Description
Title of Invention: MICROORGANISM PRODUCING OPHOSPHOSERINE
AND METHOD OF PRODUCING LCYSTEINE
OR DERIVATIVES THEREOF FROM OPHOSPHOSERINE
USING THE SAME
Technical Field
[1] The present invention relates to a method for production of cysteine or its derivatives
using O-phosphoserine as an intermediate and recombinant microorganism for use in
production of O-phosphoserine.
[2]
Background Art
[3] L-cysteine is an amino acid that plays an important role in sulfur metabolism of all
living organisms. It is used in the biosynthesis of proteins, such as hair keratin, glu
tathione, biotin, methionine and other sulfur-containing metabolites as well as serving
as a precursor of coenzyme A. In addition, the biosynthesis of cysteine is known to be
closely associated with the biosynthesis of other amino acids including L-serine, Lglycine,
and L-methionine. Industrially, L-cysteine and its derivatives find applications
in a variety of fields including the pharmaceutical industry (for treatment of bronchial
diseases), the cosmetics industry (in hair shampoo, compositions for permanent
waves), and the food industry (antioxidants, flavorant enhancers, dough aids, etc.).
[4] L-cysteine was once obtained industrially by acid hydrolysis of human hairs or
animal feathers (Biotechnology of the Amino Acids Production edited by Ko Aida, p
217-223, 1986). However, not only does the production of cysteine from hairs or
feathers ensure a yield of as low as 7-8 , but also the use of hydrochloric acid or
sulfuric acid produces a lot of waste resulting in environmental pollution. Further, ex
traction from hairs or feathers may induce the user to have a strong adversion thereto.
These problems have caused a push for the development of environmentally friendly
production processes of L-cysteine. The main contemporary route involves fer
mentation utilizing microorganisms.
[5] Representative among the microbial production of L-cysteine is 1) the biological
conversion of D, L-ATC using a microorganism (Ryu OH, Ju JY and Shin CS, Process
Biochem., 32:201-209, 1997). This conversion process is, however, difficult to apply
industrialy due to the low solubility of the precursor D, L-ATC. 2) Another method of
L-cysteine production is direct fermentation using E. coli (Patent No. EP0885962B;
Wada M and Takagi H, Appl. Microbiol. Biochem., 73:48-54, 2006). Excessive accumulation
of L-cysteine within microorganisms incurs intracellular toxicity, exhibiting a
limitation in the production of L-cysteine at a high concentration. To overcome this
drawback, L-cysteine-exporting proteins are employed, but there have been no sig
nificant improvements in productivity.
[6] Referring to the biosynthesis pathway of L-cysteine in microorganisms and plants,
O-acetyl- serine (OAS) acts as an intermediate precursor providing the carbon
backbone of L-cysteine (Kredich NM and Tomkins GM, J. Biol. Chem., 241:
4955-4965, 1966). The enzyme O-acetylserine sulfhydrylase (OASS), using hydrogen
sulfide as a sulfur donor, catalyses the conversion of O-acetylserine to cysteine. Alter
natively, S0 4 may be reduced to thiosulfate for use as a sulfur donor in cysteine
production (Nakamura T, Kon Y, Iwahashi H and Eguchi Y, J . Bacterid., 156:
656-662, 1983). Therefore, cystein may be produced using microorganisms accu
mulating OAS and OASS using various sulfur donors (US6,579,705). The cysteine
biosynthesis pathway via OAS uses the two enzymes of serine acetyltransferase
(CysE), which catalyzes the conversion of OAS from serine, and cysteine synthase
(CysK), which catalyzes the conversion of OAS to cysteine. Among them, serine
acetyltransferase (CysE) is highly sensitive to feedback inhibition by the final product
cysteine (Wada M and Takagi H, Appl. Microbiol. Biochem., 73:48-54, 2006).
[7]
Disclosure of Invention
Technical Problem
[8] Leading to the present invention, the present inventors found out the existence of Ophosphoserine
sulfhydrylase (OPSS) in Aeropyrum pernix, Mycobacterium tu
berculosis, and Trichomonas vaginalis that takes an O-phospho-L-serine
(OPS)-specific pathway, rather than the OAS-specific pathway, to synthesize Lcysteine
through intensive research (Mino K and Ishikawa K, FEBS letters, 551:
133-138, 2003; Burns KE, Baumgart S, Dorrestein PC, Zhai H, McLafferty FW and
Begley TP, J. Am. Chem. Soc, 127: 11602-11603, 2005; Westrop GD, Goodall G,
Mottram JC and Coombs GH, J. Biol. Chem., 281: 25062-25075, 2006) and that the
OPSS of M. tuberculosis, can use Na2S as a sulfur donor in converting OPS to cysteine
even in the absence of the additional enzymes when five C-terminal amino acid
residues are removed therefrom (Argen D, Schnell R and Schneider G, FEBS letters,
583: 330-336, 2009). In the present invention, a microorganism is mutated to ac
cumulate OPS therein, following incubation to convert OPS into cysteine in the
presence of the OPSS enzyme. Nowhere has this method been previously described.
[9]
Solution to Problem
[10] It is an object of the present invention to provide a method for producing cysteine or
a derivative thereof.
[11] It is another object of the present invention to provide a recombinant microorganism
for the production of O-phosphoserine.
[12]
Advantageous Effects of Invention
[13] The method of the present invention in which O-phosphoserine is produced at high
yield by a recombinant microorganism and is used for conversion into cysteine, as it is,
is more friendly to the environment and ensures higher efficiency in the production of
cysteine than do chemical synthesis methods. The cysteine and its derivatives produced
by the fermentation and bioconversion of the present invention can be widely used in
the production of animal and human foods and food additives.
[14]
Brief Description of Drawings
[15] FIG. 1 is a schematic diagram showing the accumulation of O-phosphoserine by
microbial fermentation and the enzymatic conversion of the accumulated Ophosphoserine
into L-cysteine.
[16] FIG. 2 is a graph showing the activity of OPS sulfhydrylase according to tem
peratures.
[17] FIG. 3 is a set of graphs showing pH sensitivity of OPS sulfhydrylase.
[18] FIG. 4 is a photograph showing the expression level of Msm-T in a pET system and
a pCL-Pcj 1 system as analyzed by SDS PAGE.
[19] FIG. 5 is a graph showing the enzymatic activity of OPS sulfhydrylase to convert
purified OPS fermentation broth into cysteine.
[20] FIG. 6 is a graph showing the enzymatic activity of OPS sulfhydrylase to convert
OPS fermentation broth into cysteine.
[21]
Best Mode for Carrying out the Invention
[22] As used herein, the term "cysteine conversion" is intended to refer to the catalytic
reaction of O-phosphoserine sulfhydrylase (OPSS) which results in the conversion of
the substrate O-phosphoserine (OPS) into the product cysteine, that is, it refers to the
catalytic reaction of converting OPS into cyteine.
[23] As used herein, the term "cysteine conversion rate" refers to the percentage of the
amount of the product cysteine to the amount of the starting material OPS. Under
optimal reaction conditions, 1 mole of OPS is conveted into 1 mole of cysteine. For
example, if 100 moles of OPS is converted into 100 moles of cysteine, the cysteine
conversion rate is 100 %.
[25] In accordance with an aspect thereof, the present invention provides a method for
producing cysteine or a derivative thereof, comprising:
[26] 1) culturing a recombinant microorganism which is modified to have decreased endogeneous
phosphorerine phosphatase (SerB) activity to produce O-sphosphoserine
(OPS); and 2) reacting the OPS of step 1) with a sulfide in the presence of Ophosphoserine
sulfhydrylase (OPSS) or a microorganism expressing OPSS, to produce
cysteine or a derivative thereof.
[27] In the method, step 1) is related to culturing a recombinant microorganism which is
the activity of endogenous phosphoserine phosphatase (SerB) is reduced.
[28] The SerB is a protein that has the activity of hydrolyzing OPS into L-serine. Thus, a
microorganism which has reduced endogeneous SerBactivity is characterized by the
accumulation of OPS therein. The SerB is not limited to, may comprise any amino acid
sequences, which exhibits SerB activity, and may have preferably the amino acid
sequence of SEQ ID NO: 1 or 2. However, as long as it exhibits SerB activity, any
amino acid sequence is used, which preferably has a homology of 90% or higher, more
preferaby 96% or higher, far more preferably 98% or higher, and most preferably 99%
or higher with that of SEQ ID NO: 1 or 2. The reduced SerB activity means a decrease
in SerB activity, compared to that of a prior-modified strain, and encompasses the
disrupting of SerB. Various techniques for reduction of SerB activity are well known
in the art. Illustrative examples include the deletion of a chromosomal serB, the in
troduction of mutation into the chromosomal serB to reduce endogenouse SerB
activity, the introduction of mutation into a regulatory region for the serB to reduce en
dogenouse SerB activity, the substitution of the chromosomal serB with a gene
mutated to reduce the endogenouse SerB activity and the introduction of an antisense
oligonucleotide complementary to a transcript of the serB to inhibit the translation of
the mRNA, but methods for reducing the SerB activity are not limited to these. These
techniques may be applied to the reducing the activity of other enzymes in the present
invention.
[29] The disruption of endogenous SerB results in the introduction of serine auxotrophy
into the recombinant microorganism so that the medium must be supplemented with
glycine or serine. Glycine may be provided in the form of purified glycine, a glycinecontaining
yeast extract, or tryptone. Glycine is contained at a concentration of from
0.1 to 10 g/L, and preferably at a concentration of from 0.5 to 3 g/L. As for serine, it
may be provided in the form of purified serine, a serine-containing yeast extract or
tryptone. Its concentration in the culture medium ranges from 0.1 to 5 g/L, and
preferably from 0.1 to 1 g/L.
[30] In one embodiment of the present invention, when cultured in a glycine- or serinecontaining
medium, mutant Corynebacterium glutamicum or E. coli in which the
activity of endogeneous SerB was distrupted was found to produce a higher amount of
OPS, compared to the wild-type (see Tables 2, 3, 6 and 7).
[31] In addition, the recombinant microorganism of the present invention may have
enhanced phosphoglycerate dehydrogenase (SerA) or phosphoserine aminotransferase
(SerC) activity. The SerA is a protein that has the activity of converting
3-phosphoglycerate to 3-phosphohydroxypyruvate. The SerA may have wild-type
amino acids or a mutant amino acid sequence which shows resistance to feedback in
hibition by serine, but is not limited to these. Preferably, the SerA may have one
selected from the group consisting of amino acid sequences of SEQ ID NOS: 3 to 7. So
long as it exhibits wild-type SerA activity or the mutant SerA activity resistant to
serine feedback inhibition, any amino acid sequence may be used, although it
preferably shares a homology of 90% or higher, more preferaby 96% or higher, far
more preferably 98% or higher, and most preferably 99% or higher with that of one of
SEQ ID NO: 3 to 7. A "mutant SerA resitant to feedback inhibition" means the mutant
showing a maintained or enhanced SerA activity irrespective of the feedback inhibition
by serine or glycine. The feedback-resistant mutants are well known in the art (Grant
GA et al., J. Biol. Chem., 39: 5357-5361, 1999; Grant GA et al., Biochem., 39:
7316-7319, 2000; Grant GA et al., J . Biol. Chem., 276: 17844-17850, 2001; Peters-
Wendisch P et al., Appl. Microbiol. Biotechnol., 60: 437-441, 2002; EP0943687B). In
one embodiment of the present invention, when a feedback-resistant serA was further
introduced thereinto, Corynebacterium glutamicum or E. coli having a disrupted serB
was found to produce a higher amount of OPS, as compared to the wild-type (see
Tables 4 and 9).
[32] The SerC is a protein that has the activity of converting 3-phosphohydroxypyruvate
to O-phosphoserine. The SerC is not limited to, may comprise the sequences which
exhibits SerC activity, and may have preferably the amino acid sequence of SEQ ID
NO: 8. However, as long as it exhibits SerC activity, any amino acid sequence may be
employed, but it should preferably share a homology of 90% or higher, more preferaby
96% or higher, far more preferably 98% or higher, and most preferably 99% or higher
with that of SEQ ID NO: 8. Furthermore, a mutation may be introduced into the serC
so that the enzyme activity can be increased. In one embodiment of the present
invention, when an serC was further introduced thereinto, Corynebacterium
glutamicum or E. coli having a disrupted serB and a feeback-resistant serAwas found
to produce a higher amount of OPS, compared to the wild-type (see Table 9).
[33]
[34] Further, the capacity of the recombinant microorganism of the present invention to
perform the intracellular uptake of or the degradation of O-phosphoserine may be
decreased. In detail, the recombinant microorganism may be modified to reduce the
activity of PhnC/PhnD/PhnE alkylphosphonate ABC transporter (PhnCDE operon, that
is, ATP-binding component of phosphonate transport (PhnC; EG 10713)-periplasmic
binding protein component of Pn transporter (PhnD; EG 10714)-integral membrane
component of the alkylphosphonate ABC transporter (PhnE; EG 11283)), alkaline
phosphatase (PhoA) or acid phosphatase (AphA).
[35] In one embodiment of the present invention, the further deletion of the phnCDE
operon from the recombinant mutant was observed to lead to an increase in OPS
production (Table 10). In the recombinant microorganism which was further disrupted
of PhoA or AphA activity, OPS degradation started to decrease at the time when the
concentration of phosphoric acid in the culture medium is decreased (Table 12).
Moreover, the introduction of a feedback-resistant serA or a serC raised OPS
production (Tables 14 to 16).
[36] Also, the recombinant microorganism of the present invention may be further char
acterized by the enhancement of pyrimidine nucleotide transhydrogenase (PntAB; EC
1.6.1.1) activity. As described previously (Sauer U P et al., J Biol Chem. 20;
279(8):6613-9. Epub 2003), PntAB participates in NADPH metabolism to regulate intracelluar
redox balance. In one embodiment of the present invention, the recombinant
microorganism in which PntAB activity was further enhance by overexpression of
pntAB was found to increase OPS production (Table 17).
[37] Moreover, the recombinant microorganism of the present invention may be char
acterized by the enhancement of O-acetylserine/cysteine efflux permease (YfiK), homoserine/
homoserine lactone efflux protein (RhtB; EG 11469) or threonine/homoserine
lactone efflux protein (RhtC; EG1 1468). The YfiK is known as an exporter
for exporting cysteine and OAS extracellularly (Franke I et al., J. Bacteriology, 185:
1161-1 166, 2003) and RhtB is reported to act as an extracellular exporter of homoserine/
homoserine lactone, a threonine precursor. Further, the RhtC is known as an
exporter of threonine and homoserine. The enhancement of the activity of YfiK, RhtC
and RhtB showed an increase in the growth and OPS production of the OPS accu
mulation strain (Table 18).
[38]
[39] The enhancement of the enzyme activity may be achieved using various well-known
methods, including, but not being limited to, increasing the copy number of a gene
encoding an enzyme of interest, introducing a mutation into a regulatory region for the
gene to enhance the enzyme activity, substituting the chromosomal gene with a gene
mutated to enhance the enzyme activity, and intoroducing a mutation into the
chromosomal gene to enhance the enzyme activity.
[40]
[41] In addition, the recombinant microorganism of the present invention is further char
acterized by the reduced activity of phosphoglycerate mutase. The phosphoglycerate
mutase exists as three isozymes: Gpml, GpmA and GpmB and is responsible for the
conversion of 3-phosphoglycerate into 2-phosphoglycerate in the glycolysis process.
For the use of 3-phosphoglycerate as a substrate, these enzymes are in competition
with SerA that catalyzes the synthesis of 3-phosphohydroxypyruvate. Therefore, the
decreasing activity of each of the enzymes was observed to cause an abundance of
3-phosphoglycerate, a precursor of OPS, resulting in the production of an increased
level of OPS (Table 19).
[42]
[43] In the recombinant microorganism of the present invention, L-serine dehydratase I
(SdaA; EC 4.3.1.17) or 2-amino-3-ketobutyrate coenzyme A ligase (Kbl) may be also
reduced. Thus, the recombinant microorganism is characterized by the OPS production
maintained or increased even when it is cultured in a medium containing a low con
centration of glycine or serine (Table 20).
[44] Further, the recombinant microorganism of the present invention may be further
characterized by the reduced activity of IclR. IclR is a transcription factor that
functions to repress the expression of aceBAK, a glyoxylate bypass operon (L Gui et
al., J. Bacterid., Vol 178, No. 1, 321-324, 1996). When it was deleted, the production
of OPS was observed to increase (Table 21).
[45]
[46] Also, the recombinant microorganism of the present invention may be further char
acterized by the enhancement of an enzyme activity selected from the group consisting
of i) acetyl-CoA synthetase (Acs), ii) acetic acid kinase (AckA)-phosphotransacetylase
(Pta), iii) malate synthase G (GlcB), iv) malate dehydrogenase (MaeB), v) glutamate
dehydrogenase (GdhA), vi) glyoxylate carboligase (Glc), vii) tartronate semialdehyde
reductase 2 (GlxR), viii) glycerate kinase II (GlxK), and a combination thereof.
[47] In a concrete embodiment of the present invention, when i) Acs (EC No.6.2.1.1; J
Bacteriol. 1995 May; 177(10):2878-86) or pyruvate oxidase monomer (PoxB; EC
1.2.2.2) or ii) AckA and Pta (EC 2,3,1,8), all of which aim to effectively reuse ac
cumulated acetate with the concomitant consumption of produced NADH, were further
enhanced, the recombinant microorganism of the present invention was found to
increase production of OPS (Table 22). Functioning to catalyze the synthesis of malate
from glyoxylate and the conversion of malate into pyruvate, iii) GlcB (EC No.2.3.3.9)
and iv) MaeB (EC 1.1.137) can weaken the TCA cycle and thus be used to increase the
glucose comsumption and the production of O-phosphoserine (Table 23). According to
one embodiment of the present invention, the enhancementof v) GdhA; (EC 1.4.1.2),
which catalyzes the synthesis of glutamate, a substrate of SerC, from 2-oxoglutarate
and NADPH, bestowed a much higher potential for producing OPS on the mi
croorganism (Table 17). All of vi) Glc(EC 4.1.1.47), vii) GlxR(EC 1.1.1.60) and viii)
GlxK(EC 2.7.1.31) are known to convert glycoxylate into 3-phosphoglycerate, that is,
to increase the level of the substrate of phosphoglycerate dehydrogenase (Kim HJ et al
., J. Bacterid., 186(11), 3453-3460, 2004; Eva Cusa t al., J . Bacteriol., 181(24),
7479-7484, 1999; Chnag YY et al, J. Biol. Chem. 268(6): 3911-3919, 1993). The re
combinant microorganism of the present invention, when further enhanced in the
activity of Glc, GlxR and GlxK, was improved in sugar consumption and growth
(Table 24).
[48]
[49] The recombinant microorganism of the present invention refers to any mi
croorganism in which there is the reduction of SerB activity, thus producing OPS at an
elevated level. If this condition is satisfied, any microorganism, whether prokaryotic or
eukaryotic, falls within the scope of the present invention. Representative among them
are enterobacteria or coryneform bacteria. Examples of the microorganisms useful in
the present invention include Escherichia sp., Erwinia sp., Serratia sp., Providencia
sp., Corynebacterium sp., and Brevibacterium sp. Preferable are Escherichia sp. and
Corynebacterium sp, with more preference given for Escherichia sp. and with the
highest preference being for E. coli.
[50] In an embodiment, the recombinant strain capable of producing OPS was named E.
coli CA07-0012, and deposited with the Korean Culture Center of Microorganisms,
located at 361-221, Hongje 1, Seodaemun, Seoul, Korea, on October. 12, 2011 under
accession number KCCM1 1212P.
[51] In addition, in an embodiment, the recombinant strain capable of producing OPS was
named E. coli CA07-0022/pCL-prmf-serA*(G336V)-serC, and deposited with the
Korean Culture Center of Microorganisms, located at 361-221, Hongje 1, Seodaemun,
Seoul, Korea, on September. 28, 2010 under accession number KCCM11103P. Herein,
the term "CA07-0022/pCL-prmf-serA*(G336V)-serC" is used interchangerably with
CA07-0022 serA*(G336V)/pCL-prmf-serA*(G336V)-serC.
[52] After the strain was cultured for 80 hours in a 1 L fermenter, O-phosphoserine was
produced at a concentration of 19.5 g/L (Example 35).
[53]
[54] As used herein, the term "culturing" is intended to mean growing microorganisms
under artificially controlled conditions. A culturing procedure may be conducted using
a suitable medium and culturing conditions well known in the art. Those skilled in the
art can readily control the culturing procedure to correspond to the strains employed.
For example, it may be performed in a batch type, in a continuous type, or in a fedbatch
type, but is not limited thereto.
[55] In addition, the culture medium contains a carbon source. Examples of the carbon
source include saccharides and carbohydrates such as glucose, sucrose, lactose,
fructose, maltose, starch and cellulose, oils and fats such as soybean oil, sunflower oil,
castor oil and coconut oil, fatty acis such as palmitic acid, stearic acid and linoleic acid,
alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These
carbon sources may be present solely or in combination in the culture medium. As a
nitrogen source, an organic material such as peptone, yeast extract, meat juice, malt
extract, corn steep liquor, soybean, and wheat protein, or an inorganic nitrogen
compound such as urea, ammonium sulfate, ammonium chloride, ammonium
phosphate, ammonium carbonate and ammonium nitrate may be contained in the
culture medium. These nitrogen sources may be used solely or in combination. The
medium may contain potassium dihydrogen phosphate, potassium phosphate, or corre
sponding sodium salts as a phosphorous source. The medium may contain metallic
salts such as magnesium sulfate or iron sulfate. The culture medium may also contain
amino acids, vitamins and suitable precursors. The nutrients may be added in a batch
manner or a continous manner to the medium.
[56] A compound such as ammonium hydroxide, potassium hydroxide, ammonia,
phosphoric acid and sulfuric acid may be added in a suitable manner to the culture
medium during culturing to adjust the pH of the culture. In addition, during culturing,
an anti-foaming agent such as fatty acid polyglycol ester is used to suppress the
formation of foam. Further, in order to maintain the culture medium in an aerobic
condition, oxygen or oxygen-containing gas can be injected into the culture medium.
For an anaerobic or microaerobic condition, nitrogen, hydrogen, or carbon dioxide is
provided without aeration. The culture medium may be typically maintained at a tem
perature of from 27°C to 37°C and preferably at a temperature of from 30°C to 35°C.
As for the culture period, it may be maintained until the product of interest is obtained
in a desired amount, and preferably it ranges from 10 to 100 hours.
[57] For further collection and recovery of the OPS produced during the culturing step
from the culture medium, a suitable method well known in the art may be selected
depending on the type of culture, be it a batch, continuous or fed-batch culture.
[58] In the method of the present invention, step 2) addresses the reaction of the OPS of
step 1) with a sulfide in the presence of O-phosphoserine sulfhydrylase (OPSS) or a
microorganism expressing OPSS, to induce the conversion of O-phosphoserine into
cysteine or its derivatives.
[59]
[60] The sulfide may be provided in a liquid or gas form as well as in a solid form
typically used in the art, because of the difference in pH, pressure and/or solubility. So
long as it may be converted to a thiol group (SH), any sulfur compound such as sulfide
(S2 ) or thiosulfate (S20 3
2 ) may be used in the present invention. Preferably, Na2S,
NaSH, H2S, (NH4)2S, NaSH and Na2S20 3, all of which can provide a thiol group for
OPS, may be used. In the reaction, one thiol group is supplied to one OPS molecule to
afford one molecule of cysteine or a derivative thereof. In this enzymatic conversion, a
sulfide may be preferably added at a molar concentration 0.1 to 3 times and more
preferably 1 to 2 times as high as that of OPS used. In light of the economy, a thiol
group-providing sulfide and OPS are most preferably used at a molar ratio of 1:1. In
one embodiment of the present invention, Na2S was used as the source of sulfur. Na2S
was added at a molar concentration 1 to 3 times as high as that of OPS used in the
conversion reaction. Preferably, it is fed at a molar concentration twice as high as that
of OPS to effectively convert OPS into cysteine (Table 34).
[61] As used herein, the term "O-phosphoserine sulfhydrylase (OPSS)" refers to an
enzyme that catalyzes the transfer of a thiol group (SH) to OPS (O-phosphoserine) to
convert OPS into cysteine. The enzyme was first found in Aeropyrum pernix, My
cobacterium tuberculosis, and Trichomonas vaginalis (Mino K and Ishikawa K, FEBS
letters, 551: 133-138, 2003; Burns KE et al, J . Am. Chem. Soc, 127: 11602-11603,
2005).
[62] As used herein, the term "mutant" refers to a culture or an individual that shows an
inheritable or non-heritable alteration in phenotype. When used in conjunction with
OPSS, the term "mutant" is intended to mean an OPSS enzyme which is genetically
altered such that its activity can be effectively enhanced, compared to the wild-type.
[63] In the present invention, the OPSS mutant can be constructed by deleting, sub
stituting or adding a part of a nucleotide sequence encoding OPSS. According to one
embodiment of the present invention, an OPSS enzyme with enhanced activity was
prepared by deleting five C-terminal amino acid residues of the OPSS enzyme of My
cobacterium smegmatis.
[64] The OPSS mutant can be obtained in E. coli, widely used for enzyme expression,
using gene synthesis techniques based on codon optimization by which enzymes of
interest can be obtained in high yield. Alternatively, screening methods of useful
enzyme resources based on the bioinformatics of massive amounts of genetic in
formation about microorganisms may be used to obtain the OPSS mutant. In one em
bodiment of the present invention, OPSS enzymes that utilize OPS as a substrate to
synthesize cysteine were selected from various microbes by screening the homology of
amino acid sequences. In this regard, cell pellets obtained using a medium and culture
conditions that were suitable in the art were lyzed, followed by the purification of the
supernatant containing the enzyme to afford the OPSS enzyme (Table 26).
[65] In addition, a high-yield expression system was developed for obtaining the OPSS
enzyme economically. A pET vector employing a T7 promoter is well known in the
art. However, the present inventors developed an enzyme expression system, named
the CJl system (Korean Patent 10-0620092 Bl), instead of employing the typical
system. In one embodiment of the present invention, the expression levels of OPSS
between a pET system comprising a T7 promoter and the CJl system comprising a CJl
promoter were compared given the same conditions. As a result, the CJl system
showed a higher expression level of OPSS than the pET system. In addition, the overexpression
of OPSS required a low temperature (18°C) and a long period of time in the
pET system, but a high temperature (37°C) and a short period of time in the pCL-pCJl
system. Preferably, the pCL-pCJl system is used to obtain OPSS (Example 46).
[66] The enhancement of the enzyme activity may be achieved using various well-known
methods. For example, it can be performed by increasing the number of copies of a
gene encoding OPSS, using a strong promoter, or introducing a genetic mutation.
[67]
[68] Optimization of the enzymatic conversion of OPSS may be achieved using various
methods known in the art. For example, the optimization may be based on a full under
standing of the characteristics of OPSS, such as the optimal temperature and pH, in
hibition against substrates, substrate concentration, heat stability, etc. In addition, the
optimization may be determined by optimal conditions for the enzymatic conversion,
such as the optimal OPSS concentration, the optimal balances of the substrates used in
terms of concentrations, a preference for sulfur compounds providing SH for the
enzymatic conversion, a preference for certain buffers, the influence of ions generated,
and cofactors and their optimal concentrations.
[69] In one embodiment of the present invention, the OPSS enzyme obtained using the
above-mentioned method was characterized and on the basis of the determined charac
teristics, an economically beneficial enzymatic conversion process that has a high
conversion rate of cysteine from OPS, with the guarantee of enzyme stability, was
developed. In the enzymatic conversion process, the reaction temperature can be set
from 37°C to 80°C. In detail, Ape-OPSS (SEQ ID NO: 12), belonging to Archea,
exhibits increased enzymatic activity at 60°C compared to 37°C, and the enzyme itself
is highly stable to heat, with optimal reactivity at 60°C. On the other hand, Msm-T
(SEQ ID NO: 10) exhibits optimal activity at 37°C and is relieved the activity to heat
treatment at 60°C. The OPSS enzyme was observed to have enzymatic activity over a
pH range of 6.0 to 10.0. Ape-OPSS showed optimal activity at pH 7.4. With the ap
pearance of optimal activity at a pH of from 8.0 to 9.0, Msm-T showed stability over a
wider pH range, compared to Ape-OPSS (Tables 28 and 31, and FIGS. 2 and 3).
[70] As a cofactor, 0.001 - 2 mM PLP (pyridoxal-5' -phosphate) or 0.001 - 100 mM DTT
may be used in the enzymatic conversion. In one embodiment of the present invention,
the cysteine conversion rate was 2.3-fold increased in the presence of 25 mM DTT or
0.2 mM PLP. As such, treatment with DTT or PLP brought about an improvement in
the cysteine conversion rate of step 2). The addition of the cofactor was set to a
reasonable level in consideration of the increased production cost and the increased
conversion rate (Table 30).
[71] The reaction conditions for OPSS may vary depending on the kinds and con
centration of the OPS used. In one embodiment of the present invention, pure OPS
(commercially available), OPS purified from the culture prepared in step 1), and the
OPS-containing culture of step 1) were used under various conditions to provide the
optimal conversion rates. As a result, the cysteine conversion rate varied depending on
the kind and concentration of OPSS and the reaction temperature and the kind and con
centration of OPS (FIGS. 5 and 6, and Table 35).
[72]
[73] The method of the present invention may further comprise isolating and purifying the
cysteine produced in step 2). After the enzymatic conversion, cysteine can be isolated
and purified from the culture medium using a method well known in the art.
[74]
[75] Those skilled in the art may chemically synthesize cysteine derivatives from cysteine
using a well known method. Cysteine may be readily reacted with an acetylation agent
to give NAC (N-acetylcysteine) and with haloacetic acid under basic conditions to give
SCMC (S-carboxymetylcysteine). These cysteine derivatives are used as materials in
medicines that treat coughs, bronchitis, bronchial asthma, and sore throat.
[76]
[77] In the present invention, the OPS broth obtained through microbial fermentation is
used as a substrate for synthesizing cysteine. The OPS broth obtained by microbial fer
mentation has economical advantages over commercially available pure OPS in that
the OPS broth can be used without having to be additionally purified and the cofactor
PLP necessary for the conversion can be obtained from the fermented culture.
[78] In one embodiment of the present invention, a conversion process was developed
which ensures a cysteine conversion rate of as high as 80% when 50 mg/ml Msm-T was
used under reaction conditions of a 50 mM OPS broth or a 60 mM purified OPS broth,
100 mM Na2S or 120 mM Na2S, and 0.2 mM PLP. It should be appreciated to those
skilled in the art that the enzymatic conversion using highly active enzymes can easily
be optimized and scaled up.
[79]
[80] In accordance with another aspect thereof, the present invention provides a r e
combinant microorganism which is reduced the activity of SerB for the production of
OPS. In one embodiment, the recombinant microorganism shows an enhancement of
serine feedback-resistant serA or serC or deletion of at least one selected from among
PhnC/PhnD/PhnE alkylphosphonate ABC transporter (phnCDE operon), alkaline
phosphatase (phoA) and acid phosphatase (aphA). Preferably, the recombinant mi
croorganisms for the production of OPS are the microorganism deposited under
accession No. KCCMl 1103P or KCCMl 12 12P. More preferably, the recombinant mi
croorganism for the production of OPS is the microorganism deposited under
accession No. KCCMl 1103P.
[81]
Mode for the Invention
[82] A better understanding of the present invention may be obtained through the
following examples which are set forth to illustrate, but are not to be construed to limit
the present invention.
[83]
[84]
[85]
[86] EXAMPLE 1: Preparation of Phosphoserine Phosphatase (serB) deficient
Corynebacterium Strain
[87]
[88] Corynebacterium glutamicum 13032 was modified by deleting the serB gene (SEQ
ID NO: 13, EC 3.1.3.3) encoding phosphoserine phosphatase, which catalyses the
synthesis of L-serine from O-phosphoserine, therefrom. To this end, a fragment for inactivation
of serB was constructed. In this regard, primers were designed for the
preparation of the recombinant strain 13032-AserB of the present invention. First, the
serB sequence of Corynebacterium glutamicum 13032 was obtained with reference to
the data of the NIH GenBank, and primers SEQ ID NOS: 22 to 27 were synthesized on
the basis of the serB sequence. For the site- specific gene disruption, a pDC vector
which cannot replicate in Corynebacterium glutamicum was employed. A pDC-AserB
plasmid in which the open reading frame of serB was internally disrupted was con
structed and adopted for the preparation of a site-specific serB gene deletion in
Corynebacterium glutamicum mutant strain. The internal gene distruption of the pDCAserB
was generated by crossover PCR using primer pairs of SEQ ID NOS: 22 and 23
and SEQ ID NOS: 24 and 25, with the genomic DNA of Corynebacterium glutamicum
ATCC 13032 serving as a template, and introducing the PCR product into a pDC
vector. The resulting recombinant plasmid was transformed into wild-type
Corynebacterium glutamicum by electroporation (van der Rest et al. 1999). The
plasmid was introduced into the chromosome by primary recombination (crossing
over), followed by secondary recombination (crossing over) to excise the original serB
from the chromosome.
[89] After completion of the secondary recombination, the Corynebacterium glutamicum
transformants containing the deletion mutation of serB was analyzed by diagnostic
PCR using a pair of gene-specific primers SEQ ID NOS: 26 and 27. The recombinant
strain was named CBO1-0047.
[90]
[91] EXAMPLE 2 : Assay for O-Phosphoserine Productivity in the Phosphoserine
Phosphatase deficient Corynebacterium Strain
[92]
[93] The mutant strain CBOl-0047, resulting from the deletion of serB from
Corynebacterium glutamicum 13032, which was anticipated to accumulate Ophosphoserine,
was spread over BHIS plates and incubated overnight in a 30°C
incubator. Afterwards, the colonies appearing on the BHIS plates were inoculated in 25
mL of a titer medium shown in Table 1 using a platinum loop and then incubated at
30°C for 48 hours with shaking at 200 rpm. The results are summarized in Table 2,
below.
[94]
[95] Table 1
[Table 1]
[96]
[97]
[99] The CBO1-0047 strain was observed to grow very slowly in the titer medium. This
growth retardation was not improved even upon the addition of an L-glycine
supplement. However, the growth was increased in the presence of L-serine, but a
slight increase in the production of O-phosphoserine compared to the wild-type was
observed. The results are summarized in Table 3, below.
[100]
[101] Table 3
[Table 3]
[102]
[103] EXAMPLE 3 : Construction of mutated Phosphoglycerate Dehydrogenase
(SerA*) Gene derived from Corynebacterium
[104]
[105] The Corynebacterium glutamicum-dem/ed genes serA*(E235K) (SEQ ID NO: 14)
and serA*(197A) (SEQ ID NO: 15) were constructed, which encode respective
mutants of 3-phosphoglycerate dehydrogenase, an enzyme catalyzing the synthesis of
3-phosphohydroxypyruvate from 3-phosphoglycerate. The mutants were reported to be
feedback resistant (FBR) to serine (Peters-Wendisch P et al., Appl. Microbiol.
BiotechnoL, 60: 437-441, 2002; EP0943687B). serA*(E235K) was obtained by sewing
PCR on the genomic DNA of ATCC13032 using primers of SEQ ID NOS: 28 to 3 1
while serA*(197A) was constructed by PCR using pairs of primers of SEQ ID NOS:
28 to 32. The PCR products thus obtained were inserted into respective T vectors to
construct recombinant vectors called Tblunt-serA*(E235K) and Tblunt-serA*(197A).
Subsequently, the two vectors were treated with restriction enzymes EcoRV and Xbal
to give two DNA fragments serA*(E235K) and serA*(197A). These fragments were
inserted to respective pECCGl 17-Pcj7-GFP-terminator vectors which had been
disgested with the same restriction enzymes. As a result, two recombinant vectors
pECCG117-Pcj7-serA*(E235K), and pECCG117-Pcj7- serA*(197A) were obtained.
[106]
[107] EXAMPLE 4 : Preparation of serA* overexpressing Corynebacterium Strain
and Assay for O-Phosphoserine Productivity
[108]
[109] The two Corynebacterium-dem/ed FBR-serA* plasmids constructed in Example 3
were introduced into Corynebacterium glutamicum CBO1-0047. To evaluate Ophosphoserine
productivity, the transformants were spread over BHIS plates and
incubated overnight at 30°C. Afterwards, the colonies appearing on the BHIS plates
were inoculated in 25 mL of a titer medium shown in Table 1 additionally contained 2
g/L L-serine using a platinum loop and then incubated at 30°C for 48 hours with
shaking at 200 rpm. The results are summarized in Table 4, below.
[HO]
[111] Table 4
[Table 4]
[112]
[113] In the Corynebacterium glutamicum strains transformed with the corynebacterium -
derived FBR-serA*, as shown in Table 4, The accumulations of O-phosphoserine at a
concentration of from 0.1 to 0.3 g/L were observed.
[114]
[115]
[116]
[117] EXAMPLE 5 : Preparation of E. coli Strain having the reduced activity of Phosphoserine
Phosphatase (SerB)
[118]
[119] E. coli was modified by deleting the serB gene (SEQ ID NO: 16) encoding phosphoserine
phosphatase, which catalyses the synthesis of L-serine from Ophosphoserine,
therefrom. The deletion mutant E. coli K12 was prepared using the
one-step inactivation method (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci.,
97: 6640-6645, 2000) to delete an antibiotic-resistant maker gene. To prepare the serB
deletion strain, first, PCR was performed on a pKD3 plasmid (Datsenko KA and
Wanner BL, Proc. Natl. Acad. Sci., 97: 6640-6645, 2000; GenBank No. AY048742)
using a pair of primers of SEQ ID NOS: 33 and 34. The PCR product was (indroduced
into competent cells of pKD46 containing E. coli K12 (Datsenko KA and Wanner BL,
Proc. Natl. Acad. Sci., 97: 6640-6645, 2000; GenBank No. AY048746) by electroporation.
Thereafter, strains that showed resistance to chloramphenicol were subjected
to PCR to confirm the deletion of serB, and then transformed with pCP20 (Datsenko
KA and Wanner BL, Proc. Natl. Acad. Sci., 97: 6640-6645, 2000) to remove the an
tibiotic-resistant marker. The resulting mutant strain was named CA07-0012.
[120] In addition, the initiation codon of serB was modified to lower phosphoserine
phosphatase activity as follows. The wild-type serB gene with ATG as an initiation
codon was obtained by PCR with the genomic DNA of E. coli W31 10 serving as a
template. A mutant serB with CTG as an initiation codon was constructed by sewing
PCR. A pair of primes of SEQ ID NOS: 35 and 36 was used in the PCR for amplifying
the wild-type serB while pairs of primers of SEQ ID NOS: 37 to 38 were employed for
PCR amplification of the mutant serB. The PCR products was treated with Hindlll and
cloned into pccBACl (Epicentre) at the Hindlll restriction site to construct
pccBACl-Pself-ATG-serB, and pccBACl-Pself-CTG-serB respectively. The wildtype
and the mutant serB vector was introduced into CA07-0012 to compare the phos
phoserine phosphatase activity.
[121]
[122] EXAMPLE 6 : Assay of Strain having the reduced activity of SerB for OPhosphoserine
Productivity
[123]
[124] The phosphoserine phosphatase deficient mutant strain CA07-0012 that was an
ticipated to accumulate O-phosphoserine, was spread over LB plates and incubated
overnight in a 33°C incubator. Afterwards, the colonies appearing on the LB plates
were inoculated in 25 mL of a titer medium shown in Table 5 using a platinum loop
and then incubated at 33°C for 48 hours with shaking at 200 rpm. The results are
summarized in Table 6, below.
[125]
[126] Table 5
[Table 5]
[127]
[128]
[129]
[130] To enhance the growth and O-phosphoserine productivity thereof, CA07-0012 was
cultured for 48 hours in the titer medium of Table 5 additionally contained 1 g/L Lglycine.
The results are summarized in Table 7, below.
[131]
[132] Table 7
[Table 7]
[133]
[134] As shown in Table 7, the addition of L-glycine to the culture medium allowed the
strain to increase the growth rate and the O-phosphoserine productivity.
[135]
[136] EXAMPLE 7 : Construction of the Vector Harvoring the Mutated Phosphoglycerate
Dehydrogenase (SerA*) Gene derived from E. coli
[137]
[138] The E. li-derived genes serA*(G336V) (SEQ ID NO: 18), serA*(G336V, G337V)
(SEQ ID NO: 19), and serA*(G336V, R338G) (SEQ ID NO: 20) encoding respective
mutants of 3-phosphogly cerate dehydrogenase, an enzyme catalyzing the synthesis of
3-phosphohydroxypyruvate from 3-phosphogly cerate were constructed. The mutants
were reported to be feedback resistant (FBR) to serine (Grant GA, Xu XL and Hu Z,
Biochem., 39: 7316-7319, 2000; Grant GA, Hu Z and Xu XL, J. Biol. Chem., 276:
17844-17850, 2001). The introduction of the mutant genes into the chromosome of E.
coli was carried out using the sewing PCR method. The DNA fragments containing
mutations were prepared using following primers.
[139] Primers of SEQ ID NOS: 39 and 4 1 were used commonly in SerA* gene. To
introduce mutations into the serA gene, PCR was performed with a pair of primers of
SEQ ID NOS: 42 and 43 for serA*(G336V), with a pair of primers of SEQ ID NOS:
44 and 45 for serA*(G336V, G337V), and with a pair of primers of SEQ ID NOS: 46
and 47 for serA*(G336V, R338G). The primers were synthesized on the basis of in
formation about the K12 W31 10 gene (GenBank accession number AP 003471) and its
neighboring nucleotide sequences, registered in the NIH GenBank.
[140]
[141] EXAMPLE 8 : Cloning of E. coli-Derived serA Gene, serA* Gene, and
3-Phosphoserine Aminotransferase (serC) Gene
[142]
[143] serA(SEQ ID NO: 17, EC 1.1.1.95), serC(SEQ ID NO: 21, EC 2.6.1.52),
serA*(G336V), serA*(G336V, G337V) and serA*(G336V, R338G) were cloned as
follows. serA and serC were obtained by performing PCR on the genomic DNA of E.
coli W3110 while serA*(G336V), serA*(G336V, G337V), and serA*(G336V, R338G)
were constructed by PCR with the DNA fragments of Example 7 serving as templates.
PCR primers were SEQ ID NOS: 48 and 49 for serA and SEQ ID NOS: 50 and 5 1 for
serC. After treatment with EcoRV and Hindll, the PCR products were cloned into the
recombinant vector pCL-Prmf , constructed by inserting the E. coli rmf promoter into
the pCL1920 vector (GenBank No AB236930) to produce respective recombinant
vectors named pCL-Prmf-serA, pCL-Prmf-serC, pCL-Prmf-serA*(G336V), pCLPrmf-
serA*(G336V, G337V), and pCL-Prmf-serA*(G336V, R338V) respectively.
[144] In addition, plasmids in which serA, one of the three serA mutants, and/or serC form
an operon, that is, pCL-Prmf-serA-(RBS)serC, pCL-Prmf-serA*(G336V)-(RBS)serC,
pCL-Prmf-serA*(G336V, G337V)-(RBS)serC, and pCL-Prmf-serA*(G336V,
R338V)-(RBS)serC were constructed. In this regard, an (RBS)serC fragment was
obtained using primers of SEQ ID NOS: 5 1 and 52 and cloned at a Hindlll site into
pCL-Prmf-serA, pCL-Prmf-serA*(G336V), pCL-Prmf-serA*(G336V, G337V), and
pCL-Prmf-serA*(G336V, R338V).
[145]
[146] EXAMPLE 9 : Preparation of E. coli-Derived serA, serA* and serC enhanced
Strains and Assay for O-Phosphoserine Productivity
[147]
[148] The eight plasmids constructed in Example 8 were transformed into CA07-0012 and
the resulting recombinant strains were assayed for the productivity of Ophosphoserine.
Each strain was spread over LB plates and incubated overnight at 33°C.
Afterwards, colonies appearing on the LB plates were inoculated into 25 mL of titer
media of Table 8 and cultured at 33°C for 48 hours with shaking at 200 rpm. The
results are summarized in Table 9, below.
[149]
[150] Table 8

[Table 9]
[153]
[154] As apparent from the data of Table 9, the E. coli CA07-0012 strain increased in the
productivity of O-phosphoserine when it was transformed with serA, and the pro
ductivity of O-phosphoserine was increased to a greater extent upon the introduction of
one of the three serA* mutants. The strains in which serA, or one of three serA*
mutants and serC that were activated simultaneously showed higher productivity of Ophosphoserine
than did those in which there was the sole activation of serA or serA*.
The highest productivity of O-phosphoserine was detected in a strain in which the
mutant serA* and serC were activated simultaneously.
[155]
[156] EXAMPLE 10: Preparation of PhnC/PhnD/PhnE Alkylphosphonate ABC
Transporter (phnCDE operon) deficient E. coli Strain
[157] In E. coli, PhnC/PhnD/PhnE alkylphosphonate ABC transporter is reported to
translocate O-phosphoserine into the cytoplasm (Wanner BL and Metcalf WW. FEMS
Microbiol. Lett, 15:133-139, 1992). The phnCDE operon encoding a PhnC/
PhnD/PhnE alkylphosphonate ABC transporter protein was deleted from a serB
deletion strain to prepare the CA07-0016 strain. For the deletion of phnCDE, a pair of
primers of SEQ ID NOS: 53 and 54 were employed. The deletion was performed in a
manner similar to that of Example 5.
[158] In addition, pCL-Prmf-serA*(G336V)-(RBS)serC, constructed in Example 8, was in
troduced into CA07-0016.
[159]
[160] EXAMPLE 11: Assay of phnCDE operon deficient E. coli Strain for OPhosphoserine
Productivity
[161]
[162] The strains CA07-0016 and CA07-0016/pCL-Prmf-serA*(G336V)-(RBS)serC,
prepared in Example 10, were evaluated for O-phosphoserine productivity. Each strain
was spread over LB plates or LB(spectinomycine) plates and incubated overnight at
33°C. Afterwards, colonies appearing on the LB plates or the LB (spectinomycine)
plates were inoculated into 25 mL of titer media of Table 8 using a platinum loop and
cultured at 33°C for 48 hours with shaking at 200 rpm. The results are summarized in
Table 10, below.
[163]
[164] Table 10
[Table 10]
[165]
[166] As seen in Table 10, the phnCDE operon deletion strain showed only a slight
increase in O-phosphoserine productivity.
[167]
[168] EXAMPLE 12: Preparation of Alkaline Phosphatase (phoA), Acid Phosphatase
(aphA) deficient E. coli Strain
[169]
[170] The phosphoserine phosphatase deletion E. coli strain was additionally deleted the
phoA gene coding for alkaline phosphatase and the aphA gene coding for acid
phosphatase. A DNA fragment for use in deleting phoA was obtained by performing
PCR on a pkD3 plasmid with a pair of primers of SEQ ID NOS: 55 and 56. On the
other hand, a DNA fragment for use in deleting aphA was obtained using a pair of
primers of SEQ ID NOS: 57 and 58 in the same manner. Each deletion strain was
prepared in the same manner as in Example 5. The strain which deleted both phoA and
aphA was prepared by electroporating the DNA fragment for aphA deletion into a
competent cell of the phoA deletion strain which had been transformed again with
pKD46. Thereafter, the transformants which were resistant to chloramphenicol were
subjected to PCR to confirm the deletion of aphA, and then transformed with pCP20 to
remove the antibiotic-ressitant marker. The resulting mutant strains and their
genotypes are summarized in Table 11, below.
[171]
[172] Table 1 1
[Table 11]
[173]
[174] To each deletion strain, pCL-Prmf-serA*(G336V)-(RBS)serC, constructed in
Example 8, was introduced in the same manner as in Example 10.
[175]
[176] EXAMPLE 13: Assay of Alkaline Phosphatase (phoA), Acid Phosphatase
(aphA) deficient E. coli Strain for Ability to Degrade O-phosphoserine
[177]
[178] The strains prepared in Example 12 were assayed for the productivity of OPS and the
incapability of degrading OPS. Each strain was spread over LB plates or LB
(spectinomycine) plates and incubated overnight at 33°C. Afterwards, colonies
appearing on the LB plates or the LB (spectinomycine) plates were inoculated into 25
mL of titer media of Table 8 using a platinum loop and cultured at 33°C for 72 hours
with shaking at 200 rpm. The results are summarized in Table 12, below. Incapability
of degrading OPS was evaluated by a change in phosphate ion level as determined by
phosphate ion analysis.
[179]
[180] Table 12
[Table 12]
[181]
[182] As seen in Table 12, the aphA deletion strain showed an abnormal growth
phenomenon whereas the strains which lacked phoA or both phoA and aphA
somewhat increased in O-phosphoserine productivity and decreased in the capability of
degrading O-phosphoserine. On the other hand, the strain in which neither phoA nor
aphA was deleted degraded the O-phosphoserine accumulated for 72 hours, with the
concomitant increase of the P0 4 level.
[183]
[184] EXAMPLE 14: Preparation of phnCDE operon, phoA and aphA deficient
Strains
[185]
[186] The serB deficient strain (CA07-0012) was modified to further delete phnC/
phnD/phnE alkylphosphonate ABC transporter-encoding phnCDE, alkaline
phosphatase-encoding phoA, and acid phosphatase-encoding aphA. The strains thus
prepared are given in Table 13, below. The one-step inactivation method described in
Example 5 was employed to prepare the deletion mutants.
[187]
[188] Table 13
[Table 13]
[189]
[190] Into each of the deletion strains, the pCL-Prmf-serA*(G336V)-(RBS)serC, con
structed in Example 8, was introduced in the same manner as in Example 10.
[191]
[192] EXAMPLE 15: Assay of phnCDE operon, phoA and aphA deficient E. coli
Strains for O-Phosphoserine Productivity
[193]
[194] The strains prepared in Example 14 were assayed for OPS productivity. Each strain
was spread over LB plates or LB (spectinomycine) plates and incubated overnight at
33°C. Afterwards, colonies appearing on the LB plates or the LB (spectinomycine)
plates were inoculated into 25 mL of titer media of Table 8 using a platinum loop and
cultured at 33°C for 72 hours with shaking at 200 rpm. The results are summarized in
Table 14, below.
[195]
[196] Table 14
[Table 14]
[197]
[198] CA07-0020 and CA07-0022 were found to have increased OPS productivity and
decreased ability to degrade O-phosphoserine, compared to CA07-0012. This property
was also detected in the strains transformed further with pCLPrmf-
serA*(G336V)-(RBS)serC.
[199]
[200] EXAMPLE 16: Preparation of E. coli Mutants deficient of phnCDE operon,
phoA, and aphA Genes and Having Substitutition of Phosphoglycerate Dehy¬
drogenase (serA*)
[201]
[202] In CA07-0022, 3-phosphoglycerate dehydrogenase-encoding serA was substituted
with serA*(G336V), serA*(G336V, G337V), or serA*(G336V, R338G), all being
reported to have feedback resistance to serine, on the chromosome, as follows.
[203] To introduce mutations into the serA gene on the chromosome, vectors were con
structed as follows. PCR was performed with a pair of primers of SEQ ID NOS: 40
and 4 1 on serA*(G336V), serA*(G336V, G337V), and serA*(G336V, R338G),
prepared in Example 7. After treatment with both Sacl and BamHI, the PCR products
thus obtained were cloned into pSG76C at the Sacl and BamHI site. The resulting re
combinant vector was transformed into E. coli BW which was then spread over LB
plates. The colonies appearing on the plates were subjected to base sequencing, and the
transformants into which mutations were introduced were selected. From them,
plasmids were prepared using a typical miniprep method. According to the introduced
mutations, the plasmids were named pSG76C-serA*(G336V), pSG76C-serA*(G336V,
G337V) and pSG76C-serA*(G336V, R338G).
[204] Each of the E. coli mutants was prepared as described previously (Posfai G, Kolisnychenko
V, Bereczki Z and Blattner FR, Nucleic Acids Res. 27: 4409-4415, 1999),
and the antibiotic-resistant marker gene was removed from them. To prepare the
serA*(G336V) mutant, pSG76C-serA*(G336V) was introduced into a competent cell
of CA07-0022 by electroporation. The strains resistant to chlorampenicol were
subjected to PCR to confirm the introduction of serA*(G336V). The strain was
transformed with pST76-ASceP (Posfai G, Kolisnychenko V, Bereczki Z and Blattner
FR, Nucleic Acids Res. 27: 4409-4415, 1999) to remove the antibiotic -resistant marker
gene. The resulting strain was named CA07-0022 serA*(G336V). The CA07-0022
serA*(G336V) strain was transformed with pSG76C-serA*(G336V, G337V) and
pSG76C-serA*(G336V, R338G) in a similar manner to give serA*(G336V, G337V)
and serA*(G336V, R338G) mutants, named CA07-0022 serA*(G336V, G337V) and
serA*(G336V, R338G), respectively.
[205]
EXAMPLE 17: Assay of E. coli Mutants deficient of phnCDE operon, aphA,
and aphA Genes and Having Substitutition of Phosphoglycerate Dehydrogenase
(serA*) for O-Phosphoserine Productivity
The strains prepared in Example 16 were assayed for O-phosphoserine productivity
Each strain was spread over LB plates or LB (spectinomycine) plates and incubated
overnight at 33°C. Afterwards, colonies appearing on the LB plates or the LB
(spectinomycine) plates were inoculated into 25 mL of titer media of Table 8 using a
platinum loop and cultured at 33°C for 48 hours with shaking at 200 rpm. The results
are summarized in Table 15, below.
] Table 15
[Table 15]
]
] The strains in which serA had been altered to sereine feedback-resistant genes
showed somewhat decreased growth rates, but an increase in O-phosphoserine pro
ductivity.
]
] EXAMPLE 18: Preparation of Mutant E. coli Strains deficient of phnCDE
operon, phoA and aphA and HavingSubstituted Phosphoglycerate Dehydrogenase
(serA*) and enhanced 3-Phosphoserine Aminotransferase and Assay for OPhosphoserine
Productivity
]
] Into the strains prepared in Example 16, that is, CA07-0022 serA*(G336V),
CA07-0022 serA*(G336V, G337V), and CA07-0022 serA*(G336V, R338G) was in
troduced in the plasmid prepared in Example 8, that is, pCL-Prmf-serC. The resulting
mutants were evaluated for O-phosphoserine productivity in the same manner as in
Example 9. The results are summarized in Table 16, below.
[217]
[218] Table 16
[Table 16]
[219]
[220] As seen in Table 16, the serC-activated strains were found to be improved in Ophosphoserine
productivity. This phenomenon was more apparent in the strain in
which serA was modified into a serine feedback-resistant gene.
[221]
[222] EXAMPLE 19: Pyrimidine Nucleotide Transhydrogenase (PntAB)-enhanced
Strain and construction of Glutamate Dehydrogenase (GdhA) containing vector
[223]
[224] To prepare a strain in which pntAB encoding for pyrimidine nucleotide transhy
drogenase is upregulated, the pntAB promoter was changed with a trc promoter using a
mutant loxP system (Arakawa H et al.,BMC Biotechnol. 1: 7, 2001). In this regard,
PCR was performed on the pmlox-trc(ref) plasmid using a pair of primers of SEQ ID
NOS: 59 and 60, and the PCR product thus obtained was introduced into a competent
cell of CA07-0022 serA*(G336V) anchoring pKD46 by electroporation. The transformants
which showed resistance to chloramphenicol were subjected to PCR to
confirm the replacement of the promoter, followed by transformation with pJW168(Le
Borgne S et al, Methods Mol Biol. 267: 135-43, 2004) to remove the antibioticresistant
marker gene. The resulting strain was named CA07-0022 serA*(G336V)
P(trc)-pntAB. The primers used for the PCR were designed on the basis of the in
formation about the K12 W31 10 gene (GenBank accession number AP002223,
AP002224) and its neighboring nucleotide sequences, registered in the NHI GenBank.
[225] The glutamate dehydrogenase-encoding gdhA gene was amplified using PCR with a
pair of primers of SEQ ID NOS: 6 1 and 62 to give a single polynucleotide. Both the
primers of SEQ ID NOS: 6 1 and 62 have the restriction enzyme site Hindlll. The
primers were designed on the basis of the information about the K12 W31 10 gene
(GenBank accession number AP 002380) and its neighboring nucleotide sequences,
registered in the NHI GenBank.
[226] PCR started with denaturation at 94°C for 3 min and proceeded with 25 cycles of de
naturing at 94°C for 30 sec, annealing at 56°C for 30 sec and extending at 72°C for 2
min, followed by extending at 72°C for 7 min. As a result, a 1714 bp-long polynu
cleotide was obtained. After treatment with Hindlll, the PCR product was cloned into
pCClBAC at the Hindlll site and introduced into E. coli DH5a which was then spread
over LB plates. Base sequencing allowed the selection of the developed colonies that
had no mutations in their gdhA gene. The plasmid was isolated using a typical
miniprep method and named pCClBAC-P(native)-gdhA.
[227]
[228] EXAMPLE 20: Introduction of pntAB and gdhA into OPS-Producing Strain
and Assay for OPS Productivity
[229]
[230] To prepare an OPS-producing strain in which pntAB and gdhA were upregulated, the
CA07-0022 serA*(G336V) strain or the CA07-0022 serA*(G336V) P(trc)-pntAB
strain was transformed with pCL-P(trc)-serA*(G336V)-serC and
pCClBAC-P(native)-gdhA individually or in combination, as shown in the following
table. Each transformant was incubated overnight at 33°C on LB plates. The colonies
were inoculated into the 25 mL of titer media of Table 8 using a platinum loop and
cultured at 33°C for 48 hours with shaking at 200 rpm.
[231]
[232] Table 17
[Table 17]
[233]
[234] As seen in Table 17, the strain was improved in O-phosphoserine productivity when
pntAB was upregulated therein. The upregulation of both pntAB and gdhA brought
about a bigger increase in O-phosphoserine productivity, as compared to the control.
Hence, pntAB and gdhA are understood to play an important role in the production of
OPS.
[235]
[236] EXAMPLE 21: Construction of Vectors Carrying Genes Encoding E. coli OAcetylserine/
Cysteine Efflux Protein (ydeD), O-Acetylserine/Cysteine Efflux
Permease (yfiK), Homoserine/Homoserine Lactone Efflux Protein (rhtB),
Threonine/Homoserine Efflux Protein (rhtC), Arsenite/Antimonite Transporter
(asrB), and Leucine/Isoleucine/Valine Transport Subunit (livHM)
[237]
[238] The release of the produced O-phosphoserine out of the cell requires a suitable export
factor none of which have, however, been reported previously. In this context, six
genes, that is, O-acetylserine/cysteine efflux protein-encoding ydeD, Oacetylserine/
cysteine efflux permease-encoding yfiK (Franke I, Resch A, Dassler T,
Maier T and Bock A, J. Bacteriology, 185: 1161-166, 2003), homoserine/homoserine
lactone efflux protein-encoding rhtB, threonine/homoserine efflux protein-encoding
RhtC, arsenite/antimonite transporter-encoding asrB, and leucine/isoleucine/valine
transport subunit-encoding livHM were selected from among the previously reported
variety of transporter genes, and were cloned and evaluated.
[239] Each gene was obtained by performing PCR on the genomic DNA of E. coli W31 10,
with a pair of primers of SEQ ID NOS: 63 and 64 for ydeD, with a pair of primers of
SEQ ID NOS: 65 and 66 for yfiK, with a pair of primers of SEQ ID NOS: 67 and 68
for rhtB, with a pair of primers of SEQ ID NOS: 69 and 70 for rhtC, with a pair of
primers of SEQ ID NOS: 7 1 and 72 for asrB, and with a pair of primers of SEQ ID
NOS: 73 and 74 for livHM. After treatment with EcoRV and Hindlll, each of the PCR
products thus obtained was cloned at the EcoRV and Hindlll site into the pCLPrmf-
GFP, to give recombinant vectors, named pCL-Prmf-ydeD, pCL-Prmf-yfiK,
pCL-Prmf-rhtB, pCL-Prmf-rhtC, pCL-Prmf-arsB, and pCL-Prmf-livHM.
[240]
[241] EXAMPLE 22: introduction of Vectors Carrying Genes Encoding E. coli YdeD,
YfiK, RhtB, RhtC, AsrB, livHM into O-Phosphoserine-Producing Strain and
Assay for O-Phosphoserine Productivity
[242]
[243] The CA07-0022 serA*(G336V) strain was transformed with the six plasmids con
structed in Example 2 1 and evaluated for O-phosphoserine productivity in the same
manner as in Example 9. The results are given in Table 18, below.
[244]
[245] Table 18
[Table 18]
[246]
[247] As shown in Table 18, the strains transformed with ydeD, mdtG or livHM exhibited
decreased growth rate and decreased OPS productivity whereas transformation with
yfiK, rhtB or rhtC increased growth rate and OPS productivity (Table 18).
[248]
[249] EXAMPLE 23: Preparation of Phosphoglycerate Mutase (gpml, gpmA and
gpmB) deficient Strain
[250]
[251] gpml, gpmA, and gpmB, each encoding phosphoglycerate mutase, were deleted
solely or in combination from CA07-0022 serA*(G336V) to produce the mutant
strains named CA07-0022 serA*(G336V)AgpmI, CA07-0022 serA*(G336V)AgpmA,
CA07-0022 serA*(G336V)AgpmB, CA07-0022 serA*(G336V)AgpmIAgpmA,
CA07-0022 serA*(G336V)AgpmAAgpmB, and CA07-0022
serA*(G336V)AgpmIAgpmAAgpmB, respectively. The gpmA- and gpmB-deletion
strains were prepared in a manner similar to that of Example 5, using a pair of primers
of SEQ ID NOS: 75 and 76 for gpmA and a pair of primers of SEQ ID NOS: 81 and 82
for gpmB. For the construction of a gpml deletion strain, as described in Example 16, a
gpml mutation containing a stop codon was introduced using pSG76C. A gpml mutant
containing a stop codon was amplified by sewing PCR using primers of SEQ ID NOS:
77 to 81, with the genomic DNA of K12 W31 10 serving as a template, and cloned into
pSG76 at the SacI/BamHI site.
[252]
[253] EXAMPLE 24: Assay of gpml, gpmA and gpmB deficient Strains for OPS Pro¬
ductivity
[254]
[255] The strains prepared in Example 23 were evaluated for OPS productivity in the same
manner as in Example 9. The results are summarized in Table 19, below.
[256]
[257] Table 19
[Table 19]
Strain OD562nm Sugar OPS(g/L)
consumed
(g L)
CA07-0022 serA*(G336V) 23 40 2.4
CA07-0022 serA*(G336V)AgpmI 22 38 2.5
CA07-0022 serA*(G336V)AgpmA 20 34 2.8
CA07-0022 serA*(G336V)AgpmB 20 34 2.7
CA07-0022 serA*(G336V)AgpmIAgpmA 19 32 2.6
CA07-0022 serA*(G336V)AgpmAAgpmB 2 1 35 3.3
[258]
[259] As can be seen in Table 19, when each of gpml, gpmA and gpmB was deleted and
the others not deleted, the sugar consumption of the mutant strains decreased, but their
OPS productivity increased, compared to the mother strain. Particularly, the strain
devoid of both gpmA and gpmB had similar sugar consumption, but increased OPS
productivity, compared to the strains devoid of either gpmA or gpmB. Therefore, the
deletion of gpml, gpmA and gpmB is understood to produce an increased amount of
3-phosphoglycerate, a precursor of OPS, thus leading to increased OPS production.
[260]
[261] EXAMPLE 25: Preparation of 2-Amino-3-ketobutyrate CoA Ligase (kbl), LSerine
Deaminase I (sdaA) deficient Strains
[262]
[263] The kbl gene coding for 2-amino-3-ketobutyrate CoA ligase and the sdaA gene
coding for L-serine deaminase I were deleted from CA07-0022 serA*(G336V) to yield
CA07-0022 serA*(G336V) Akbl, and CA07-0022 serA*(G336V) AsdaA, respectively.
The kbl- and the sdaA-deletion strain were prepared in a manner similar to that of
Example 5, using a pair of primers of SEQ ID NOS: 83 and 84 for kbl and a pair of
primers of SEQ ID NOS: 85 and 86 for sdaA.
[264]
[265] EXAMPLE 26: Assay for OD and Sugar Consumption of kbl/sdaA deficient
Strains According to Glycine concentration
[266]
[267] The strains prepared in Example 25 were evaluated for OD, sugar consumption, and
O-phosphoserine productivity when they were incubated in the same medium
condition as described in Table 8 of Example 9, with the exception that glycine was
used in an amount of from 0 to 2.5 g/L.
[268]
[269] Table 20
[Table 20]
[270]
[271] As can seen in Table 20, the OD and the rate of sugar consumption in all three of the
strains increased when the glycine level in the medium was increased. Particularly, the
sdaA-deletion strain showed a significant increase in OD and sugar consumption rate
at a glycine concentration of 1 g/L. The OPS productivity of the kbl deletion strain
greatly improved in the presence of 2.5 g/L glycine.
[272]
[273] EXAMPLE 27: Preparation of iclR deficient Strain
[274]
[275] The transcription factor iclR was deleted from CA07-0022 serA*(G336V) to produce
CA07-0022 serA*(G336V) AiclR. The deletion mutant strain was prepared using the
one-step inactivation method as in Example 5 and the antibiotic-resistant marker gene
was removed. For the preparation of the iclR deletion strain, PCR was performed with
a pair of primers of SEQ ID NOS: 87 and 88.
[276]
[277] EXAMPLE 28: Assay of the iclR deficient Strain for OPS Productivity
[278]
[279] The strain prepared in Example 27 was evaluated for OPS productivity in the same
manner as in Example 9.
[280]
[281] Table 2 1
[Table 21]
[282]
[283] As is apparent from the data of Table 21, the OPS productivity of the iclR deletion
strain was found to increase.
[284]
[285] EXAMPLE 29: Construction of Vectors Carrying E. coli Acetyl CoA Synthetase
(acs), Pyruvate Oxidase Monomer (poxB), Acetate Kinase (ackA) and Phosphate
Acetyltransferase (pta)
[286]
[287] To enhance the production and reuse of acetate in the O-phosphoserine-producing
strain, expression plasmids carrying acetyl CoA synthetase-encoding acs, pyruvate
oxidase monomer-encoding poxB, acetate kinase-encoding ackA and phosphate acetyltransferase-
encoding pts, respectively, were constructed.
[288] Each gene was obtained by performing pfu PCR on the genomic DNA of E. coli
W31 10 with a pair of primers of SEQ ID NOS: 89 and 90 for acs, with a pair of
primers of SEQ ID NOS: 9 1 and 92 for poxB, and with a pair of primers of SEQ ID
NOS: 93 and 94 for ackA and pta. After treatment with Hindlll, each of the PCR
products thus obtained was cloned at the EcoRV and Hindlll site into the pCLPrmf-
GFP vector constructed by inserting an E. coli rmf promoter into pCL1920, so as
to give pCL-Prmf-acs, pCL-Prmf-poxB, and pCL-Prmf-ackA-pta. Subsequently, these
plasmids were treated with EcoRI to obtain DNA inserts, that is, Prmf-acs, Prmf-poxB,
and Prmf-ackA-pta, which were then introduced into pCClBAC (EcoRI)
(CopyControl™ pcclBAC™ Vector, Epicentre. Cat. Nos. CBAC311) to construct
pCClBAC-Prmf-acs, pCClBAC-Prmf-poxB, and pCClBAC-Prmf-ackA-pta, r e
spectively.
[289]
[290] EXAMPLE 30: Preparation of E. coli acs, poxB, ackA, pta-enhanced OPSProducing
Strain and Assay for OPS Productivity
[291]
[292] The CA07-0022 serA*(G336V) strain was transformed with the three vectors
prepared in Example 29 and assayed for OPS productivity in the same manner as in
Example 9.
[293]
[294] Table 22
[Table 22]
[295]
[296] As can be seen in Table 22, the growth rate of the strain transformed with poxB
decreased whereas the introduction of acs or ackA-pta increased the growth rate and
OPS productivity.
[297]
[298] EXAMPLE 31: Construction of Vectors Carrying E. coli malate synthase A
(aceB), Isocitrate Lyase Monomer (aceA), Phosphoenolpyruvate Carboxykinase
(pckA), Malate Synthase G (glcB), and Malate Dehydrogenase (maeB)
[299]
[300] Plasmids which allow the expression of both malate synthase A-encoding aceB and
isocitrate lyase monomer-encoding aceA, phosphoenolpyruvate carboxykinaseencoding
pckA, malate synthase G-encoding glcB, and malate dehydrogenaseencoding
maeB in E. coli, respectively, were constructed.
[301] The genes were prepared by performing pfu PCR on the genomic DNA of E. coli
W31 10 with a pair of primers of SEQ ID NOS: 95 and 96 for aceBA, with a pair of
primers of SEQ ID NOS: 97 and 98 for pckA, with a pair of primers of SEQ ID NOS:
99 and 100 for glcB, and with a pair of primers of SEQ ID NOS: 101 and 102 for
maeB. After treatment with Hindlll, each of the PCR products thus obtained was
cloned at the EcoRV and Hindlll site into the pCL-Prmf-GFP vector constructed by
inserting an E. coli rmf promoter into pCL1920, so as to give pCL-Prmf-aceBA, pCLPrmf-
pckA, pCL-Prmf-glcB, and pCL-Prmf-maeB.
EXAMPLE 32: Preparation of E. coli aceB, aceA, pckA, glcB and maeBenhanced
OPS-Producing Strain and Assay for O-phosphoserine Productivity
The CA07-0022 serA*(G336V) strain was transformed with the four vectors
prepared in Example 3 1 and assayed for O-phosphoserine productivity in the same
manner as in Example 9.
] Table 23
[Table 23]
]
] As can be seen in Table 23, the sugar consumption rate and OPS productivity of the
strain somewhat decreased when transformed with aceBA and the growth rate sig
nificantly decreased when transformed with pckA whereas the introduction of glcB or
maeB increased OPS productivity.
]
] EXAMPLE 33: Construction of Vectors Carrying Glyoxylate Carboligase (glc),
Tartronate Semialdehyde Reductase 2 (glxR), and Glycerate Kinase II (glxK)
]
] Glyoxylate carboligase-encoding gel, tartronate semialdehyde reductase 2-encoding
glxR, and glycerate kinase II-encoding glxK, all of which are involved in the
conversion of glyoxylate into 3-phosphogly cerate, were cloned as follows. The genes
were obtained by performing PCR on the genomic DNA of E. coli W31 10 with a pair
of primers of SEQ ID NOS: 103 and 104 for gel, and with pairs of primers of SEQ ID
NOS: 105 to 108 for glxR-glxK. After digestion with EcoRV and Hindlll, each of the
PCR products was cloned at the EcoRV and Hindlll sites into the pCL-Prmf-GFP
vector constructed by inserting an E. coli rmf promoter into pCL1920 to afford r e
combinant plasmids, named pCL-Prmf-gcl, pCL-Prmf-glxR-glxK, and pCLPrmf-
glxR-glxK-Prmf-gcl, respectively.
[314]
[315] EXAMPLE 34: Introduction of Vectors Carrying gle, glxR, glxK into OPhosphoserine-
Producing Strain and Assay for O-Phosphoserine Productivity
[316]
[317] The three plasmids constructed in Example 33 were introduced into CA07-0022
serA*(G336V) which were then evaluated for O-phosphoserine productivity in the
same manner as in Example 9. The results are summarized in Table 24, below.
[318]
[319] Table 24
[Table 24]
[320]
[321] As can be seen in Table 24, the final O-phosphoserine productivity of the strains
transformed respectively with gel, glxR-glxK and glxR-glxK-gcl was decreased, but
growth rate and sugar consumption rate were increased, compared to the CA07-0022
serA*(G336V) strain itself. Particularly, the introduction of glxR-glxK was found to
have the greatest increase on growth rate and sugar consumption rate.
[322]
[323] EXAMPLE 35: Evaluation of O-phosphoserine-Producing Strain in a
Fermentor
[324]
[325] CA07-0022 serA*(G336V)/pCL-Prmf-serA*(G336V)-serC strains were incubated at
33°C for 24 hours on MMYE agar plates (2 g/L glucose, 2 mM magnesium sulfate, 0.1
mM calcium chloride, 6 g/L sodium pyrophosphate, 0.5 g/L sodium chloride, 3 g/L
potassium dihydrogen phosphate, 10 g/L yeast extract, 18 g/L agar) containing 50 mg/
mL spectinomycine. The resulting colonies were scraped from 1/10 of the area of each
agar plate, inoculated into a 50 mg/mL spectinomycine-containing seed medium, 10 g/
L glucose, 0.5 g/L magnesium sulfate, 3 g/L potassium dihydrogen phosphate, 10 g/L
yeast extract, 0.5 g/L sodium chloride, 1.5 g/L ammonium chloride, 12.8 g/L sodium
pyrophosphate, 1 g/L glycine) in a baffle flask, and incubated at 30°C for six hours
while shaking at 200 rpm. To 300 mL of a main medium in a 1 L fermentor, the
resulting seed culture in an amount as large as 16% of the volume of the main medium
was added, followed by incubation at 33°C and pH 7.0. The main medium had the
composition given in Table 25, below.
[326]
[327] Table 25
[Table 25]
[328]
[329] During incubation, the pH of the culture medium was adjusted to 7.0 with ammonia
water. Upon the depletion of glucose from the culture medium, fed-batch-type fer
mentation was conduced by adding a 520 g/L glucose solution. Following fermentation
for 80 hours, O-phosphoserine was produced at a concentration of 19.5 g/L as
measured by HPLC.
[330]
[33 1]
[332]
[333] EXAMPLE 36: Development of OPS Sulfhydrylase (OPSS)
[334]
[335] Aeropyrum pernix, Mycobacterium tuberculosis, and Trichomonas vaginalis are
reported to have O-phosphoserine sulfhydrylase (OPSS), an enzyme that employs Ophospho-
L- serine (OPS), instead of O-acetyl serine (OAS) in E. coli, as a substrate for
the synthesis of cysteine (Mino K and Ishikawa K, FEBS letters, 551: 133-138, 2003;
Burns KE, Baumgart S, Dorrestein PC, Zhai H, McLafferty FW and Begley TP, J. Am.
Chem. Soc, 127: 11602-11603, 2005; Westrop GD, Goodall G, Mottram JC and
Coombs GH, J. Biol. Chem., 281: 25062-25075, 2006). Based on the report, the
present inventors found two types of OPS sulfhydrylase, which converts OPS into
cysteine, from Aeropyrum pernix and Mycobacterium tuberculosis H37Rv. Of them,
the Mycobacterium tuberculosis H37Rv-derived OPSS enzyme was used for screening
amino acid homology. As a result, three types of OPSS were secured from My
cobacterium smegmatis str. MC2 155, Rhodococcus jostii RHA1, and Nocardia
farcinica IFM 10152.
[336] To obtain OPSS from each strain, a pET28a vector system (Novagen), which is
typically used for enzyme expression, was constructed. Each templates and primers for
use in cloning the five different OPS sulfhydrylase genes and the resulting recombinant
plasmids are summarized in Table 26, below. Suitable combinations of the templates
and the primers, as given in Table 26, were used for PCR for amplifying respective
OPSS genes. The PCR products and the pET28a vector were digested with Ndel and
Hindlll (37°C for 3 hours). Each of the gene fragments was ligated to the digested
pET28a vector (Novagen). Base sequencing confirmed the construction of the ex
pression vectors carrying the each OPSS genes. The enzyme expression vectors were
introduced into E. coli (DE3) to produce strains capable of expressing five OPSS
enzymes. Enzyme names are given in Table 26, below.
[337]
[338] Table 26
[Table 26]
[339]
[340] Expression of the enzymes was conducted according to the instructions of the pET
system manufacturer (Novagen). Single colonies of each strain from the LB plates
were inoculated into 5 mL of LB broth and incubated at 37°C for 16 hours while
shaking at 200 rpm. The cultures were transferred to 25 mL of fresh LB broth (in 250
mL flasks) and incubated to an OD600 of 0.5 - 0.6 (for 2 - 3 hours) in the same
condition, immediately after which 1mM IPTG was added to the media to induce the
enzymes to be expressed during incubation at 18°C for 18 hours while shaking at 120
rpm. The enzymes were purified using Ni-NTA columns for His-tag, with the aid of
His SpinTrap (GE Healthcare). Of the five OPSS enzymes thus isolated, four were
found to be in soluble forms, with one (Rjo-OPSS) being an inclusion body, as
analyzed by 14% SDS-PAGE electrophoresis.
[341]
[342] EXAMPLE 37: Assay of OPS sulfhydrylase (OPSS) for Cysteine Synthesis
Activity
[343]
[344] The OPS sulfhydrylase enzymes obtained from the four microorganism strains were
assayed for ability to catalyze the conversion of O-phosphoserine (OPS) to cysteine.
With regard to assay conditions and methods (cysM enzyme assay), reference was
made to previous reports (Mino K and Ishikawa K, FEBS letters, 551: 133-138, 2003;
Burns KE, Baumgart S, Dorrestein PC, Zhai H, McLafferty FW and Begley TP, J. Am.
Chem. Soc, 127: 11602-11603, 2005; Westrop GD, Goodall G, Mottram JC and
Coombs GH, J. Biol. Chem., 281: 25062-25075, 2006). The amount of the substrate
used is represented by a unit of mL. Assay conditions for enzyme activity are
summarized in Table 27, below.
[345]
[346] Table 27
[Table 27]
[347]
[348] Reaction solutions excepting of the enzymes were incubated at 37°C for 5 min, after
which 50 mg of purified OPS sulfhydrylase was added to the reaction solution. At pre
determined times during incubation at 37°C, 100 mL of the enzyme reactions was
taken and mixed with 100 mL of 33.2% TCA to stop the enzymatic reaction. The
cysteine concentrations of the enzyme reactions were quantitatively analyzed by
measuring absorbance at OD560 according to the Gaitonde method. Cysteine synthesis
activities of the four different OPS sulfhydrylase enzymes are summarized in Table 28,
below. The cysteine synthesis titers of the OPSS enzymes are expressed as cysteine
conversion rates with reaction time.
[349]
[350] Table 28
[Table 28]
[351]
[352] The OPS sulfhydrylase enzymes derived from Aeropyrum pernix and Mycobacterium
tuberculosis H37Rv, which were previously reported (Mino K and Ishikawa K, FEBS
letters, 551: 133-138, 2003; Westrop GD, Goodall G, Mottram JC and Coombs GH, J.
Biol. Chem., 281: 25062-25075, 2006), were confirmed to have the activity of using
OPS as a substrate to synthesize cysteine. The cysteine synthesis activity of the novel
Mycobacterium smegmatis str. MC2 155-derived OPS sulfhydrylase, which was
obtained by screening amino acid homology with the Mtb-OPSS enzyme, was first
found. As seen in the data of Table 28, the conversion rate from OPS into cysteine of
Ape-OPSS reached near 100% in one hour. The final conversion rate of the Msm-
OPSS enzyme, which was newly selected through enzyme screening on the basis of
previously reported Mycobacterium tuberculosis H37Rv-derived OPSS, was 43.7%
that was 4.3 times as high as that of Mtb-OPSS. On the other hand, the novel Nocardia
farcinica IFM 10152-derived OPS sulfhydrylase, obtained by the homology screening,
exhibited insufficient activity of converting O-phosphoserine into cysteine.
[353]
[354] EXAMPLE 38: Preparation of Mtb-T and Msm-T that encode C-Terminally 5
Amino Acid Residues truncated Mtb-OPSS and Msm-OPSS
[355]
[356] Mycobacterium tuberculosis H37Rv-derived OPSS (Mtb-OPSS), which catalyzes the
conversion of OPS to cysteine with the aid of the additional enzymes mec-i- and cysO,
is reported to be able to use an S2 containing sulfur source in converting OPS to
cysteine even in the absence of the additional enzymes when five C-terminal amino
acid residues are removed therefrom (Agren D, Schnell R and Schneider G, FEBS
letters, 583: 330-336, 2009). On the basis of this report, Mtb-T (SEQ ID NO: 11),
which can rapidly convert OPS in the presence of S2 _ as a sulfur source, was obtained.
Msm-T was also obtained from Msm-OPSS (SEQ ID NO: 9) that shares an amino acid
homology with Mtb-OPSS. Expression vectors carrying the two enzyme mutants were
constructed. In this regard, pfu PCR was performed on the genomic DNA of My
cobacterium tuberculosis H37Rv or Mycobacterium smegmatis in the presence of a
pair of primers of SEQ ID NOS: 119, 120, 121 and 122. The OPSS gene fragments
thus obtained were treated with Ndel and Hindlll and were cloned into the pET28a
vector digested with the same restriction enzymes to construct recombinant expression
vectors named pET28a-Mtb-T and pET28a-Msm-T, respectively. The recombinant ex
pression vectors were introduced into E. coli (DE3). The expression of the two mutant
OPSS enzymes was confirmed by 14% SDS PAGE. The two mutant OPSS enzymes
are purified and expressed in the same conditions as in Example 36. As a result, Mtb-T
(SEQ ID NO: 11) and Msm-T (SEQ ID NO: 10) were obtained.
[357]
[358] EXAMPLE 39: Assay of Mtb-T and Msm-T for Cysteine Conversion Activity
[359]
[360] On the basis of the report that Mycobacterium tuberculosis H37Rv-derived OPSS
mutants devoid of five C-terminal amino acid residues show increased affinity for an S
2 _ group-containing sulfur source even in the absence of subsidiary enzymes (Agren D,
Schnell R and Schneider G, FEBS letters, 583: 330-336, 2009), Mtb-T and Msm-T
were obtained. They were evaluated for enzymatic activity by measuring final cysteine
conversion rates. Enzymatic activity was assayed in the same condition and manner as
in Example 37. The produced cysteine was quantitatively analyzed using the Gaitonde
method.
[361]
[362] Table 29
[Table 29]
[363]
[364] As seen in Table 29, Msm-T, being devoid of the five C-terminal amino acid residues
of Mycobacterium smegmatis str. MC2 155-derived OPSS allowed the conversion of
cysteine from the substrate at a rate of 100% in one hour.
[365] When its amino acid sequence was modified, the O-phosphoserine sulfhydrylase
(OPSS) can more effectively catalyze the biosynthesis of L-cysteine.
[366]
[367] EXAMPLE 40: Requirement of Cofactor for OPS Sulfhydrylase Activity
[368]
[369] To examine the effect of cofactors on the cysteine conversion of OPSS, the cysteine
conversion rate of Msm-T was measured in the absence or presence of PLP
(pyridoxal-5' -phosphate) and DTT (dithiothreitol). In this regard, the substrates of 50
mM OPS broth and 100 mM Na2S were reacted at 37°C for 30 min in the presence of
25 mM DTT or 0.2 mM PLP. The cysteine thus produced was quantitatively analyzed
using the Gaitonde method. As seen in Table 30, the cysteine conversion rate in the
presence of both PLP and DTT was 2.3 times as large as that in the absence of both
PLP and DTT. Thus, both PLP and DTT were observed to have a positive influence on
the conversion.
[370]
[371] Table 30
[Table 30]
[372]
[373] EXAMPLE 41: The Influence of Temperature on the Activity of OPS
Sulfhydrylase
[374]
[375] The cysteine conversion rates of Ape-OPSS and Msm-T according to temperatures
were examined. The enzymatic activity at 37°C and 60°C was measured 2, 5, 10, 30,
and 60 min after reaction. The reaction was conducted under the condition of 100 mM
HEPES (pH 7.4), 5 mM OPS, 10 mM Na2S, 0.2 mM PLP, and CysM 50 mg/mL. The
amount of produced cysteine was determined using the Gaitonde method. In the
condition of a buffer, as shown in FIG. 2, Ape-OPSS showed a faster initial reaction
rate at 37°C as well as higher reactivity at 60°C than did Msm-T.
[376]
[377] EXAMPLE 42: Heat Stability of OPS Sulfhydrylase
[378]
[379] Ape-OPSS and Msm-T were analyzed for heat stability. Each of the enzymes was
diluted to a concentration of 2 mg/mL in an OPS broth and thermally treated at 37°C
and 60°C for 10, 30, 60, 120, and 240 min, followed by reaction at 37°C for 30 min
under the condition of 5 mM OPS, 10 mM Na2S, 0.2 mM PLP, and 100 mM HEPES
(pH 7.4). For this reaction, 10 mg/mL Ape-OPSS and 50 mg/mL Msm-T were
employed. The amounts of the produced cysteine were measured using the Gaitonde
method. Ape-OPSS was observed to retain its intact activity in spite of heat treatment
at 60°C for 4 hours while the activity of Msm-T was maintained at 37°C, but decreased
by 50% upon heat treatment at 60°C for 30 min. The results are given in Table 31,
below.
[380]
[381] Table 3 1
[Table 31]
[382]
[383] An examination was made of the retention of enzymatic activity at 37°C when Msm-
T was used in an amount of 50 mg/mL, which is a practical concentration in OPS broth.
In the absence of Na2S, 50 mg/mL Msm-T was treated, together with 50 mM OPS broth
and 0.2 mM PLP, at 37°C for 0.5, 1, 2, 4, and 6 hours, after which Na2S was added to
induce the enzymatic reaction. After the reaction for 30 min, the activity of Msm-T
was measured. The amounts of the produced cysteine were determined using the
Gaitonde method. As a result, the activity of Msm-T was decreased below 50% 2 hours
after reaction at 37°C in OPS broth (Table 32).
[384]
[385] Table 32
[Table 32]
[386]
[387] EXAMPLE 43: The Infulence of pH on the OPS Sulfhydrylase
[388]
[389] The cysteine conversion rates of Ape-OPSS and Msm-T according to pH were
measured. In 100 mM buffer, Ape-OPSS and Msm-T, each having a concentration of
50 mg/mL, were subjected to reaction at 37°C for 10 min. In this regard, K-phosphate
buffer with a pH of 6.4 / 7.0 / 7.4 / 8.0, Tris-HCl buffer with a pH of 7.0 / 7.4 / 8.0 / 8.5
/ 8.8, and Na-carbonate buffer with a pH of 8.0 / 8.5 / 9.0 / 10.0 were used. The quan
titative analysis of the produced cysteine was conducted using the Gaitonde method.
As seen in FIG. 3, Msm-T exhibited the highest activity at a pH of from 8.0 to 9.0 irre
spective of buffer. As for Ape-OPSS, its highest activity was detected in K-phosphate
(pH 7.4), with an optimal pH differing from one buffer to another.
[390]
[39 1] EXAMPLE 44: Effect of Ions on the Activity of OPS Sulfhydrylase
[392]
[393] Effects of ions on the activity of the OPSS enzymes were examined as follows. In a
reaction mixture containing 5 mM OPS, 10 mM Na2S, 0.2 mM PLP, and 100 mM
HEPES (pH 7.4), the enzymes were subjected to reaction at 37°C for 30 min in the
presence of (NH4)2S0 4 (1, 3, 5, 10, 20 g/L), KH2P0 4 (0.5, 1, 2, 4, 8 g/L), or NH4C 1
(0.2, 0.5, 1, 2 g/L). Ape-OPSS and Msm-T were used at a concentration of 10 mg/mL
and 50 mg/mL, respectively. The amounts of the produced cysteine were determined
using the Gaitonde method.
[394] No changes were detected in the cysteine conversion rate when (NH4)2S0 or KH2PO
4 was added to the reaction mixture. On the other hand, as seen in Table 33, the
cysteine conversion rate was decreased with an increase in NH4C 1 concentration. Par
ticularly, the maximal enzyme activity was decreased by more than 70% when 2 g/L
NH4C 1 was added. Therefore, NH4C 1 was observed to have a negative effect on the
conversion activity of OPS sulfhydrylase.
[395]
[396] Table 33
[Table 33]
[397]
[398] EXAMPLE 45: Effect of Sulfur Source on the Cysteine Synthesis Activity of
OPS Sulfhydrylase
[399]
[400] An experiment was conducted to examine the effect of sulfur sources on the cysteine
synthesis activity of each enzyme. In a reaction mixture containing 5 mM OPS, 0.2
mM PLP, and 100 mM HEPES, each enzyme (50 mg/mL Ape-OPSS, 50 mg/mL Msm-
T) was subjected to reaction at 37°C for 1 hour in the presence of 10 mM Na2S, NaSH,
or Na2S20 3. The amounts of the produced cysteine were measured using the Gaitonde
method. Ape-OPSS was observed to prefer Na2S20 3 as a sulfur source, whereas Msm-
T prefers Na2S. The results are summarized in Table 34, below.
[401]
Table 34
[Table 34]
[403]
[404] EXAMPLE 46: Construction of the Expression Vector Carrying OPS
Sulfhydrylase (pCL-Pcjl System) and Expression in E. coli
[405]
[406] PCR was performed using primers of SEQ ID NOS: 123 and 124, with the
pET28a-Msm-T vector serving as a template. The PCR product thus obtained was
treated with EcoRV and Hindlll and cloned into pCL-P(CJl) to construct a r e
combinant vector named pCL-P(CJl)-Msm-T. To examine a difference in the ex
pression level of Msm-T between the pET system and the pCL-Pcjl system, strains for
expressing the enzyme were prepared. The pET system was introduced into Rosetta
(DE3) while the pCL-Pcjl system used the K12G strain. Single colonies taken from
LB plates were inoculated into 5 mL of LB broth and cultured at 37°C for 16 hours
while shaking at 200 rpm. These cultures were transferred to 25 mL of fresh LB broth
containing kanamycine or spectinomycine and 0.2% glucose (in 250 mL flasks) and
incubated to an OD600 of 0.5 - 0.6, immediately after which 1mM IPTG was added to
the media to induce the enzymes to be expressed. During incubation at 37°C while
shaking at 200 rpm, the expression levels of the enzyme were measured at various
culture times (8, 16, 24 hours). The enzyme expression levels of the two systems were
analyzed on 14% SDS PAGE (FIG. 4).
[407]
[408] EXAMPLE 47: Cysteine Synthesis by OPS Sulfhydrylase with the Purified OPS
Fermentation Broth
[409]
[410] The conversion rates from purified OPS to cysteine of Msm-T and Ape-OPSS were
determined. In the presence of 75 mg/mL of each of the enzymes and 0.2 mM PLP, 60
mM OPS purified from OPS fermentation broth was reacted with 120 mM Na2S at
37°C or 70°C for 30, 60, 90, and 120 min. The reaction was conducted only at 37°C for
Msm-T, but at both 37°C and 70°C for Ape-OPSS. The amounts of the produced
cysteine were measured using the Gaitonde method. As seen in FIG. 5, a purified OPS
fermentation broth served well as a substrate for the enzymatic conversion into
cysteine. Particularly, the conversion rate of Ape-OPSS was increased at 70°C even
upon the use of the purified OPS fermentation broth.
[411]
[412] EXAMPLE 48: Cysteine Synthesis by OPS Sulfhydrylase with the OPS Fer¬
mentation Broth
[413]
[414] When an OPS fermentation broth was used as a substrate, the cysteine conversion
rates of Msm-T and Ape-OPSS were measured according to the concentrations of the
enzymes. In the presence of 100 mM Na2S and 0.2 mM PLP, 50 mM of OPS fer
mentation broth was reacted with 5 mg/mL or 50 mg/mL of each of Msm-T and Ape-
OPSS at 37°C. The amounts of the produced cysteine were measured using the
Gaitonde method. As seen in FIG. 6, the highest conversion rate was detected in 50 mg/
mL Msm-T. In addition, upon the use of OPS fermentation broth as a substrate, the
activity of Msm-T was higher than that of Ape-OPSS.
[415]
[416] EXAMPLE 49: Cysteine Conversion Rate According to OPS Concentration
[417]
[418] To examine the effect of OPS concentration on the conversion rate of Msm-T, prede
termined amounts of purified OPS were added to OPS fermentation broth to induce the
conversion reaction. The enzyme was used in an amount of 50 mg. The amounts of
cysteine in the reaction solution were measured using the Gaitonde method. Msm-T
exhibited a conversion rate of as high as 100% when the concentration of OPS was
about 30 g/L.
[419] When the concentration of OPS exceeded 50 g/L, both the conversion rate and the
conversion percentage were found to decrease. From these results, it is understood that
when OPS fermentation broth is used as a substrate, there is an optimal concentration
ratio between OPS and the enzyme.
[420]
[421] Table 35
[Table 35]
Cysteine Conversion Rate (Msm-T 50 ug)
[422]

We Claim:
1. A method for producing cysteine or a derivative
thereof, comprising:
1) culturing a recombinant microorganism in which the
5 activity of endogeneous phosphoserine phosphatase (SerB) is
reduced, to produce 0-sphosphoserine (OPS); and
2) reacting the OPS of step 1) with a sulfide in
presence of 0-phosphoserine sulfhydrylase (OPSS) or a
microorganism expressing OPSS, to produce cysteine or a
10 derivative thereof.
2. The method of claim 1, wherein the phosphoserine
phosphatase has an amino acid sequence of SEQ ID NO: 1 or 2.
15 3. The method of claim 1, wherein the the level of enzyme
activity is reduced by using a technique selected from the
group consisting of deletion of the chromosomal gene encoding
the enzyme, the introduction of mutation into the chromosomal
gene to reduce the endogenouse geneactivity, the substitution
20 of the chromosomal gene with a gene mutated to reduce the
endogenouse enzyme activity, the introduction of mutation into
a regulatory region for the gene to reduce endogenouse gene
activity, and the introduction of an antisense oligonucleotide
coitplementary to a transcript of the gene to inhibit the
25 translation of the mRNA.
90
4. The method of claim 3, wherein the recombinant
microorganism in which the activity of endogenous SerB is
disrupted is cultured in a mediimi containing glycine or
5 serine.
5. The method of claim 4, wherein the mediimi contains
glycine in an amount of from 0.1 to 10 g/L.
10 6. The method of claim 4, wherein the medium contains
serine in an amount of from 0.1 to 5 g/L.
7. The method of claim 1, wherein the recombinant
microorganism has been further modified to enhance the
15 activity of phosphoglycerate dehydrogenase (SerA) or
phosphoserine aminotransferase (SerC).
8. The method of claim 7, wherein the SerA is a wild-type
or a mutant resistant to serine feedback inhibition'.
20
9. The method of claim 7, wherein:
i) the SerA has one selected from the group consisting
of amino acid sequences of SEQ ID NOS: 3 to 7; and
ii) the SerC has an amino acid sequence of SEQ ID NO:
25 8.
91
10. The method of claim 1, wherein the recombinant
microorganism has been further modified to reduce the activity
of a PhnCDE (phnC (ATP-binding component of phosphonate
5 transport, EG 10713)-phnD (periplasmic binding protein
component of Pn transporter, EG 10714)-phnE (integral membrane
component of the alkylphosphonate ABC transporter, EG 11283)).
11. The method of claim 1, wherein the recombinant
10 microorganism has been further modified to reduce the activity
of alkaline phosphatase (PhoA) or acid phosphatase (AphA) .
12. The method of claim 1, wherein the recombinant
microorganism has been further modified to enhance the
15 activity of nucleotide transhydrogenase (PntAB).
13. The method of claim 1, wherein the recombinant
microorganism has been further modified to enhance the
activity of at least one enzyme selected from the group
20 consisting of o-acetylserine/cysteine efflux permease (YfiK),
homoserine/homoserine lactone efflux protein (RhtB), and
threonine/homoserine efflux protein (RhtC).
14. The method of claim 7, 12 or 13, wherein the the level
25 of enzyme activity is enhanced by using a technique selected
92
from the group consisting of increasing a copy number of a
gene encoding the enzyme, introducing a mutation into a
regulatory region for the gene to enhance the enzyme activity,
substituting the chromosomal gene with a gene mutated to
5 enhance the enzyme, and introducing a mutation into the
chromosomal gene to enhance the enzyme activity.
15. The method of claim 1, wherein the recombinant
microorganism has been further modified to reduce the activity
10 of at least one selected from the group consisting of
phosphoglycerate mutase isozymes (GpmA, Gpml or GpmB).
16. The method of claim 1, wherein the recombinant
microorganism has been further modified to reduce the activity
15 of L-serine dehydratase I (SdaA).
17. The method of claim 1, wherein the recombinant
microorganism has been further modified to reduce the activity
of 2-amino-3-ketobutyrate coenzyme A ligase (Kbl) or a
20 transcription factor (IclR).
18. The method of claim 1, wherein the recombinant
microorganism has been further modified to enhance the
activity of at least one enzyme selected from the group
25 consisting of acetyl-CoA synthetase (Acs), acetic acid kinase
93
(AckA)-phosphotransacetylase (Pta), malate synthase G (GlcB),
malate dehydrogenase (MaeB), glutamate dehydrogenase (GdhA),
glyoxylate carboligase (Glc), tartronate semialdehyde
reductase 2 (GlxR) and glycerate kinase II (GlxK).
5
19. The method of claim 18, wherein the recombinant
microorganism is improved in sugar consuitption and growth by
enhancement of the acitivity of at least one enzyme selected
from the group consisting of Glc, GlxR, and GlxK.
10
20. The method of claim 1, wherein the recombinant
microorganism is Escherichia sp. or Coryneform bacteria.
21. The method of claim 1, wherein the sulfide of step 2)
15 is selected from the group consisting of NaaS, NaSH, (NH4)2S,
H2S, Na2S203 and a combination thereof.
22. The method of claim 1, wherein the sulfide of step 2)
is used at a molar concentration 0.1 to 3 times as high as
20 that of OPS used in the enzymatic conversion.
23. The method of claim 1, wherein the OPSS of step 2) is
derived from at least one species selected from the group
consisting of Aeropyrum pernix, Mycobacterium tuberculosis,
25 Mycobacterium smegmatis and Trichomonas vaginalis.
94
24. The method of claim 23, wherein the OPSS is further
modified to increase a conversion rate of step 2).
5 25. The method of claim 1, wherein the conversion of step
2) is carried out in presence of a cofactor selected from
0.001 ~ 2 mM PLP (pyridoxal-5-phosphate), 0.001 ~ 100 mM DTT
(dithiothreitol), and a combination thereof.
10 26. The method of claim 1, further comprising isolating and
purifying the cysteine or its derivatives.
27. A recombinant microorganism in which the activity of
endogenous SerB is reduced.
15
28. The recombinant microorganism of claim 27, has been
further modified to enhance the activity of SerA or SerC,
wherein the SerA is resistant to serine feedback inhibition.
20 29. The recombinant microorganism of claim 27, has been
further modified to reduce the activity of at least one
selected from among PhnCDE, PhoA, and AphA.
30. The recombinant microorganism of claim 27, deposited
25
95
under accession No. KCCM11103P.