although the
amino acid may have deletion, modification, substitution, or addition in part of the sequence.
Additionally, it is obvious that any amino acid constituting each of the above proteins of
the present disclosure must also belong to the scope of the present disclosure although the amino
acid may have deletion, modification, substitution, or addition in part of the sequence, as long as
the amino acid has the same sequence represented by each of the SEQ ID NOS. or has a
homology thereto while having an effect substantially the same as or corresponding to that of
each of the proteins.
The effect of uniformly increasing O-acetylhomoserine productivity by the enhancement
of YjeH activity was confirmed based on microorganisms having various genetic backgrounds
and O-acetylhomoserine productivities. This may occur by promoting the production of a
specific intermediate material by YjeH in the biosynthetic pathway. However, considering the
characteristics of YjeH, which is an inner membrane and a kind of amino acid transporter family,
the increase of O-acetylhomoserine productivity is thought to be achieved by increasing the
ability of exporting O-acetylhomoserine, which is a final product and a kind of amino acid,
thereby promoting intracellular reactions.
In still another aspect of the present disclosure, the present disclosure provides a method
for producing O-succinylhomoserine including culturing a microorganism of the genus
Escherichia producing O-acetylhomoserine of the present disclosure and recovering the cultured
medium.
WO/2015/186990 PCT/KR2015/005659
11
The medium used for culturing the microorganism of the present disclosure and other
culture conditions are not particularly limited but any medium used for the conventional
culturing of the microorganism of the genus Escherichia may be used. Specifically, the
microorganism of the present disclosure may be cultured in a conventional medium containing
appropriate carbon sources, nitrogen sources, phosphorous sources, inorganic compounds, amino
acids and/or vitamins, etc., in an aerobic condition while adjusting temperature, pH, etc.
In the present disclosure, the carbon sources may include carbohydrates (e.g., glucose,
fructose, sucrose, maltose, mannitol, sorbitol, etc.); alcohols (e.g., sugar alcohol, glycerol,
pyruvic acid, lactic acid, citric acid, etc.); amino acids (e.g., glutamic acid, methionine, lysine,
etc.); etc., but are not limited thereto. Additionally, the carbon sources may include natural
organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava,
sugar cane molasses, corn steep liquor, etc. Specifically, carbohydrates such as glucose and
sterilized pretreated molasses (i.e., molasses converted to reducing sugar) may be used, and in
addition, various other carbon sources in an appropriate amount may be used without limitation.
These carbon sources may be used alone or in combination of at least two kinds.
Examples of the nitrogen sources may include inorganic nitrogen sources (e.g., ammonia,
ammonium sulfate, ammonium chloride, ammonium carbonate, ammonium phosphate,
ammonium nitrate, etc.); amino acids (glutamic acid, methionine, glutamine, etc.); and organic
nitrogen sources (e.g., peptone, N-Z amine, meat extract, yeast extract, malt extract, corn steep
liquor, casein hydrolysate, fish or decomposition product thereof, defatted soybean cake or
decomposition product thereof, etc.). These nitrogen sources may be used alone or in
combination of at least two kinds, but are not limited thereto.
Examples of the phosphorus sources may include monopotassium phosphate,
dipotassium phosphate, and corresponding sodium-containing salts. Examples of inorganic
compounds to be used may include sodium chloride, calcium chloride, iron chloride, magnesium
sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. Additionally, amino acids,
vitamins, and/or appropriate precursors may be included. These media or precursors may be
added in a batch culture process or a continuous culture process to a culture, but are not limited
thereto.
In the present disclosure, the pH of a culture may be adjusted during the cultivation by
adding a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric
acid, and sulfuric acid to the culture in an appropriate manner. During the cultivation, an
WO/2015/186990 PCT/KR2015/005659
12
antifoaming agent, such as fatty acid polyglycol ester, may also be added to prevent foam
generation. Additionally, oxygen or an oxygen-containing gas may be injected into the culture
in order to maintain an aerobic state of the culture; or nitrogen, hydrogen, or carbon dioxide gas
may be injected without the injection of a gas in order to maintain an anaerobic or microaerobic
state of the culture.
The culture temperature may generally be in a range from 27°C to 37°C, and more
specifically, from 30°C to 35°C, but is not limited thereto. The cultivation may be continued
until the desired amount of useful materials is obtained, and specifically for from 10 hours to 100
hours, but is not limited thereto.
The method of producing O-acetylhomoserine of the present disclosure may further
include a method of recovering O-acetylhomoserine from the cultured microorganism or the
cultured medium.
Specifically, the recovering of O-acetylhomoserine may be performed by a method of
culturing microorganisms of the present disclosure, for example, an appropriate method known
in the art such as a batch process, a continuous batch process, or a fed-batch process.
The recovering may include a purification process.
The thus-recovered O-acetylhomoserine may be used to produce methionine by a
two-step process developed by the present inventors (Korean Patent No. 10-0905381), i.e., a
two-step process.
The two-step process includes a process for producing L-methionine and organic acids
by an enzymatic reaction using an enzyme having the activity of O-acetylhomoserine
sulfhydrylase or a strain containing the enzyme by utilizing O-acetylhomoserine produced by a
strain producing the L-methionine precursor and methyl mercaptan as substrates.
More specifically, the present disclosure provides a method for producing L-methionine
by an enzyme reaction using an enzyme such as O-acetylhomoserine sulfhydrylase by utilizing
O-acetylhomoserine accumulated by the above method as a substrate.
In the two-step process, when O-acetylhomoserine is used as a precursor of
L-methionine, specifically, O-acetylhomoserine sulfhydrylase derived from a microorganism
strain belonging to the genus Leptospira, the genus Chromobacterium, and the genus
Hyphomonas, and more specifically, from a microorganism strain belonging to Leptospira
meyeri, Pseudomonas aurogenosa, Hyphomonas neptunium, and Chromobacterium violaceum
may be used.
WO/2015/186990 PCT/KR2015/005659
13
The reaction is shown below.
CH3SH + O-acetyl-L-homoserine <=> acetate + methionine
Such additional methionine-producing process is disclosed in Korean Patent No.
10-0905381, and the entire specification of the above Korean patent may be included as a
reference to the present disclosure.
Mode for Invention
Hereinafter, the present disclosure will be described in more detail with reference to the
following Examples. However, these Examples are for illustrative purposes only, and the
disclosure is not intended to be limited by these Examples.
Example 1: Preparation of strains having O-acetylhomoserine productivity
1-1. Deletion of metB gene in wild-type E. coli
For the preparation of a strain producing O-acetylhomoserine, E. coli, a representative
microorganism of the genus Escherichia, was used. To this end, a wild-type E. coli (K12)
W3110 (ATCC27325) was obtained from the American Type Culture Collection (ATCC) and
used. First, for blocking the pathway of producing cystathionine from
O-succinyl-L-homoserine, the metB gene (SEQ ID NO: 4) encoding cystathionine synthase was
deleted.
Specifically, an FRT one-step PCR deletion method was used for the deletion of metB
gene (Wanner BL., Proc. Natl. Acad. Sci. USA 97: 6640 - 6645, 2000). A deletion cassette was
prepared by performing PCR based on the pKD3 vector (Wanner BL., Proc. Natl. Acad. Sci.
USA 97: 6640 - 6645, 2000) as a template using the primers TKd of SEQ ID NOS: 13 and 14.
In particular, PCR was performed for a total of 30 cycles under the following conditions:
denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 1 min.
The thus-obtained PCR product was electrophoresed on a 1.0% agarose gel, and the
DNA obtained from a 1.2 kb band was purified. The recovered DNA fragment was introduced
into the E. coli (K12) W3110 strain, which was already transformed with the pKD46 vector
(Wanner BL., Proc. Natl. Acad. Sci. USA 97: 6640 - 6645, 2000), by electroporation. The
WO/2015/186990 PCT/KR2015/005659
14
W3110 strain transformed with pKD46 was cultured using LB medium containing ampicillin
(100 μg/L) and L-arabinose (5 mM) until the culture reached an OD600 = 0.6 at 30°C, and used
after washing twice with sterile distilled water and once with 10% glycerol. The
electroporation was performed at 2500 V. The recovered strain was spread on LB plate
medium containing chloramphenicol (25 μg/L), cultured overnight at 37°C, and the resistant
strains were selected.
PCR was performed using the thus-selected strain as a template along with the same
primers (SEQ ID NOS: 13 and 14), and the deletion of the metB gene was confirmed by
observing the presence of a 1.2 kb gene band on a 1.0% agarose gel. The strain, in which the
deletion of metB gene was confirmed, was transformed with pCP20 vector (Wanner BL., Proc.
Natl. Acad. Sci. USA 97: 6640 - 6645, 2000) and cultured in the same LB medium. Then, PCR
was performed under the same conditions followed by electrophoresis on a 1.0% agarose gel to
observe the presence of a 1.2 kb gene band, thus confirming the deletion of metB gene. The
strain, upon confirmation of the deletion, was transformed with pCP20 vector (Wanner BL.,
Proc. Natl. Acad. Sci. USA 97: 6640 - 6645, 2000) and cultured in the same LB medium. Then,
PCR was performed under the same conditions followed by electrophoresis on a 1.0% agarose
gel to select the final strain with the deletion of metB gene represented by the gene band, which
was reduced to a size of 150 bp, thereby confirming the removal of the chloramphenicol marker
from the strain.
The thus prepared and selected strain, with the deletion of metB gene, encoding
cystathionine synthase was named as “W3-B”.
1-2. Deletion of thrB gene
For increasing the amount of O-succinylhomoserine synthesis from the W3-B strain
prepared in Example 1-1, thrB gene (SEQ ID NO: 6) encoding homoserine kinase was deleted
from the strain. For the deletion of thrB gene, the same FRT one-step PCR deletion method,
which was used in the deletion of metB gene in Example 1-1, was used.
First, for the preparation of a thrB deletion cassette, PCR was performed using the pKD4
vector (Wanner BL., Proc. Natl. Acad. Sci. USA 97: 6640 - 6645, 2000) as a template and
thereby a deletion cassette was prepared. Specifically, PCR was performed using the primers of
SEQ ID NOS: 15 and 16 for a total of 30 cycles under the following conditions: denaturation at
94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 1 min.
WO/2015/186990 PCT/KR2015/005659
15
The thus-obtained PCR product was electrophoresed on a 1.0% agarose gel, and the
DNA obtained from a 1.6 kb band was purified. The recovered DNA fragment was introduced
into the W3-B strain, which was already transformed with the pKD46 vector by electroporation.
The recovered strain was spread on LB plate medium containing kanamycin (50 μg/L), cultured
overnight at 37°C, and the resistant strains were selected.
PCR was performed under the same conditions using the strain selected through the
above process as a template along with primers (SEQ ID NOS: 15 and 16), and the deletion of
the thrB gene was confirmed by observing the presence of a 1.6 kb gene on a 1.0% agarose gel.
The strain confirmed was transformed again with the pCP20 vector and cultured on LB medium,
and then, PCR was performed under the same conditions followed by electrophoresis on a 1.0%
agarose gel to select the final strain with the deletion of thrB gene represented by the gene band,
which was reduced to a size of 150 bp, thereby confirming the removal of the kanamycin marker
from the strain. The thus-prepared and selected strain, with the deletion of thrB gene, encoding
homoserine kinase was named as “W3-BT”.
Exemplary embodiments with respect to the strains with the deletion of metB gene and
thrB gene, etc., are disclosed in Korean Patent No. 10-0905381 or International Publication No.
WO 2008/013432, and the entire specification of the Korean patent or the international patent
publication may be included herein as a reference to the present disclosure.
1-3. Preparation of a strain with modified metA gene having an activity of homoserine
acetyltransferase
For enhancing the activity of homoserine acetyltransferase in the strain prepared in
Example 1-2, an attempt was made to introduce a modified metA gene (SEQ ID NO: 8), which
encodes homoserine acetyltransferase with an enhanced activity, to the strain.
In this regard, for the preparation of a modified metA gene with an enhanced activity, the
metA gene was first amplified and obtained by PCR using the chromosome of the W3110 strain
as a template along with primers (SEQ ID NOS: 17 and 18). The primers (SEQ ID NOS: 17
and 18) used in the PCR were prepared to include an EcoRV restriction site and a HindIII
restriction site, respectively, based on the nucleotide sequence of E. coli chromosome of
NC_000913 registered in the NIH GenBank.
The thus-obtained PCR product and the pCL1920 plasmid including pcj1 were treated
with EcoRV and HindIII and cloned. E. coli DH5α was transformed with the cloned plasmid
WO/2015/186990 PCT/KR2015/005659
16
and cultured in LB plate medium containing spectinomycin (50 μg/L), and the transformed E.
coli DH5α was selected and thereby the plasmid was obtained. The thus-obtained plasmid was
named as “pCL_Pcj1_metA”.
A modified metA gene was prepared using the site-directed mutagenesis kit (Stratagene,
USA) based on the pCL_Pcj1_metA obtained above. Specifically, the 111th amino acid (Gly) of
homoserine acetyltransferase was substituted with glutamic acid (Glu) (G111E). The
thus-prepared plasmid was named as “pCL_Pcj1_metA(EL)”.
Additionally, the 111th amino acid (Gly) of homoserine acetyltransferase was substituted
with glutamic acid (Glu), and additionally, the 112th amino acid (Leu) was substituted with
histidine (His) using the primers (SEQ ID NOS: 21 and 22). Accordingly, the plasmid
including the metA gene, in which the 111th amino acid was changed from glycine to glutamic
acid and the 112th amino acid was changed from leucine to histidine, was named as
“pCL_Pcj1_metA(EH)”.
Then, for preparing a replacement cassette to substitute with the modified metA gene
after introducing it into a strain, PCR was performed using the pKD3 vector as a template along
with primers (SEQ ID NOS: 27 and 28) for a total of 30 cycles under the following conditions:
denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 2 min.
The metA(EH) part of the replacement cassette was obtained as a PCR product using
pCL-Pcj1-metA(EH) as a template along with primers (SEQ ID NOS: 23 and 24) and the part of
the metA wild-type was obtained using primers (SEQ ID NOS: 25 and 26), respectively. The
three PCR products were used for the preparation of the metA(EH) replacement cassette, which
includes a chloramphenicol marker, using primers (SEQ ID NOS: 23 and 26), and the modified
metA gene was introduced into the W3-BT strain, which was already transformed with pKD46
vector, prepared in Example 1-2 by electroporation. The strain, in which the introduction was
confirmed, was again transformed with pCP20 vector, cultured in LB medium, and the strain in
which the chloramphenicol marker was removed and the metA gene was substituted with
metA(EH) was named as “W3-BTA”.
Exemplary embodiments with respect to the strains with enhanced activity of
homoserine acetyltransferase, etc., are disclosed in Korean Patent No. 10-1335841 or
International Publication No. WO 2012/087039, and the entire specification of the Korean patent
or the international patent publication may be included herein as a reference to the present
WO/2015/186990 PCT/KR2015/005659
17
disclosure.
1-4. Preparation of strains including 2 copies of ppc, aspC, and asd genes
For increasing the O-acetylhomoserine productivity of W3-BTA prepared in Example
1-3, the existing strategy for enhancing biosynthetic pathway was introduced. Efforts were
made to prepare a strain, in which phosphoenolpyruvate carboxylase involved in the biosynthesis
of oxaloacetate from phosphoenolpyruvate, aspartate aminotransferase involved in the
biosynthesis of aspartate from oxaloacetate, and aspartate-semialdehyde dehydrogenase involved
in the biosynthesis of homoserine from aspartate-semialdehyde dehydrogenase (i.e., ppc gene,
aspC gene, and asd gene) were amplified into 2 copies.
Accordingly, ppc gene was amplified into 2 copies using the primers of SEQ ID NOS:
29, 30, 31, and32; aspC gene was amplified into 2 copies using the primers of SEQ ID NOS: 33
and 34; and asd gene was amplified into 2 copies using the primers of SEQ ID NOS: 35, 36, 37,
and 38, respectively.
The strain, in which the biosynthetic pathway for O-acetylhomoserine was enhanced
based on W3-BTA strain by the above process, was named as “W3-BTA2PCD (= WCJM)”.
Exemplary embodiments with respect to the strains with enhanced activity of
homoserine acetyltransferase, etc., are disclosed in Korean Patent No. 10-0905381 or
International Publication No. WO 2008/013432, and the entire specification of the Korean patent
or the international patent publication may be included herein as a reference to the present
disclosure.
1-5. Experiment of flask culture
For the experiment of the amount of O-acetylhomoserine production by the strains
prepared in Examples 1-3 and 1-4, an Erlenmeyer flask culture was performed. The W3110,
W3-BTA, and WCJM strains were inoculated into LB medium, cultured at 33°C overnight.
Then, a single colony was inoculated into LB medium (3 mL), cultured at 33°C for 5 hours,
again diluted 200-fold in a 250 mL Erlenmeyer flask containing 25 mL of medium for producing
O-acetylhomoserine, cultured at 33°C at a rate of 200 rpm for 30 hours, and the amount of
O-acetylhomoserine production was confirmed by HPLC analysis. The medium composition
used is summarized in Table 1 below.
WO/2015/186990 PCT/KR2015/005659
18
[Table 1] Composition for flask medium for producing O-acetylhomoserine
Composition Concentration
(per Liter)
Glucose 40 g
Ammonium sulfate 17 g
KH2PO4 1.0 g
MgSO4·7H2O 0.5 g
FeSO4·7H2O 5 mg
MnSO4·8H2O 5 mg
ZnSO4 5 mg
Calcium carbonate 30 g
Yeast extract 2 g
Methionine 0.15 g
Threonine 0.15 g
The strains were cultured in the above medium for 30 hours and the amounts of
O-acetylhomoserine production were confirmed by HPLC analysis. The results are shown in
Table 2 below.
[Table 2] O-acetylhomoserine production by flask culture
OD
(562 nm)
Glucose
Consumption (g/L)
O-AH
(g/L)
W3110 14.2 40 0
W3-BTA 8.4 36 0.9
WCJM 9.6 35 1.2
As can be confirmed from Table 2, wild-type W3110 strain did not produce
O-acetylhomoserine at all, however, the W3-BTA strain produced O-acetylhomoserine in a
concentration of 0.9 g/L, and the WCJM strain, in which the biosynthetic pathway is enhanced,
produced O-acetylhomoserine in a concentration of 1.2 g/L.
Example 2: Identification of membrane proteins increasing the O-acetylhomoserine
productivity
The present inventors have applied yjeH (SEQ ID NO: 1), of which the association with
WO/2015/186990 PCT/KR2015/005659
19
O-acetylhomoserine exportation and O-acetylhomoserine productivity has never been disclosed.
The yjeH gene of a strain was enhanced by cloning the yjeH gene into the bac vector
using the HindIII restriction site present in the bac vector. For the bac vector, the CopyControl
BAC Cloning kit (Cat. No. CCBAC1H-HindIII, Epicentre) was used.
First, for obtaining the yjeH gene, PCR was performed using primers (SEQ ID NOS: 9
and 10) for a total of 30 cycles under the following conditions: denaturation at 94°C for 30 sec,
annealing at 55°C for 30 sec, and extension at 68°C for 1 min. The resulting PCR product was
electrophoresed on a 1.0% agarose gel, and the DNA obtained from a 1.2 kb band was purified.
The purified DNA was treated with HindIII at 37°C overnight, purified one more time, and yjeH
and the BAC vector were cloned using T4 ligase. E. coil DH5α was transformed with the
cloned plasmid, and the transformed E. coil DH5α was selected from LB plate medium
containing chloramphenicol 50 μg/mL. The thus-prepared plasmid was introduced into
W3-BTA and WCJM strains, which produce O-acetylhomoserine, and the flask evaluation on
their O-acetylhomoserine productivity was performed.
The resulting PCR product was electrophoresed on a 1.0% agarose gel, and the DNA
obtained from a 1.2 kb band was purified. The purified DNA was treated with HindIII at 37°C
overnight, purified one more time, and yjeH and the BAC vector were cloned using T4 ligase. E.
coil DH5α was transformed with the cloned plasmid, and the transformed E. coil DH5α was
selected from LB plate medium containing chloramphenicol 50 μg/mL and a plasmid was
obtained therefrom. The thus-prepared plasmid was introduced into W3-BTA and WCJM
strains, which produce O-acetylhomoserine, and the flask evaluation on their
O-acetylhomoserine productivity was performed.
Specifically, each strain was spread on solid LB medium and cultured in a 33°C
incubator overnight. Single colonies for each strain cultured on solid LB medium overnight
were inoculated to LB medium (3 mL), cultured at 33°C for 5 hours. Again, the resultant was
diluted 200-fold in a 250 mL Erlenmeyer flask containing 25 mL of medium for producing
O-acetylhomoserine, cultured at 33°C at a rate of 200 rpm for 30 hours, and the amount of
O-acetylhomoserine production was confirmed by HPLC analysis. The results are summarized
in Table 3 below.
[Table 3] Measurement of O-acetylhomoserine production by flask culture
OD Glucose O-AH
WO/2015/186990 PCT/KR2015/005659
20
(562 nm) Consumption (g/L) (g/L)
W3-BTA/pBAC 9.5 35 0.8
WCJM/pBAC 9.6 35 1.2
W3-BTA/ pBAC-yjeH 9.8 36 1.5
WCJM/pBAC-yjeH 10.1 37 2.3
As can be confirmed from Table 3, the introduction of the yjeH plasmid into the WCJM
strain resulted in a higher OD value compared to that of the control strain, and also a higher rate
of glucose consumption. The WCJM/pBAC-yjeH strain produced O-acetylhomoserine in a
concentration of 2.3 g/L, thus confirming that the strain can have an increased
O-acetylhomoserine productivity by the yjeH introduction.
Example 3: Preparation of a plasmid with enhanced yjeH promoter and evaluation
of O-acetylhomoserine productivity
3-1. Preparation of a plasmid with enhanced yjeH promoter
An experiment was performed to replace the endogenous yjeH promoter with 3 different
promoters having strong expression-inducing activities compared to that of the endogenous yjeH
promoter, based on the plasmid prepared in Example 2.
Specifically, PCL vectors with promoters having an enhanced activity were prepared
using pro, cysk, or icd promoter. The PCL vectors were prepared using the SmaI restriction site
of the PCL vector, and the icd promoter (SEQ ID NO: 51) was prepared by PCR amplification
using the primers of SEQ ID NOS: 39 and 40; the pro promoter (SEQ ID NO: 52) using the
primers of SEQ ID NOS: 41 and 42; and the cysk promoter (SEQ ID NO: 53) using the primers
of SEQ ID NOS: 43 and 44. The thus-prepared plasmids were introduced into the WCJM strain,
respectively, and their O-acetylhomoserine productivities were evaluated in flasks.
Specifically, each strain was spread on LB plate medium and cultured in a 33°C
incubator overnight. Each strain cultured overnight on LB plate medium was inoculated into
titer medium 25 mL and cultured in a 33°C incubator at 200 rpm overnight. The results are
shown in Table 4 below.
[Table 4] Measurement of O-acetylhomoserine production by flask culture
WO/2015/186990 PCT/KR2015/005659
21
OD
(562 nm)
Glucose Consumption
(g/L)
O-AH
(g/L)
WCJM/pCL1920 9.6 35 1.2
WCJM/pCL-yjeH 10.1 37 2.3
WCJM/pCL-Picd-yjeH 10.5 38 3.1
WCJM/pCL-Ppro-yjeH 10.7 38 3.5
WCJM/pCL-Pcysk-yjeH 9.4 39 4.4
As can be confirmed from Table 4, the strain introduced with the pCL-Pcysk-yjeH
plasmid showed a decrease in OD value compared to that of the self-promoter, but the strain
showed a higher rate of glucose consumption and the highest O-acetylhomoserine production
(4.4 g/L).
3-2. Preparation of a plasmid for enhancing a gene and promoter in a biosynthetic
pathway
For maximizing O-acetylhomoserine productivity, a plasmid for enhancing the
biosynthetic pathway up to homoserine was prepared. For cloning aspartate kinase, homoserine
O-acetyltransferase, and yjeH into the PCL vector, the pCL-thrA-metX plasmid, which was
already prepared, was used.
Specifically, for obtaining the yjeH gene, PCR was performed using the primers (SEQ
ID NOS: 11 and 12) for a total of 30 cycles under the following conditions: denaturation at 94°C
for 30 sec, annealing at 55°C for 30 sec, and extension at 68°C for 1 min.
The resulting PCR product was electrophoresed on a 1.0% agarose gel, and the DNA
obtained from a 1.2 kb band was purified. The purified DNA was treated with KpnI at 37°C
overnight, purified one more time, and yjeH and the BAC vector were cloned using T4 ligase. E.
coil DH5α was transformed with the cloned plasmid, and the transformed E. coil DH5α was
selected from LB plate medium containing spectinomycin 50 μg/mL and plasmid was obtained
therefrom. The thus-prepared plasmid was introduced into the WCJM strain, which produces
O-acetylhomoserine, and the flask evaluation on their O-acetylhomoserine productivity was
performed. The thus-prepared plasmids were all 3 kinds and they were prepared using the 3
different promoters prepared in Example 3-1. The 3 different kinds of plasmids were
introduced into the WCJM strain and the flask evaluation was performed in the same manner as
in Example 3-1. The results are shown in Table 5 below.
WO/2015/186990 PCT/KR2015/005659
22
[Table 5] Measurement of O-acetylhomoserine production by flask culture
OD
(562 nm)
Glucose Consumption
(g/L)
O-AH
(g/L)
WCJM/pC2 9.6 35 1.5
WCJM/pC2-yjeH 10.1 37 2.8
WCJM/pC2-Picd-yjeH 10.5 38 4.2
WCJM/pC2-Ppro-yjeH 10.7 38 4.5
WCJM/pC2-Pcysk-yjeH 9.4 39 5.4
As can be confirmed from Table 5, when the biosynthetic pathway and yjeH were
enhanced simultaneously, the O-acetylhomoserine production was improved further. The order
of the increase of the O-acetylhomoserine production was as follows: in the same manner as
described above, the strain introduced with the pC2-Pcysk-yjeH plasmid showed a decrease in
OD value compared to that of the strain, in which the self-promoter was used, but the strain
introduced with the pC2-Pcysk-yjeH plasmid showed the highest glucose consumption rate, and
also the highest O-acetylhomoserine production (5.4 g/L).
Example 4: Preparation of a strain with enhanced endogenous yjeH promoter and
evaluation of O-acetylhomoserine productivity
4-1. Preparation of a strain with enhanced endogenous yjeH and evaluation thereof
For preparing a strain with an enhanced activity of the endogenous yjeH gene of the
WCJM strain, which produces O-acetylhomoserine, an experiment of replacing a promoter was
performed. The WCJM strain of the present disclosure has one copy of the yjeH gene, and the
strain was prepared to increase the yjeH gene by enhancing the promoter instead of increasing
the copy number of the yjeH gene.
Specifically, as a promoter for the replacement, the icd, cysK, and pro promoters (Picd,
Pcysk, and Ppro), whose activities were confirmed in Example 3, and the FRT one-step PCR
deletion method specified above was used (Wanner BL., Proc. Natl. Acad. Sci. USA 97: 6640 -
6645, 2000). Insertion cassettes were prepared using the pKD4 vector as a template along with
the primers of SEQ ID NOS: 45 and 46 for the icd promoter, the primers of SEQ ID NOS: 47
and 48 for the cysk promoter, and the primers of SEQ ID NOS: 49 and 50 for the pro promoter.
WO/2015/186990 PCT/KR2015/005659
23
PCR was performed for a total of 30 cycles under the following conditions: denaturation at 94°C
for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 1 min.
The thus-obtained PCR product was electrophoresed on a 1.0% agarose gel, and the
DNA obtained from a 2.5 kb band was purified. The recovered DNA fragment was introduced
into the WCJM strain, which was already transformed with the pKD46 vector (Wanner BL., Proc.
Natl. Acad. Sci. USA 97: 6640 - 6645, 2000), by electroporation. The WCJM strain transformed
with pKD46 was cultured using LB medium containing ampicillin 100 μg/L and L-arabinose 5
mM until the culture reached an OD600 = 0.6 at 30°C, and used after washing twice with sterile
distilled water and once with 10% glycerol. The electroporation was performed at 2500 V.
The recovered strain was spread on LB plate medium containing chloramphenicol 25 μg/L,
cultured overnight at 37°C, and the resistant strains were selected.
PCR was performed using the thus-selected strain as a template along with the primers
of SEQ ID NOS: 45 and 46 for the icd promoter, the primers of SEQ ID NOS: 47 and 48 for the
cysk promoter, and the primers of SEQ ID NOS: 49 and 50 for the pro promoter under the same
conditions described above, and the replacement of the endogenous promoter of the yjeH gene
with each of the foreign promoter was confirmed by observing the presence of a 2.5 kb gene
band on a 1.0% agarose gel. The strains, in which the promoter replacement was confirmed,
were transformed with the pCP20 vector (Wanner BL., Proc. Natl. Acad. Sci. USA 97: 6640 -
6645, 2000) and cultured in LB medium. Then, PCR was performed under the same conditions
followed by electrophoresis on a 1.0% agarose gel to prepare the final strains with the promoter
replacement represented by the gene band, which was reduced to a size of 1 kb, thereby
confirming the removal of the kanamycin marker from the strains. The thus-prepared strains
were named according to their respective promoter: i.e., “WCJM-PIY” for the strain with the icd
replaced promoter of icd, “WCJM-PCY” for the strain with the replaced promoter of cysk, and
“WCJM-PCY” for the strain with the replaced promoter of pro. The O-acetylhomoserine
productivities of the strains, in which the promoter of the yjeH gene was replaced were measured
by performing flask culture evaluation and the results are shown in Table 6 below.
[Table 6] Measurement of O-acetylhomoserine production by flask culture
OD
(562 nm)
Glucose
Consumption (g/L)
O-AH
(g/L)
WCJM 9.6 35 1.2
WO/2015/186990 PCT/KR2015/005659
24
WCJM-PIY 9.2 38 1.8
WCJM-PCY 10.5 38 3.1
WCJM-PPY 10.1 38 1.9
As can be confirmed from Table 6, when the yjeH gene expression of the WCJM strain
was enhanced by replacing the endogenous promoter of the yjeH gene within the chromosome
with a promoter having a strong expression activity, the O-acetylhomoserine productivity was
not rapidly changed compared to the result of Example 3, in which the strain was transformed by
introducing with a plasmid (5 copies), but the O-acetylhomoserine productivity of each of the
strains was shown to increase compared to that of the WCJM strain, which is the strain producing
O-acetylhomoserine.
4-2. Preparation of a strain with an enhanced promoter of the yjeH gene in a strain with a
high yield of O-acetylhomoserine and evaluation of O-acetylhomoserine productivity of the
strain
The method of preparing a strain producing O-acetylhomoserine using a wild-type
W3110-derived strain, which has threonine productivity by NTG mutation, is known
(International Publication No. WO 2012/087039). In particular, the thus-prepared strain
producing O-acetylhomoserine in high yield was deposited at Korean Culture Center of
Microorganisms (KCCM) with Accession No. KCCM11468P.
The strain with Accession No. KCCM11146P has high-yield O-acetylhomoserine
productivity, which consumes 40 g/L of glucose and produces about 15 g/L to 16 g/L during
flask culture. In this regard, the present inventors examined whether the strains, which already
have high O-acetylhomoserine productivity, can further improve their O-acetylhomoserine
productivity by enhancing the yjeH gene in the strains.
Specifically, the promoter of the yjeH gene was replaced with a promoter with a high
expression-inducing activity, and this was performed in the same manner as in Example 4-1.
The thus-prepared strains, in which the promoter of the yjeH gene of the KCCM11146P strain,
were named as “KCCM11146P-PIY” for the strain with the replaced promoter of icd,
“KCCM11146P-PCY” for the strain with the replaced promoter of cysk, and
“KCCM11146P-PPY” for the strain with the replaced promoter of pro, respectively.
The O-acetylhomoserine productivities of the strains, in which the promoter of the yjeH
gene was replaced, were measured by flask culture evaluation. Specifically, KCCM11146P,
WO/2015/186990 PCT/KR2015/005659
25
KCCM11146P- PIY, KCCM11146P- PCY, and KCCM11146P- PPY strains were inoculated into
LB medium and cultured at 33°C overnight. The resulting single colonies were inoculated into
3 mL of LB medium, cultured at 33°C for 5 hours, diluted 200-fold in a 250 mL Erlenmeyer
flask containing 25 mL of medium for producing O-acetylhomoserine, cultured at 33°C for at a
rate of 200 rpm for 30 hours, and the amount of O-acetylhomoserine production was analyzed by
HPLC. The results are shown in Table 7 below.
[Table 7] Measurement of O-acetylhomoserine production by flask culture
OD
(562 nm)
Glucose
Consumption (g/L)
O-AH
(g/L)
KCCM11146P 18.3 40 14.2
KCCM11146P- PIY 16.2 40 16.3
KCCM11146P- PCY 19.2 40 18.2
KCCM11146P- PPY 18.8 40 16.2
As can be confirmed from Table 7, the KCCM11146P strain produced 14.2 g/L of
O-acetylhomoserine, whereas, in the cases of the strains with a replaced promoter, the PCY
strain showed the highest production (18.2 g/L) and the PIY and PPY strains showed an
increased production of O-acetylhomoserine compared to that of the original strain.
The present inventors have confirmed that the strain based on the KCCM11146P strain,
in which the yjeH gene activity is enhanced, can increase O-acetylhomoserine production. As a
result, the inventors have named the strain as “CA05-4008”, and deposited the strain with the
Korean Culture Center of Microorganisms (KCCM), under the Budapest Treaty, on November
22, 2013, with Accession No. KCCM11484P.
From the foregoing, a skilled person in the art to which the present disclosure pertains
will be able to understand that the present disclosure may be embodied in other specific forms
without modifying the technical concepts or essential characteristics of the present disclosure.
In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and
should not be construed as limiting the scope of the present disclosure. On the contrary, the
present disclosure is intended to cover not only the exemplary embodiments but also various
alternatives, modifications, equivalents, and other embodiments that may be included within the
spirit and scope of the present disclosure as defined by the appended claims.
WO/2015/186990 PCT/KR2015/005659
26
WO/2015/186990 PCT/KR2015/005659
27
We Claim:
Claim 1
A microorganism of the genus Escherichia producing O-acetylhomoserine, wherein an
activity of the protein comprising the amino acid sequence of SEQ ID NO: 1 is enhanced
compared to a non-modified microorganism.
Claim 2
The microorganism of the genus Escherichia according to claim 1, wherein the
microorganism is Escherichia coli.
Claim 3
The microorganism of the genus Escherichia according to claim 1, wherein an activity
of cystathionine synthase is further weakened or inactivated.
Claim 4
The microorganism of the genus Escherichia according to claim 1, wherein an activity
of homoserine kinase is further weakened or inactivated.
Claim 5
The microorganism of the genus Escherichia according to claim 1, wherein an activity
of homoserine acetyltransferase is further enhanced compared to a non-modified microorganism.
Claim 6
The microorganism of the genus Escherichia according to claim 1, wherein the activity
of at least one enzyme selected from the group consisting of phosphoenolpyruvate carboxylase,
aspartate aminotransferase, and aspartate semialdehyde dehydrogenase is further enhanced.
Claim 7
The microorganism of the genus Escherichia according to claim 1, wherein an activity
of aspartate kinase is enhanced compared to a non-modified microorganism.
WO/2015/186990 PCT/KR2015/005659
28
Claim 8
A method for producing O-succinylhomoserine comprising
culturing the microorganism of the genus Escherichia producing O-acetylhomoserine of
any of claims 1 to 7 to obtain a cultured medium.
Claim 9
The method according to claim 8, further comprising recovering O-acetylhomoserine
from the cultured microorganism or the cultured medium.