Note: Descriptions are shown in the official language in which they were submitted.
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Regulation of Carbon Assimilation
Background of the Invention
Field of the Invention
This invention relates to a polypeptide having phosphoenolpyruvate
carboxylase activity which does not require acetyl coenzyme A for activation
and
is desensitized to feedback inhibition by aspartic acid, and to genes coding
for this
polypeptide. The invention also relates to recombinant DNA molecules
containing
these genes, to bacteria transformed with these genes, and to methods of
producing amino acids using the transformed bacteria.
Related Art
Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31 ) is an enzyme
which is found in almost all bacteria and all plants. PEP carboxylase
catalyzes the
condensation reaction between the three carbon glycolytic intermediate PEP and
carbon dioxide resulting in the formation of the four carbon oxaloacetate
(OAA),
a metabolic intermediate common to the tricarboxylic acid (TCA) cycle and to L-
aspartic acid biosynthesis. The TCA cycle requires continuous replenishment of
C4 molecules in order to replace the intermediates withdrawn for amino acid
biosynthesis, and by playing an anaplerotic role in supplying OAA to the TCA
cycle, the biotin-independent PEP carboxylase aids in fulfilling this
function.
OAA is a very important substrate for the production of cell metabolites
such as amino acids, especially the glutamate family, i. e. , glutamate,
arginine and
proline, and the aspartate family, i. e. , aspartate, lysine, methionine,
threonine and
isoleucine. By catalyzing the reaction which results in the formation of OAA,
PEP
carboxylase plays an important role in supplying organic acids by metabolic
processes. For example, fermentive production of succinic acid from glucose by
Escherichia coli was significantly increased by the over-expression of PEP
carboxylase. See Millard, C., et al., Appl. Environ. Microbiol. 62:1808-1810
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(1996). Accordingly, PEP carboxylase also plays an important role in the
production of amino acids which are formed from glutamate and aspartate.
The amino acid is a compound which universally exists in cells as
components of proteins. However, for the sake of economic energy metabolism
and substance metabolism, its production is strictly controlled. This control
is
principally feedback control, in which the final product of a metabolic
pathway
inhibits the activity of an enzyme which catalyzes an earlier step of the
pathway.
PEP carboxylase also undergoes various regulations in expression of its
activity.
For example, in the case of PEP carboxylase of microorganisms belonging
to the genus Brevibacterium, Corynebacterium or the genus Escherichia, PEP
carboxylase activity is inhibited by aspartic acid. See e.g., Mori, M., et
al., J.
Biochem. 98:1621-1630 (1985); O'Regan, M., et al., Gene 77:237-251 (1989).
Therefore, the aforementioned amino acid biosynthesis, in which PEP
carboxylase
participates, is also inhibited by aspartic acid. However, PEP carboxylase
activities from Corynebacterium microorganisms having decreased sensitivity to
aspartic acid have been described. See Eikmanns, B.J., et al., Mol. Gen.
Genet.
218:330-339 (1989).
In addition to being allosterically inhibited by aspartic acid, acetyl co-
enzyme A (acetyl-CoA) is an allosteric activator of PEP carboxylase from
Brevibacterium flavum and Escherichia coli, for example. See Mori, M., et al.,
J. Biochem. 98:1621-1630 (1985); Morikawa, M., et al., J. Biochem. 81:1473
1485 ( 1977). PEP carboxylases from other organisms that are not regulated by
aspartic acid or acetyl-CoA have been reported. See Valle, F., et al., J.
Indus.
Microbiol. 17:458-462 (1996); O'Regan, M., et al., Gene 77:237-251 (1989);
Vance, C., et al., Plant Physiol. 75:261-264 (1984).
Since the anaplerotic enzyme PEP carboxylase is critical to the
maintenance of an optimal pool of OAA, and consequently determines the
biosynthetic levels of amino acids deriving from OAA, one way of improving
amino acid production by fermentation would be to manipulate the corresponding
ppc gene. For example, the amplification of the ppc gene from Brevibacterium
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lactofermentum has been shown to improve the production of proline and
threonine. See Sano, K., et al., Agric. Biol. Chem. 51:597-599 (1987).
Various techniques have been developed for efficient production in amino
acid fermentation by using mutant strains converted to be insensitive to
feedback
control. However, there has been no report of utilizing a PEP carboxylase
derived
from a plant for fermentative production of amino acids of the aspartic acid
or
glutamic acid families or of utilizing a ppc gene derived from a coryneform
bacterium which is integrated into microbial chromosomal DNA for fermentative
production of amino acids of the same families in which the PEP carboxylase is
not substantially regulated by acetyl-CoA or aspartic acid.
U.S. Patent No. 4,757,009 (Sano et al.; Ajinomoto Company) discloses
a process for producing an amino acid by fermentation which comprises
cultivating in a culture medium a Corynebacterium or Brevibacterium strain
carrying a recombinant DNA molecule comprising a plasmid having operationally
inserted therein a gene coding for PEP carboxylase, wherein the gene is a
chromosomal gene isolated from a Corynebacterium or a Brevibacterium strain
carrying a PEP carboxylase gene and has a chromosomal gene coding for an
amino acid, and isolating the amino acid from the culture medium. The
Corynebacterium or Brevibacterium strain from which the gene coding for PEP
carboxylase is isolated is a strain which exhibits weakened feedback
inhibition by
aspartic acid.
European Patent No. 358,940 (Bachmann et al.; Degussa
Aktiengesellschaft) discloses a plasmid pDM6 that is introduced into
Corynebacterium glutamicum DM58-1, which is deposited at the Deutsche
Sammlung von Mikroorganismen (DSM) under DSM 4697, wherein the plasmid
contains a genetic sequence comprising information coding for the production
of
a protein having PEP carboxylase activity. The ppc gene is isolated from a
genomic bank of Corynebacterium glutamicum ATCC 13032, and the PEP
carboxylase is not stimulated by acetyl-CoA. Also disclosed is a method of
producing L-lysine, L-threonine, and L-isoleucine by fermentation which
comprises culturing in an appropriate medium a host bacterium belonging to the
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genus Corynebacterium or Brevibacterium which contains plasmid pDM6, and
recovering the L-amino acid from the medium.
U.S. Patent No. 5,876,983 (Sugimoto et al.; Ajinomoto Company)
discloses a method of producing an amino acid which comprises selecting a
microorganism of the genus Escherichia containing a DNA sequence encoding a
mutant PEP carboxylase desensitized to feedback inhibition by aspartic acid by
growing Escherichia microorganisms in the presence of a wild-type PEP
carboxylase inhibitor selected from the group consisting of 3-bromopyruvate,
aspartic acid-~3-hydrazide and DL-threo-(3-hydroxyaspartic acid; culturing a
microorganism of the genus Escherichia or coryneform bacteria transformed with
the DNA sequence encoding a mutant PEP carboxylase in a suitable medium; and
separating from the medium an amino acid selected from the group consisting of
L-lysine, L-threonine, L-methionine, L-isoleucine, L-glutamic acid, L-arginine
and
L-proline.
Although there are many examples of culturing amino acid-producing
bacteria by recombinant DNA techniques, high levels of amino acid productivity
are not always achieved. Therefore, a need still continues to exist for a
method
of producing amino acids by fermentation in high titre and yields. A PEP
carboxylase that is not substantially regulated by acetyl-CoA or aspartic acid
could
improve carbon flow from the three carbon intermediate PEP to the four carbon
intermediate OAA. The improved flow could contribute to compounds derived
from OAA and increase amino acid biosynthesis.
Summary of the Invention
Accordingly, the present invention relates to a DNA fragment comprising
a gene encoding a polypeptide having PEP carboxylase activity, wherein the
gene
is capable of being expressed in a host microorganism, and wherein the
polypeptide does not require acetyl-CoA for activation and is desensitized to
feedback inhibition by aspartic acid.
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The present invention also relates to a recombinant DNA molecule
comprising a plasmid and a gene encoding a polypeptide having PEP carboxylase
activity operationally inserted therein, wherein the recombinant DNA molecule
is
capable of propagating and the gene is capable of being expressed in a host
microorganism comprising the genus Escherichia, Corynebacterium and
Brevibacterium, and wherein the polypeptide does not require acetyl-CoA for
activation and is desensitized to feedback inhibition by aspartic acid.
The present invention further relates to a host microorganism belonging
to the genus Escherichia, Corynebacterium or Brevibacterium transformed with
a DNA fragment comprising a gene encoding a polypeptide having PEP
carboxylase activity, wherein the gene is derived from a plant belonging to
the
class Monocotyledonae or Dicotyledonae or from a microorganism belonging to
the genus Corynebacterium or Brevibacterium, wherein the polypeptide does not
require acetyl-CoA for activation and is desensitized to feedback inhibition
by
aspartic acid, and wherein the host microorganism transformed with the DNA
fragment expresses the gene.
In another aspect of the present invention there is provided a method of
producing an amino acid by fermentation. The method comprises cultivating a
host microorganism belonging to the genus Escherichia, Corynebacterium or
Brevibacterium in a suitable medium and isolating from the culture medium an
amino acid, wherein the host microorganism is transformed with a DNA fragment
comprising a gene encoding a polypeptide having PEP carboxylase activity,
wherein the host microorganism expresses the gene, and wherein the polypeptide
does not require acetyl-CoA for activation and is desensitized to feedback
inhibition by aspartic acid.
In addition, the present invention relates to a method of selecting a DNA
fragment comprising a gene encoding a polypeptide having PEP carboxylase
activity wherein the polypeptide does not require acetyl-CoA for activation
and
is desensitized to feedback inhibition by aspartic acid, to a method of
increasing
the rate of conversion of PEP to OAA, to a method of recycling carbon in a
fermentation process, to a method of assimilating carbon in a fermentation
process
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which does not require biotin, to a method of increasing the production of
organic
acids in a fermentation process, and to a method of increasing the production
of
amino acids in a fermentation process.
Brief Description of the Figures
Figure 1 is a diagram of a strategy for gene replacement.
Detailed Description of the Preferred Embodiments
Before describing the invention in detail, several terms used in the
specification will be defined.
"Activator," as used herein, includes both a substance necessary for the
polypeptide to become active in the first place, as well as a substance which
merely accentuates activity.
"Amino acids" as used herein refer to the naturally occurring L amino acids
(alanine, arginine, aspartic acid, asparagine, cystine, glutamic acid,
glutamine,
glycine, histidine, isoleucine, leucine, lysine, methionine, proline,
phenylalanine,
serine, threonine, tryptophan, tyrosine, and valine).
"Chimeric gene" refers to a gene comprising heterogeneous regulatory and
coding sequences. It is a hybrid gene produced by recombinant DNA technology.
"DNA fragment" refers to a fraction of a deoxyribonucleic acid molecule.
"Expression," as used herein, is intended to mean the production of the
protein product encoded by a gene.
"Gene" refers to a nucleic acid fragment that expresses a specific protein,
including regulatory sequences preceding (5' non-coding) and following (3'
non-coding) the coding region. It is a discrete chromosomal region comprising
regulatory DNA sequences responsible for the control of expression, i.e.,
transcription and translation, and for a coding sequence which is transcribed
and
translated to give a distinct polypeptide.
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"Host microorganism" means the microorganism that is transformed with
the introduced genetic material.
"Inhibition" includes both the reduction of activity of the polypeptide and
the complete lack of activity as well.
"Isolated" as used herein means that the material is removed from its
original environment (e.g., the natural environment if it is naturally
occurring).
"Polypeptide" or "protein" as used herein refers to a molecule composed
of monomers (amino acids) linearly linked by amide bonds (also known as
peptide
bonds). It indicates a molecular chain of amino acids and does not refer to a
specific length of the product. Thus, peptides, oligopeptides and proteins are
included within the definition of polypeptide. This term is also intended to
refer
to post-expression modifications of the polypeptide, for example,
glycosolations,
acetylations, phosphorylations, and the like. A recombinant or derived
polypeptide is not necessarily translated from a designated nucleic acid
sequence.
It may also be generated in any manner, including chemical synthesis or
expression
of a recombinant expression system.
"Regulatory sequences" refer to nucleotide sequences located upstream
(5'), within, and/or downstream (3') to a coding sequence, which control the
transcription and/or expression ofthe coding sequences, potentially in
conjunction
with the protein biosynthetic apparatus of the cell.
"Synthetic DNA" refers to a nucleic acid molecule produced in whole or
in part by chemical synthesis methods.
"Transformation" herein refers to the transfer of a foreign gene into a host
cell either as part of the host cell genomic DNA or as an independent
molecule,
and its genetically stable inheritance.
In one aspect of the invention there is provided a DNA fragment
comprising a gene encoding a polypeptide having PEP carboxylase activity,
wherein the gene is capable of being expressed in a host microorganism, and
wherein the polypeptide does not require acetyl-CoA for activation and is
desensitized to feedback inhibition by aspartic acid.
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The ppc gene, which encodes the enzyme PEP carboxylase, may be any
one provided that it is a gene encoding for the PEP carboxylase of a plant
belonging to the class Monocotyledonae or Dicotyledonae or of a microorganism
belonging to the genus Brevibacterium or Corynebacterium, and provided the
expressed polypeptide does not require acetyl-CoA for activation and is
substantially desensitized to feedback inhibition by aspartic acid. The ppc
gene is
preferably determined for its base sequence and cloned. When it has not been
cloned, a DNA fragment containing the gene can be amplified and isolated by
using the PCR method and the like, followed by using a suitable vector to
achieve
cloning. Preferred donors of the ppc gene are strains which exhibit weakened
feedback inhibition by aspartic acid. Such strains are recognized as being
resistant
to aspartic acid-antagonistic inhibitors.
PEP carboxylase is a key enzyme of photosynthesis in C4 plants. It is
specifically localized in the cytosol of mesophyll cells and is regulated by a
phosphorylation/dephosphoylation process. See Giglioli-Guivarc'h, N., et al.,
Cytometry 23:241-249 (1996). In addition, PEP carboxylase plays a crucial role
in the assimilation of COz during symbiotic NZ fixation in legume root
nodules.
See Pathirana, S., et al., Plant J. 12:293-304 (1997).
In one embodiment, the DNA fragment containing a gene encoding a
polypeptide having PEP carboxylase activity is derived from a plant belonging
to
the class Monocotyledonae or Dicotyledonae. In a preferred embodiment, the
DNA fragment is derived from an alfalfa plant. Most preferably, the DNA
fragment is derived from a Medicago sativa strain.
It has been shown that PEP carboxylase activity from a strain of Medicago
sativa was not substantially inhibited by L-aspartic acid. See Vance, C.P., et
al.,
Plant Physiol. 75:261-264 (1984). Further, the native ppc nucleotide sequence
from Medicago sativa is known (Pathirana, S., et al., Plant Molecular Biology
20:437-450 (1992)) and provided in SEQ ID NO:1, and the amino acid sequence
of the native PEP carboxylase encoded thereby is provided in SEQ ID N0:2.
, Since these sequences are known, primers may be designed and synthesized
based
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on the nucleotide sequences, and then the genes may be obtained by PCR, using
the messenger RNA as a template.
Post-translational regulation of plant PEP carboxylase is achieved, for
example, through phosphorylation of the protein. See Jiao, J.A., et al., Arch.
Biochem. Biophys. 269:526-535 (1989); Duff, S.M., et al., Eur. J. Biochem.
228:92-95 (1995). Alfalfa PEP carboxylase contains several conserved
sequences,
one of which is proposed to be involved in phosphorylation (MASIDAQLR,
residues 8 to 16). See Pathirana, S.M., et al., Plant Molecular Biology 20:437-
450 (1992).
In another preferred embodiment, the DNA fragment containing a gene
encoding a polypeptide having PEP carboxylase activity which is derived from a
plant belonging to the class Monocotyledonae or Dicotyledonae is modified by
one or more nucleotide substitutions, deletions and/or insertions. Most
preferably,
the modification comprises deleting the nucleotides encoding the amino acid
sequence: Met - Ala - Ser - Ile - Asp - Ala - Gln - Leu - Arg.
In another embodiment, the DNA fragment containing a gene encoding a
polypeptide having PEP carboxylase activity is derived from a microorganism
belonging to the genus Brevibacterium or Corynebacterium. In a preferred
embodiment, the DNA fragment is derived from a Corynebacterium glutamicum
strain. The native ppc nucleotide sequence of Corynebacterium glutamicum is
shown in SEQ ID N0:3.
It is to be understood that the number of amino acids in the active PEP
carboxylase molecule of the present invention may vary, and all amino acid
sequences derived from an alfalfa plant or a Corynebacterium strain that have
PEP
carboxylase activity and the desired de-regulatory characteristics are
contemplated
as being included in the present invention. Polypeptide sequences which differ
from each other only by conservative substitutions are included as well. Such
conservative substitutions consist of a substitution of one amino acid at a
given
position in the sequence for another amino acid of the same class. One or more
non-conservative amino acid substitutions, deletions and/or insertions,
located at
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positions of the sequence that do not alter the polypeptide to the extent that
the
biological activity of the polypeptide is destroyed, are also included.
Modifications to the sequence, such as deletions, insertions, and/or
substitutions in the sequence which produce silent changes that do not
substantially affect the functional properties of the resulting PEP
carboxylase
protein molecule are also contemplated. For example, an alteration in the gene
sequence which reflects the degeneracy of the genetic code, or which results
in the
production of a chemically equivalent amino acid at a given site, are
contemplated.
It is therefore understood that the invention encompasses more than the
specific exemplary sequences. Each of the proposed modifications is well
within
the routine skill in the art, as is determination of retention of biological
activity of
the encoded products.
In another embodiment, the DNA fragment containing a gene encoding a
polypeptide having PEP carboxylase activity is a chimeric gene comprising an
incomplete PEP carboxylase nucleotide sequence derived from a microorganism
belonging to the genus Brevibacterium or Corynebacterium and an incomplete
PEP carboxylase nucleotide sequence derived from a plant belonging to the
class
Monocotyledonae or Dicotyledonae. Together, the two incomplete sequences
form a complete chimeric ppc gene capable of expressing a polypeptide having
PEP carboxylase activity in which the polypeptide does not require acetyl
coenzyme A for activation and is desensitized to feedback inhibition by
aspartic
acid.
In a preferred embodiment, one incomplete PEP carboxylase nucleotide
sequence is derived from a microorganism belonging to the genus
Corynebacterium, and the other incomplete PEP carboxylase nucleotide sequence
is derived from an alfalfa plant. Most preferably, one incomplete PEP
carboxylase
nucleotide sequence is derived from a Corynebacterium glutamicum strain, and
the other incomplete PEP carboxylase nucleotide sequence is derived from a
Medicago .rativa strain.
In another embodiment, the DNA fragment is complementary DNA
(cDNA), genomic DNA or synthetic DNA. A DNA fragment of the present
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invention encoding PEP carboxylase can readily be obtained in a variety of
ways,
including, without limitation, chemical synthesis, cDNA or genomic library
screening, expression library screening, and/or PCR amplification of cDNA.
These methods and others useful for isolating such DNA are set forth, for
example, by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor (1989)), by Ausubel et al.,
eds. (Current Protocols in Molecular Biology, Current Protocols Press (1994)),
and by Berger and Kimmel (Methods in Enzymology: Guide to Molecular Cloning
Techniques, Vol. 152, Academic Press, Inc., San Diego (1987)).
Isolation of the ppc gene can be conducted, for example, by the following
method. Although the following example refers to Corynebacterium for
simplicity, it is to be recognized that bacteria from the genus Brevibacterium
can
likewise be used. First, a chromosomal gene is extracted from a
Corynebacterium
strain carrying a ppc gene (utilizing, for example, the method of H. Saito and
K.
Miura, Biochem. Biophys. Acta 72:619 (1963)). The gene is cleaved with an
appropriate restriction enzyme and then sub-cloned onto a plasmid shuttle
vector
capable of propagating in coryneform bacteria or in E. coli. To cleave
chromosomal genes, a wide variety of restriction enzymes can be employed by
controlling the degree of cleavage, for example, by controlling the time of
the
cleavage reaction, the temperature, etc. Cleavage of DNA by restriction
enzymes
is well understood by those skilled in the art and need not be set forth here
in
detail.
A PEP carboxylase-deficient mutant of coryneform bacteria or E. coli is
transformed with the resulting recombinant DNA. Transformants thus obtained
can be selected and isolated by conventional methods based on characteristics
possessed by the vector DNA and/or the recipient. For example, bacterial
strains
which come to possess PEP carboxylase activity are isolated, and a ppc gene
can
be isolated therefrom.
When the microorganism transformed with the DNA fragment of the
present invention as described above is cultivated, and the DNA sequence is
expressed, then an enzyme which does not require acetyl-CoA for activation and
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is substantially desensitized to aspartic acid inhibition may be obtained. It
becomes apparent, by measuring PEP carboxylase activity in the absence and/or
presence of acetyl-CoA, for example, whether or not the enzyme requires acetyl-
CoA as an activator. It also becomes apparent, by measuring the PEP
carboxylase
activity in the presence and/or absence of aspartic acid in an enzyme reaction
system, for example, whether or not the enzyme thus obtained is substantially
inhibited by aspartic acid.
It is possible for the measurement of the enzyme activity to use a
spectrometric method (Yoshinage, T., et al., J. Biochem. 68:747-750 (1970))
and
the like. For example, when the enzyme assay is measured in a continuous or
kinetic mode while the reaction is occurring, the reaction can be measured
spectrophotometrically by following the decrease in the absorbance (usually at
340
nanometers).
In another aspect of the invention there is provided a method of selecting
a DNA fragment comprising a gene encoding a polypeptide having PEP
carboxylase activity wherein the polypeptide does not require acetyl-CoA for
activation and is desensitized to feedback inhibition by aspartic acid. The
method
comprises extracting a chromosomal gene from a Corynebacterium strain carrying
a ppc gene, cleaving the chromosomal gene with an appropriate restriction
enzyme, ligating the ppc gene with a plasmid vector capable of propagating in
Corynebacterium, transforming a Corynebacterium strain in which the ppc and
pyc genes are nonfunctional, isolating strains which show superior growth on
minimal medium with glucose as the only carbon source, and isolating a DNA
fragment from the strain.
Pyruvate carboxylase (EC 6.4.1.1 ) is an important anaplerotic enzyme that
replenishes OAA, which is consumed for biosynthesis during growth, from
pyruvate and is used in lysine and glutamic acid production in industrial
fermentations. In addition to PEP carboxylase, the biotin-dependent pyruvate
carboxylase encoded by the pyc gene has recently been found to be an
anaplerotic
enzyme in Corynebacterium glutamicum. Inactivation of both the ppc and the pyc
gene in Corynebacterium glutamicum led to the inability of the microorganism
to
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grow on glucose. See Peters-Wendisch, P., et al., Microbiology 144:915-27
(1998). By inactivating both the ppc and the pyc genes, a DNA fragment
containing a ppc gene of the invention that was cloned into a replicating
plasmid
can be identified by the ability of a strain to show growth on minimal medium
with
glucose as the only carbon source.
In another embodiment, inhibitors of PEP carboxylase activity are also
added to the medium. For example, an analog of aspartic acid may be added. The
analog compound preferably exhibits a growth inhibitory action against a
microorganism belonging to the genus Corynebacterium which produces a wild-
type PEP carboxylase, the aforementioned growth inhibitory action is recovered
by existence of L-glutamic or L-aspartic acid, and the analog compound
inhibits
wild-type PEP carboxylase activity. If a strain being resistant to the analog
compound is selected from a microorganism belonging to the genus
Corynebacterium, it is much more likely that a host microorganism which
produces PEP carboxylase with desensitized feedback inhibition by aspartic
acid
will be obtained.
In another embodiment, strains are isolating which show an increased
production of an amino acid derived from OAA. Such amino acids include
aspartate, lysine, methionine, threonine and isoleucine. In addition, strains
can be
grown on minimal medium in the absence of acetyl-CoA, and the PEP carboxylase
activity can be measured.
In another aspect of the invention there is provided a recombinant DNA
molecule comprising a plasmid and a gene encoding a polypeptide having PEP
carboxylase activity operationally inserted therein, wherein the recombinant
DNA
molecule is capable of propagating and the gene is capable of being expressed
in
a host microorganism comprising the genus Escherichia, Corynebacterium and
Brevibacterium, and wherein the polypeptide does not require acetyl-CoA for
activation and is desensitized to feedback inhibition by aspartic acid.
The plasmid vector used in the present invention can be any vector as long
as it can be propagated in cells of bacteria from Escherichia, Corynebacterium
or
Brevibacterium. The vector DNA is cleaved by the same restriction enzyme used
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for cleavage of the chromosomal gene or is connected to an oligonucleotide
having a complementary base sequence at the respective terminals of the
chromosomal DNA cleavage fragment and the cleaved vector DNA. The plasmid
vector and the chromosomal gene-containing fragment are then subjected to a
ligation reaction. When a gene is inserted by this or any other method in the
sense
direction and in proper reading frame so that the PEP carboxylase enzyme is
expressed when the plasmid is transcribed and translated by the genetic
machinery
of a cell in which the plasmid is inserted, the gene is said to be
"operationally
inserted" into the plasmid vector.
In a preferred embodiment, the gene encoding the polypeptide having PEP
carboxylase activity is derived from an alfalfa plant. Most preferably, the
gene is
derived from a Medicago sativa strain. In another preferred embodiment, the
gene is modified by one or more nucleotide substitutions, deletions and/or
insertions. Most preferably, the modification comprises deleting the
nucleotides
encoding the amino acid sequence: Met - Ala - Ser - Ile - Asp - Ala - Gln -
Leu -
Arg.
In another aspect of the invention there is provided a host microorganism
transformed with a DNA fragment of the present invention containing a gene
encoding a polypeptide having PEP carboxylase activity. As the host,
microorganisms utilized for the production of L-amino acids may be used, for
example, those belonging to the genus Brevibacterium, the genus
Corynebacterium, the genus Bacillus, the genus Escherichia, the genus Seratia,
the genus Providencia, and the genus Arthrobacter.
In a preferred embodiment, the DNA fragment containing the ppc gene is
expressed in a host microorganism belonging to the genus Escherichia,
Corynebacterium or Brevibacterium. As the host, there may be exemplified
microorganisms belonging to the genus Escherichia, for example, Escherichia
coli, preferably L-lysine-producing Escherichia coli, coryneform bacteria,
preferably L-lysine-producing strains, and the like. The coryneform bacteria
referred to in the present invention is a group of microorganisms which are
aerobic
Gram-positive non-acid-fast rods having no spore-forming ability, including
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bacteria belonging to the genus Corynebacterium, bacteria belonging to the
genus
Brevibacterium having been hitherto classified into the genus Brevibacterium
but
being united as bacteria belonging to the genus Corynebacterium at present,
and
bacteria belonging to the genus Brevibacterium closely related to bacteria
belonging to the genus Corynebacterium.
In one embodiment, when the DNA fragment is derived from a plant from
the class Monocotyledonae or Dicotyledonae, the host microorganism may be
transformed with a recombinant DNA molecule comprising a plasmid and the
DNA fragment operationally inserted therein. Alternatively, the host
microorganism may be transformed by integrating the DNA fragment of the
present invention into the host chromosomal DNA.
Preferably, the DNA fragment is derived from an alfalfa plant, and most
preferably, it is derived from a Medicago sativa strain. In another preferred
embodiment, the plant-derived DNA fragment is modified by one or more
nucleotide substitutions, deletions and/or insertions. Most preferably, the
modification comprises deleting the nucleotides encoding the amino acid
sequence: Met - Ala - Ser - Ile - Asp - Ala - Gln - Leu - Arg.
Further, as described above, it is acceptable that the DNA sequence of the
present invention is inserted into vector DNA capable of self replication and
introduced into the host. As the vector DNA, a plasmid vector is preferable,
and
those capable of self replication in a host cell are most preferable.
Alternatively,
a vector of phage DNA can be also utilized.
When the DNA fragment containing a gene is derived from a plant of the
class Monocotyledonae or Dicotyledonae or from a microorganism belonging to
the genus Corynebacterium or Brevibacterium, it is also acceptable that the
DNA
fragment is integrated into the chromosomal DNA of a host microorganism by
means of a method using, for example, transposons (Berg, D.E. and Berg, C.M.,
BiolTechnol. 1:417 (1983)), Mu phage (Japanese Patent Laid-open No. 2-
109985) or homologous recombination (Experiments in Molecular Genetics, Cold
Spring Harbor Lab. ( 1972)). In addition, in order to integrate the DNA of the
present invention into the coryneform bacteria, it is possible to utilize a
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temperature-sensitive plasmid as disclosed in Japanese Patent Laid-open No.
5-7491.
In a preferred embodiment, the DNA fragment is derived from a
Corynebacterium glutamicum strain and is integrated into the chromosomal DNA
of a host microorganism. The region flanking the ppc gene in the
Corynebacterium glutamicum chromosome has been sequenced (SEQ ID NO: 3).
According to the gene replacement strategy of the present invention, the
chromosomal copy of the ppc gene is removed and replaced with an antibiotic
resistance gene marker (Figure 1 ). The marker is in turn replaced with a
modified
ppc gene of the present invention.
The unique design of this gene replacement strategy facilitates complete
removal of the chromosomal ppc DNA sequence of a host microorganism and
substitution of a new ppc gene without altering the expression of the two
neighboring genes, the tpi gene and the sect gene. The tpi gene encodes the
glycolytic enzyme triosephosphate isomerase, and the sect gene encodes sect,
an integral membrane protein involved in protein export.
The design of this gene replacement strategy depends upon the
reconstitution of intact tpi and sect genes that flank the ppc gene. Four
oligonucleotides can be used to clone the DNA regions flanking ppc:
(1) 5' GTTGG TGAGC CACTG GAAAT CCGTG 3' (SEQ ID:NO 4)
(2) 5' GATGT CATCG CGTAA AAAAT CAGTC 3' (SEQ ID:NO 5)
(3) 5' CACTG CGCTG CGCAA CTCTA GATAG 3' (SEQ ID:NO 6)
(4) 5' GACCA CCACC TTGCC GAAAT CTTGG 3' (SEQ ID:NO 7).
In another aspect of the present invention there is provided a method of
producing an amino acid by fermentation. The method comprises cultivating a
host microorganism belonging to the genus Escherichia, Co~ynebacterium or
Brevibacterium in a suitable medium and isolating from the culture medium an
amino acid, wherein the host microorganism is transformed with a DNA fragment
comprising a gene encoding a polypeptide having PEP carboxylase activity,
wherein the host microorganism expresses the gene, and wherein the polypeptide
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does not require acetyl-CoA for activation and is desensitized to feedback
inhibition by aspartic acid.
The method for cultivating the aforementioned hosts is not especially
different from a cultivation method for amino acid-producing microorganisms in
the prior art. Namely, an ordinary medium is used containing a carbon source,
a
nitrogen source, inorganic ions, substances satisfying nutrient auxotrophy,
and
optionally organic trace nutrients such as amino acids, vitamins and the like.
As the carbon source, carbohydrates such as glucose, sucrose, lactose,
etc., as well as organic acids such as acetic acid may be used. As the
nitrogen
source, ammonia gas, aqueous ammonium, ammonium salt and the like can be
used. As inorganic ions, potassium ions, sodium ions, magnesium ions,
phosphate
ions, and the like are appropriately added to the media as required.
The cultivation is performed until the generation and accumulation of the
amino acid substantially stops while suitably controlling pH and temperature
of the
medium under an aerobic condition. In order to collect amino acids thus
accumulated in the cultivated medium, an ordinary method can be applied. For
example, after the removal of the cells by filtration, ultrafiltration,
centrifugation
or other known means, the amino acid is recovered, for example, by
concentration
of the cell-free solution and crystallization of the amino acid (or a salt
thereof).
Alternatively, the compound can be recovered by ion exchange chromatography.
In a preferred embodiment, the amino acid is one which is derived from
OAA, such as L-aspartate, L-lysine, L-methionine, L-threonine and L-
isoleucine.
Most preferably, the amino acid is L-lysine.
In another aspect of the invention there is provided a method of increasing
the rate of conversion of PEP to OAA. The method comprises transforming a
host microorganism with a DNA fragment of the present invention. In a
preferred
embodiment, the host microorganism is selected from the genus Escherichia,
Corynebacterium or Brevibacterium.
PEP carboxylase catalyzes the condensation reaction between PEP and
carbon dioxide resulting in the formation of OAA. A PEP carboxylase of the
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present invention that is not substantially regulated by acetyl-CoA or
aspartic acid
therefore increases the rate of conversion of PEP to OAA.
In the case wherein the DNA fragment is derived from a plant belonging
to the class Monocotyledonae or Dicotyledonae, transformation may be by
integration or by utilization of a recombinant DNA molecule, for example. In
the
case wherein the DNA fragment is derived from a microorganism belonging to the
genus Corynebacterium or Brevibacterium, the host microorganism is
transformed by the integration of the DNA fragment of the invention into the
chromosomal DNA of the host microorganism.
In another aspect of the invention there is provided a method of recycling
carbon in a fermentation process. The method comprises transforming a host
microorganism with a DNA fragment of the present invention. In a preferred
embodiment, the host microorganism is selected from the genus Escherichia,
Corynebacterium or Brevibacterium.
The TCA cycle requires continuous replenishment of C4 molecules in order
to replace the intermediates withdrawn for amino acid biosynthesis. PEP
carboxylase aids in fulfilling this function by playing an anaplerotic role in
supplying the four carbon OAA to the TCA cycle. By transforming a host
microorganism with the DNA fragment of the present invention which codes for
a polypeptide having PEP carboxylase activity, a method for recycling carbon
is
thereby provided.
In the case wherein the DNA fragment is derived from a plant belonging
to the class Monocotyledonae or Dicotyledonae, transformation may be by
integration or by utilization of a recombinant DNA molecule, for example. In
the
case wherein the DNA fragment is derived from a microorganism belonging to the
genus Corynebacterium or Brevibacterium, the host microorganism is
transformed by the integration of the DNA fragment of the invention into the
chromosomal DNA of the host microorganism.
L-lysine and L-glutamic acid have been hitherto industrially produced by
fermentative methods by using coryneform bacteria belonging to the genus
Brevibacterium or Corynebacterium having abilities to produce these amino
acids.
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In these methods, it is known that the coryneform bacteria require biotin for
their
growth. The enzyme PEP carboxylase does not require biotin for biological
activity. In addition, one of the major physiological roles of PEP carboxylase
is
to replenish the TCA cycle by the assimilation of carbon. The de-regulated PEP
carboxylase of the present invention improves the assimilation of carbon
dioxide.
Therefore, in another aspect of the invention there is provided a method
of assimilating carbon in a fermentation process which does not require
biotin.
The method comprises transforming a host microorganism with a DNA fragment
of the present invention. In a preferred embodiment, the host microorganism is
selected from the genus Escherichia, Corynebacterium or Brevibacterium.
In the case wherein the DNA fragment is derived from a plant belonging
to the class Monocotyledonae or Dicotyledonae, transformation may be by
integration or by utilization of a recombinant DNA molecule, for example. In
the
case wherein the DNA fragment is derived from a microorganism belonging to the
genus Corynebacterium or Brevibacterium, the host microorganism is
transformed by the integration of the DNA fragment of the invention into the
chromosomal DNA of the host microorganism.
The anaplerotic enzyme PEP carboxylase is critical to the maintenance of
an optimal pool of OAA, and consequently determines the biosynthetic levels of
organic acids deriving from it. By transforming a host microorganism with the
DNA fragment of the present invention, the rate of production of OAA is
increased. As such, the production of organic acids derived from OAA is
increased as well.
Accordingly, in yet another aspect of the present invention there is
provided a method of increasing the production of organic acids in a
fermentation
process. In a preferred embodiment, the host microorganism is selected from
the
genus Escherichia, Corynebacterium or Brevibacterium.
In the case wherein the DNA fragment is derived from a plant belonging
to the class Monocotyledonae or Dicotyledonae, transformation may be by
integration or by utilization of a recombinant DNA molecule, for example. In
the
case wherein the DNA fragment is derived from a microorganism belonging to the
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genus Corynebacterium or Brevibacterium, the host microorganism is
transformed by the integration of the DNA fragment of the invention into the
chromosomal DNA of the host microorganism.
OAA is an important substrate for the production of cell metabolites such
as amino acids. By increasing the rate of conversion of PEP to OAA, the ppc
genes of the invention thereby increase the production of amino acids.
Therefore,
in another aspect of the invention there is provided a method of increasing
the
production of amino acids in a fermentation process. The method comprises
transforming a host microorganism with a DNA fragment of the present
invention.
In a preferred embodiment, the host microorganism is selected from the
genus Escherichia, Corynebacterium or Brevibacterium. In another preferred
embodiment, the amino acid comprises L-aspartate, L-lysine, L-methionine, L-
threonine and L-isoleucine. Most preferably, the amino acid is L-lysine.
In the case wherein the DNA fragment is derived from a plant belonging
to the class Monocotyledonae or Dicotyledonae, transformation may be by
integration or by utilization of a recombinant DNA molecule, for example. In
the
case wherein the DNA fragment is derived from a microorganism belonging to the
genus Corynebacterium or Brevibacterium, the host microorganism is
transformed by the integration of the DNA fragment of the invention into the
chromosomal DNA of the host microorganism.
All patents and publications cited in this disclosure are indicative of the
level of skill of those skilled in the art to which this invention pertains
and are all
herein incorporated by reference in their entirety.
Having now generally described the invention, the same will be more
readily understood through reference to the following Examples which are
provided by way of illustration, and are not intended to be limiting of the
present
invention, unless specified.
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Example 1
A Plant ppc Gene Functions in Escherichia coli
The cDNA clone (APPC) of the ppc gene from alfalfa (Medicago sativa)
was functional in the Escherichia coli mutant CGSC3594 which lacks a
functional
S PEP carboxylase and cannot grow on M9 medium with glucose as the sole carbon
source. When transformed with the APPC plasmid (pMS2), E. coli mutant
CGSC3594 was able to grow on M9 medium with glucose as the sole carbon
source. The DNA and amino acid sequences of the alfalfa PEP carboxylase are
provided in SEQ ID NO:1 and SEQ ID N0:2, respectively.
Example 2
The ppc Gene from Alfalfa Shows Growth Stimulation in
Corynebacterium in Shake Flasks
The effect of the ppc gene from alfalfa (Medicago sativa) on growth
stimulation in the lysine-producing Corynebacterium strain BF 100 was
determined. Growth was measured as the optical density at 660nm, the titer was
measured as g lysine/liter of medium, and the yield was measured as (g
lysine/g
glucose consumed) x 100. 30 mg/L of isopropyl-beta-D-galactoside (IPTG), an
inducer, was present. The results are shown in Table 1:
Table 1
Strain Growth Titer Yield
BF100 25 25 42
BF100/pMS2 34 23 40
BF100/pMS2/IPTG40 25 43
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Example 3
The ppc Gene from a Wild Type Corynebacterium Strain Improves
Productivity of a Lysine-Producing Corynebacterium Strain
The cDNA clone (CPPC) of the ppc gene from Corynebacterium
glutamicum ATCC 13032 was inserted into the pCPPC plasmid. When lysine
producing Corynebacterium glutamicum strain BF100 was transformed with the
pCPPC plasmid in shake flasks, the productivity was improved.
Growth was measured as the optical density at 660nm, the titer was
measured as g lysine/liter of medium, and the yield was measured as (g
lysine/g
glucose consumed) x 100. The results are shown in Table 2:
Table 2
Strain Growth Titer Yield
BF100 39 27 44
BF100/pCPPC 32 29 48
Example 4
Sensitivities to Acetyl CoA and L Aspartic Acid from Wild type and Lysine
Producing Corynebacterium Strains
Different sensitivities to acetyl-CoA and L-aspartic acid were observed in
extracts from a wild-type Corynebacterium glutamicum strain (ATCC 13032) and
a lysine-producing Corynebacterium glutamicum strain (BF 100) as determined by
PEP carboxylase activity. Activity units were measured spectrophotometrically
as the change in absorbance (340 nm/min) using crude extracts. The results are
shown in Table 3:
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Table 3
Strain PEP Carboxylase
Activity
Complete -Acetyl CoA +Aspartate (5mM)
ATCC 13032 100% 56% 100%
BF100 100% 15% 17%
Example S
Replacement of a Chromosomal ppc Gene With a Modified ppc Gene
The region flanking the ppc gene in the Corynebacterium glutamicum
chromosome has been sequenced (SEQ ID NO: 3). The chromosomal copy of the
ppc gene is removed and replaced with an antibiotic resistance gene marker
(Figure 1 ). The marker is in turn replaced with a modified ppc gene of the
present
invention. The unique design of this gene replacement strategy facilitates
complete removal of the chromosomal ppc DNA sequence of a host
microorganism and substitution of a new gene without altering the expression
of
the two neighboring genes.
The design of this gene replacement strategy depends upon the
reconstitution of intact tpi and sect genes that flank the ppc gene. Four
oligonucleotides can be used to clone the DNA regions flanking ppc:
(1) 5' GTTGG TGAGC CACTG GAAAT CCGTG 3' (SEQ ID:NO 4)
(2) 5' GATGT CATCG CGTAA AAAAT CAGTC 3' (SEQ ID:NO 5)
(3) 5' CACTG CGCTG CGCAA CTCTA GATAG 3' (SEQ ID:NO 6)
(4) 5' GACCA CCACC TTGCC GAAAT CTTGG 3' (SEQ ID:NO 7).
In view of the foregoing description taken with the Examples, those skilled
in the art will be able to practice the invention in various enablements and
embodiments without departing from the spirit and scope of the invention as
defined in the appended claims.
