U.S. patent application number 10/549262 was filed with the patent office on 2006-09-14 for nucleotide sequences of coryneform bacteria coding for proteins involved in l-serine metabolism and method for producing l-serine.
Invention is credited to Lothar Eggeling, Roman Netzer, Petra Peters-Wendisch, Hermann Sahm.
Application Number | 20060204963 10/549262 |
Document ID | / |
Family ID | 32892226 |
Filed Date | 2006-09-14 |
United States Patent
Application |
20060204963 |
Kind Code |
A1 |
Peters-Wendisch; Petra ; et
al. |
September 14, 2006 |
Nucleotide sequences of coryneform bacteria coding for proteins
involved in l-serine metabolism and method for producing
l-serine
Abstract
The invention relates to the nucleotide sequences of coryneform
bacteria coding for proteins which are involved in L-serine
metabolism with reduced and switched off L-serine dehydratase. Said
invention also relates to micro-organisms and to methods for
producing L-serine.
Inventors: |
Peters-Wendisch; Petra;
(Julich, DE) ; Netzer; Roman; (Julich, DE)
; Eggeling; Lothar; (Julich, DE) ; Sahm;
Hermann; (Julich, DE) |
Correspondence
Address: |
THE FIRM OF KARL F ROSS
5676 RIVERDALE AVENUE
PO BOX 900
RIVERDALE (BRONX)
NY
10471-0900
US
|
Family ID: |
32892226 |
Appl. No.: |
10/549262 |
Filed: |
February 12, 2004 |
PCT Filed: |
February 12, 2004 |
PCT NO: |
PCT/DE04/00248 |
371 Date: |
May 10, 2006 |
Current U.S.
Class: |
435/6.14 ;
435/116; 435/190; 435/252.3; 435/471; 536/23.2 |
Current CPC
Class: |
C12P 13/06 20130101;
C12N 9/88 20130101 |
Class at
Publication: |
435/006 ;
435/116; 435/252.3; 435/190; 435/471; 536/023.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C12P 13/06 20060101
C12P013/06; C12N 9/04 20060101 C12N009/04; C12N 1/21 20060101
C12N001/21; C12N 15/74 20060101 C12N015/74 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 13, 2003 |
DE |
103 11 399.1 |
Claims
1. A nucleic acid which is replicatable in a microorganism of the
family Corynebacterium and optionally a recombinant nucleic acid,
characterized in that it has a nucleotide sequence coding for
L-serine dehydratase which is partially or completely mutated or
expressed to a lesser degree than the naturally occurring
nucleotide sequence or which is not expressed at all.
2. A nucleic acid according to claim 1, characterized in that the
sdaA gene sequence is partially or completely deleted or mutated or
expressed to a lesser extent by comparison with the naturally
occurring sequence or not expressed at all.
3. A nucleic acid according to claim 1, characterized by a
nucleotide sequence according to SEQ ID NO 1 whose nucleotides form
position 506 to position 918 are completely or partially deleted or
mutated, or an allele, homolog or derivative of this nucleotide
sequence or a nucleotide sequence hybridizing therewith.
4. A nucleic acid according to claim 1, characterized in that it is
isolated from a coryneform bacterium.
5. A nucleic acid according to claim 1, characterized in that it is
isolated from Corynebacterium or Brevibacterium.
6. A nucleic acid according to claim 1, characterized in that it is
isolated from Corynebacterium glutamicum or Brevibacterium
flavum.
7. A gene structure containing at least one nucleotide sequence
according to claim 1 and nucleotide sequences having regulatory
sequences operatively linked therewith.
8. A vector containing at least one nucleotide sequence or a gene
structure according to claim 7 and additional nucleotide sequences
for selection, for replication in the host cell or for integration
in the host cell genome.
9. L-serine dehydratase with reduced L-serine dehydratase activity
coded with a nucleic acid according to claim 1.
10. L-serine dehydratase according to claim 9 with an amino acid
sequence according to sequence ID 2 whose amino acid are altered in
positions 135 to 274 or a modified form of this polypeptide
sequence or an isoform thereof.
11. L-serine dehydratase according to claim 9, characterized in
that it derives from coryneform bacteria.
12. L-serine dehydratase according to claim 9, characterized in
that it derives from coryneform bacteria or brevibacteria.
13. L-serine dehydratase according to claim 9, characterized in
that it derives from Corynebacterium glutamicum or Brevibacterium
flavum.
14. A microorganism characterized in that it has a nucleotide
sequence which codes for an L-serine dehydratase, which is deleted
in whole or in part or is mutated or is expressed to a reduced
extent by comparison with the naturally occurring nucleotide
sequence or is not expressed at all.
15. A microorganism according to claim 14, characterized in that
its sdaA gene is wholly or partially deleted or mutated or to a
reduced extent by comparison with the naturally occurring sdaA gene
or is not expressed at all.
16. A microorganism according to containing in replicatable form a
nucleic acid according to claim 1.
17. A microorganism according to claim 14, characterized in that it
is a coryneform bacteria.
18. A microorganism according to claim 14, characterized in that it
brings to the family a coryneform bacteria or brevibacteria.
19. A microorganism according to claim 14, characterized in that it
brings to the family a Corynebacterium glutamicum or Brevibacterium
flavum.
20. A probe for identifying and/or genes for coding which
participate in the biosynthesis of L-serine characterized in that
they are produced starting with nucleic acids according to claim 1
and contain a suitable marker for detection.
21. A method for the microbial production of L-serine characterized
in that (a) a genetically altered microorganism is produced in
which the nucleic acid in the microorganism coding for the L-serine
dehydratase is partially or completely deleted or mutated or
expressed to a reduced extent by comparison with the naturally
occurring nucleic acid or is not expressed at all, (b) this
genetically altered microorganism from step (a) is used for
microbial production, and (c) the L-serine formed is isolated from
the culture medium.
22. The method according to claim 21, characterized in that the
sdaA gene sequence is partially or completely deleted or mutated or
expressed to a reduced extent by comparison with the naturally
occurring nucleotide sequence or is not expressed at all.
23. The method according to claim 21, characterized in that the
nucleotide according to Sequence ID NO 1 is completely or partially
deleted or mutated from position 506 to 918 or expressed to a
reduced extent by comparison with the naturally occurring
nucleotide sequence or not expressed at all.
24. The method according to claim 21, characterized in that a
microorganism from the group of Corynebacterium, Brevibacterium,
Arthrobacter, Pseudomonas, Nocardia, Methylobacteria,
Hyphomicrobium, Alkaligenes or Klebsiella is used.
25. A method wherein a nucleic acid according to claim 1 is used.
Description
[0001] The invention relates to nucleotide sequences of coryneform
bacteria coded for proteins which participate in L-serine
metabolism with reduced or omitted L-serine dehydratase and
microorganisms for and method of making L-serine.
[0002] The amino acid L-serine has been found to be useful in the
food industry, the animal feed industry and the pharmaceutical
industry as well as in human medicine. It serves as a building
block for the synthesis of other industrially valuable products
like for example L-tryptophan from indole and L-serine.
[0003] It is known that L-serine can be produced by the
fermentation of coryneform bacteria strains. Thus for example a
strain of Corynebacterium glycinophilum is capable of forming
L-serine from glycine and carbohydrates (Kubota K, Kageyama K,
Shiro T and Okumura S (1971) Journal of General Applications in
Microbiology, 17: 167-168; Kubota K, Kageyama K, Maeyashiki I,
Yamada K and Okumura S (1972) Journal of General Applications in
Microbiology 18: 365). The enzyme L-serine-hydroxymethyltransferase
here participates in the conversion of glycine to L-serine. (Kubota
K and Yokozeki K (1989) Journal of Fermentation and Bioengineering,
67(6):387-390). These Corynebacterium glycinophilum strains have a
defective serine dehydrataze which produces undirected mutagenesis
(Kubota K (1985) Improved production of L-serin by mutants of
Corynebacterium glycinophilum with less serine dehydratase
activity. Agricultural Biological Chemistry, 49:7-12). This
enzymatic activity is Pyridoxal 5'-Phosphate dependent and not
molecularly characterized. (Kubota K., Yokozeki K, Ozaki H. (1989)
Effects of L-serine dehydratase activity on L-serine production by
Corynebacterium glycinophilum of an examination of the properties
of the enzyme. Agric. Biol. Chem 49:7-12). From U.S. Pat. No.
4,528,273 a method of producing L-serine from glycine is known in
which the microorganism serine dehydratase is negative.
[0004] Furthermore, L-serine can be produced fermentatively from
methanol and glycine with the aid of methylotrophic bacteria like
for example Hyphomicrobium strains (Izumi Y, Yoshida T, Miyazaki S
S, Mitsunaga T, Ohshiro T, Shiamo M, Miyata A and Tanabe T (1993)
Applied Microbiology and Biotechnology, 39: 427-432). In both cases
the amino acid glycine must be introduced as a precursor for the
formation of the amino acid L-serine.
[0005] In addition, coryneform bacteria are known which can produce
the L-serine directly from carbohydrates without further addition
of precursors.
[0006] This is advantageous for industrial scale economical
production of L-serine since the L-serine can be made directly from
carbohydrates without the expensive addition of precursors, these
strains which belong to the family Corynebacterium glutamicum have
resistance to the L-serine analog serine hydroxamate and
.beta.-chloroalanine and are obtained by undirected mutagenesis
(Yoshida H and Nakamaya K (197) NIHON-Nogli-Kagakukaishi 48:
201-208).
[0007] There are also Brevibacterium flavum strains known which
have because of undirected mutagenesis defects in the breakdown of
L-serine, an enhanced activity of the serA coded 3-phosphoglycerate
dehydrogenase and an overexpression of serB and serC genes deriving
from Escherichia coli (EP0931833A2).
[0008] It is the object of the invention to make available features
which will permit improved production of L-serine or metabolic
products which derive therefrom like for example tryptophan. It is
thus also an object of the invention to provide nucleic acids which
code for proteins participating in L-serine metabolism and which by
comparison with the proteins derived from the wild type organism
show a no decomposition of L-serine to pyruvate or a reduced
decomposition of L-serine to pyruvate. In this connection it is a
further object of the invention to provide an L-serine dehydratase
as well as microorganisms with an L-serine dehydratase shown to
reduce decomposition of L-serine. Further it is an object of the
invention to provide an improved method for the microbial
production of L-serine.
[0009] Starting from the preamble of claim 1, the objects are
achieved, in accordance with the invention with the features given
in the characterizing clause of claim 1. Furthermore, the objects
are achieved starting from the preamble of claim 7 with the
features given in the characterizing part of claim 7. The objects
are also attained starting from the preamble of claim 8 according
to the invention with the features given in the characterizing part
of claim 8. The objects are also achieved starting with the
preamble of claim 9 with the features given in the characterizing
part of claim 9. The objects are also achieved starting with the
preamble of claim 14, in accordance with the invention, with the
features given in the characterizing part of claim 14. Starting
with the preamble of claim 20, the objects are also achieved
according to the invention by the features given in the
characterizing part of claim 20. Furthermore, the objects are
attained according to the invention starting from the preamble of
claim 21 by the features of the characterizing part of claim
21.
[0010] With the nucleic acids and polypeptides according to the
invention it is possible to produce an L-serine dehydratase such
that there is a reduced decomposition of L-serine or no longer any
decomposition of L-serine. Furthermore, it is possible to provide
microorganisms and a method by which L-serine production can be
obtained with higher yield by comparison with hitherto known
microbial methods.
[0011] Further advantages have been given in the dependent
claims.
[0012] According to the invention, in microorganisms of the
corynebacterium family, replicatable and optionally recombinant
nucleic acid is provided with nucleotide sequence coding for the
L-serine dehydratase, hereinafter referred to also as SDA, which is
partially or completely deleted or mutated or is expressed to a
reduced extent by comparison with the naturally occurring
nucleotide sequence or is not expressed at all.
[0013] The subject of the invention is, further, the provision of
nucleic acids whose sdaA gene sequence is partially or completely
deleted or mutated or has, relative to the naturally available
nucleotide sequence reduced expression or which does not express at
all. For example the nucleic acids with a nucleotide sequence
according to SEQ ID No 1 can have its nucleotides from position 506
to position 918, partly or completely deleted or mutated or can be
allele, homologue or derivative of this nucleotide sequence or a
nucleotide sequence which hybridizes therewith have been found to
be advantageous. In addition, it has been found to be advantageous
for the deletion or mutation of the cystein-containing sequence
required for forming the iron-sulfur clusters (Hofmeister et al.,
(1994) Iron-sulfur cluster-containing L-serine dehydratase from
Peptostreptococcus asaccharolyticus: correlation of the cluster
type with enzymatic activity. FEBS Letters 351: 416-418) has been
found to be advantageous.
[0014] The wild type L-serine-dehydratase (sdaA) gene sequence is
generally known and can be obtained by the artisan from the known
data bank (NCBI Accession Nr. AP005279) or from the attached
sequence protocol according to SEQ ID No. 1.
[0015] The complete deletion of the L-serine dehydratase (sdaA)
gene can be achieved for example by directed recombinant DNA
techniques. Suitable methods for this purpose are found in Schafer
et al. (Gene (1994) 145: 69-73) or also Link et al. (Journal of
Bacteriology (1998) 179: 6228-6237). Furthermore, only a part of
the gene can be deleted or also mutated fragments of the L-serine
dehydratase gene can be formed by replacement. By deletion or
replacement it is possible to achieve a loss or a reduction in the
L-serine dehydratase activity. An example of such a mutant is the
C. glutamicum strain ATCC133032.DELTA.sdaA which has a deletion in
the sdaA gene.
[0016] To limit the expression of the sdaA gene or achieve reduced
expression, for example, the promoter and regulatory regions which
are located upstream of the structural gene can be mutated. In a
similar manner, expression regulatory cassettes can be built onto
the structural gene, upstream thereof. By regulatable promoters it
is additionally possible to reduce the expression in the course of
fermentative L-serine formation. It is also possible to provide a
regulation of the translation in which for the example of stability
of the m-RNA is reduced. Furthermore, genes can be used which code
for the corresponding enzyme with reduced activity. Alternatively,
furthermore, a reduced expression of the L-serine dehydratase gene
can be achieved by varying the medium composition and culture
conditions. Guides thereto for the artisan can be found among
others in Martin et al. (Bio/Technology 5, 137-146 (1987)), by
Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga
(Bio/Technology 6, 428-430 (1988)), Eikmanns et al. (Gene 102,
93-98 (1991)), in the European Patent EPS 0 472 869, U.S. Pat. No.
4,601,893, Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991),
Reinscheid et al. (Applied and Environmental Microbiology 60,
126-132 (1994), LaBarre et al. (Journal of Bacteriology 175,
1001-1007 (1993)) and in patent application WO 96/15246.
[0017] The nucleic acids according to the invention are
characterized that they can be isolated from the coryneform
bacteria, preferably of corynebacterium or brevibacterium family
and especially preferably from Corynebacterium glutamicum. Examples
of the coryneform bacteria wild types, from this parental line are
for example,
[0018] Corynebacterium acetoacidophilum ATCC 13870;
[0019] Corynebacterium acetoglutamicum ATCC 15806;
[0020] Corynebacterium callunae ATCC 15991;
[0021] Corynebacterium glutamicum ATCC 13032;
[0022] Brevibacterium divaricatum ATCC 14020;
[0023] Brevibacteriium lactofermentum ATCC 13869;
[0024] Corynebacterium lilium ATCC 15990;
[0025] Brevibacterium flavum ATCC 14067;
[0026] Corynebacterium melassecola ATCC 17965;
[0027] Brevibacterium saccharolyticum ATCC 14066;
[0028] Brevibacterium immariophilum ATCC 14068;
[0029] Brevibacterium roseum ATCC 13825;
[0030] Brevibacterium thiogenitalis ATCC 19240;
[0031] Microbacterium ammoniaphilum ATCC 15354.
[0032] Examples of the production of mutants or production strains
suitable for the production of L-serine are organisms from the
group of Arthrobacter, Pseudomonas, Nocardia, Methylobacterium,
Hyphomycrobium, Alcaligenes or Klebsiella. The present invention is
characterized more particularly by the naming of the aformentioned
bacterial strains but should not be considered limited thereto.
[0033] By a "nucleic acid" or a "nucleic acid fragment" there is
meant, in accordance with the invention, a polymer of RNA or DNA
which can be single stranded or double stranded and can have
optional natural, chemically synthesized, modified or artificial
nucleotides. The term "DNA polymer" includes in this case also
genomic DNA, cDNA or mixtures thereof.
[0034] Under "alleles" are to be understood functional equivalents
in accordance with the invention, that is substantially similarly
effective nucleotide sequences. Functionally equivalent sequences
are such sequences which, in spite of different nucleotide
sequences, for example because of the degeneration of the genetic
code, still retain the desired function. Functional equivalents
thus encompass naturally occurring variants of the sequences
described therein as well as synthetic nucleotide sequences, for
example those obtained by chemical synthesis and optionally
nucleotide sequences matched to the codon requirements of the host
organism.
[0035] Under a functional equivalent is to be understood especially
also natural or synthetic mutations of the original isolated
sequence which retain the desired function. Mutations include
substitutions, additions, deletions, replacements or insertions of
one or more nucleotide residues. Included here are also sense
mutations which in the protein plane can result for example from
the replacement of conserved amino acids which however do not lead
to any basic alteration in the activity of the protein and thus can
be considered functionally neutral. This includes modifications of
the nucleotide sequence which involve in the protein plane the
N-terminus of a protein without however affecting significantly the
function of these proteins.
[0036] With the present invention, such nucleotide sequences are
encompassed which, by modification of the nucleotide sequences can
result in corresponding derivatives. The target of such
modification can, for example, be a restriction of the coding
sequence contained therein or for example also the insertion of
further restriction enzyme cutting sites.
[0037] In addition, the present invention includes artificial DNA
sequences as long as they, as described above, afford the desired
characteristics. Such artificial DNA sequences can, for example, be
those obtained by reverse translation from proteins established by
means of computer supported programming (molecular modeling) or by
in vitro selection. Especially suitable are coded DNA sequences
which, by reverse translation, can produce a polypeptide sequence
which has a specific code on utilization for the host organism. The
specific code on utilization can be easily determined by molecular
genetic methods common in the art using computer evaluations from
other previously known genes of the organism to be transformed.
[0038] "Homologous sequences" are to be understood in accordance
with the invention to be those sequences which are complementary to
the nucleotide sequences according to the invention and/or such
sequences which can hybridize with them. The hybridizing sequences
include, according to the invention, substantially similar
nucleotide sequences from the group of DNA or RNA which under
stringent conditions known per se undergo a specific interaction
(binding) of the aforementioned nucleotide sequences. In this
category are to be counted also short nucleotide sequences with a
length of for example 10 to 30 and preferably 12 to 15 nucleotides.
These include according to the invention among others, also
so-called primers or probes.
[0039] Included in the invention are also the coding regions
(structure genes) and preceding (5' or upstream) sequence regions
and/or following (3' or downstream) sequence regions. Especially in
this category are sequence regions with regulatory functions. They
can influence the transcription, the RNA stability or RNA
processing as well as the translation. Example of regulatory
sequences are, among others, promoters, enhancers, operators,
terminators or translation amplifiers.
[0040] The subject of the invention is in addition a gene structure
containing at least one of the aforedescribed nucleotide sequences
and regulatory sequences operatively linked therewith which control
expression of the coded sequences in the host cell.
[0041] In addition the present invention relates to a vector
containing a nucleotide sequence of the aforedescribed kind with
its regulator nucleotide sequence operatively linked thereto as
well as additional nucleotide sequences for the selection of host
cells capable of effecting transformation, for replication within
the host cell or for integration in the corresponding host cell
genome. In addition, the vector according to the invention can
contain a genome structure of the aforedescribed type. Suitable
vectors are those which replicate in coryneform bacteria like for
example pZ1 (Menkel E, Thierbach G, Eggeling L, Sahm H., 1989, Appl
Environ Microbiol 55(3): 684-688), pEKEx2 (Eikmanns et al., Gene
102: 93-98 (1991), or pXMJ19 (Jacoby M., Burkovski A (1999)
Construction and application of new Corynebacterium glutamicum
vectors, Biotechnol. Technique 13:437-441). Other plasmid vectors
can be used in the same manner. These identifications are however
not limiting for the present invention.
[0042] Utilizing the nucleic acid sequence according to the
invention, corresponding probes or primers can be synthesized and
used, for example, to amplify and isolate analogous genes from
other microorganisms, preferably coryneform bacteria, for example
with the aid of the PCR technique.
[0043] The subject matter of the present invention is thus also a
probe for identifying and/or isolating genes coded for proteins
participating in the biosynthesis of L-serine, whereby these probes
are produced starting from the nucleic acid sequences according to
the invention of the aforedescribed type and which contain a
suitable marker for detection. In the probe, a partial segment of
the sequences according to the invention, for example a conserved
region, can be used which for example has a length of 10 to 30 or
preferably 12 to 15 nucleotides and under stringent conditions can
hybridize with homologous nitride sequences. Numerous suitable
markers are known from the literature. The skilled worker in he art
can be guided thereto by among others the Handbook of Gait:
Oligonucleotide synthesis: a practical approach (IRL Press, Oxford,
UK, 1984) and Newton and Graham: PCR (Spektrum Akademischer Verlag,
Heidelberg, Deutschland, 1994) or for example, the Handbook "The
DIG System Users Guide for Filter Hybridization" the Firma Roche
Diagnostics (Mannheim, Deutschland) and Liebl et al. (International
Journal of Systematic Bacteriology (1991) 41: 255:260).
[0044] The subject matter of the present invention includes,
further, an L-serine dehydratase which shows reduced L-serine
decomposition by comparison with the wild type L-serine dehydratase
and which is coded by a nucleic acid sequence according to the
invention or its variants of the aforedescribed type. The present
invention thus includes an L-serine dehydratase or an L-serine
dehydratase mutant with an amino acid sequence in accordance with
sequence ID No. 2 whose amino acids from position 135 to position
274, for example, as a consequence of a directed mutagenesis in the
DNA plane, is altered or is a modified form of this polypeptide
sequence or an isoform thereof or a mixture thereof. By "altered"
in the framework of the present invention one should understand
that complete or partial removal or replacement of the amino acids
from position 135 to position 274 is contemplated.
[0045] Under isoforms we understand enzymes with the same or
comparable substrate specificity and effectiveness specificity but
which differ with respect to the primary structure.
[0046] Under modified forms are to be understood enzymes according
to the invention with changes in the sequence, for example, at the
N-terminus or C-terminus of the polypeptide or in the regions of
the conserved amino acids without however negatively affecting the
function of the enzyme. These changes can be in the form of amino
acid replacement in accordance with methods known per se.
[0047] The polypeptides according to the invention are
characterized by the fact that they derive from coryneform bacteria
and preferably are of the corynebacterium or brevibacterium family
and especially of the Corynebacterium glutamicum or Brevibacterium
types and especially preferably derive from Corynebacterium
glutamicum. Examples of the coryneform bacteria in the strain
culture of the wild type are for instance
[0048] Corynebacterium acetoacidophilum ATCC 13870;
[0049] Corynebacterium acetoglutamicum ATCC 15806;
[0050] Corynebacterium callunae ATCC 15991;
[0051] Corynebacterium glutamicum AT CC 13032;
[0052] Brevibacterium divaricatum ATCC 14020;
[0053] Brevibacterium lactofermentum ATCC 13869;
[0054] Corynebacterium lilium ATCC 15990;
[0055] Brevibacterium flavum ATCC 14067;
[0056] Corynebacterium melassecola ATCC 17965;
[0057] Brevibacterium saccharolyticum ATCC 14066;
[0058] Brevibacteriium immariophilum ATCC 14068;
[0059] Brevibacterium roseum ATCC 13825;
[0060] Brevibacterium thiogenitalis ATCC 19240; and
[0061] Microbacterium ammoniaphilum ATCC 15354.
[0062] Examples of mutants or production strands suitable for the
production of L-serine are organisms from the group of
arthrobacter, pseudomonas, nocardia, methylobacterium,
hyphomycrobium, alcaligenes or klebsiella. The present invention
has been characterized by listing the aformentioned bacteria
strains, but this list should not be considered limiting of the
invention.
[0063] The present invention comprises, further, a genetically
altered microorganism characterized in that it contains a
nucleotide sequence coding for the L-serine dehydratase which is in
part or completely deleted or mutated or expressed to a reduced
extent by comparison with the naturally occurring nucleotide
sequence or which is not expressed at all.
[0064] The invention comprises further a microorganism which is
characterized in that the sdaA gene is partially or completely
deleted or mutated or which is expressed to a reduced extent by
comparison with the naturally occurring sdaA gene or which is not
expressed at all.
[0065] The invention encompasses as well a genetically altered
microorganism containing in replicatable form a gene structure or a
vector of the aforedescribed type.
[0066] The subject of the present invention is moreover also a
genetically modified microorganism containing a polypepetide
according to the invention of the aforedescribed type and which in
comparison to the corresponding genetically unmodified
microorganism has reduced or no L-serine decomposition.
[0067] A microorganism which, according to the invention has been
genetically modified is characterized further in that it is a
coryneform bacterium, preferably of the family Corynebacterium or
Brevibacterium and especially preferably of the species
Corynebacterium glutamicum or Brevibacterium flavum.
[0068] Basically the genes can, using methods known per se like for
example the polymerase chain reaction (PCR), be amplified by the
aid of short synthetic nucleotide sequences (primers) and then
isolated. The production of the primers used can be effected
generally based upon known gene sequences from existing homologies
in conserved regions of the gene and/or taking into consideration
the GC content of the DNA of the microorganism investigated.
[0069] A further procedure for isolating coding nucleotide
sequences is the complementation of so-called defect mutants of the
organism to be investigated which at least phenotypically show a
function drop in the activity of the gene investigated or the
corresponding protein. Under a complementation is to be understood
the preservation of the gene defect of the mutant and the
substantial reproduction of the original configuration before
mutagenesis which can be achieved by the insertion of functional
genes or gene fragments from the microorganism to be investigated.
A classical mutagenesis process for producing defect mutants or
mutants with a reduced L-serine dehydratase or an L-serine
dehydratase which has been shut down is for example the treatment
of the bacteria cell with chemicals like for example
N-Methyl-N-Nitro-N-Nitrosoguanidine or the use of UV radiation.
Such methods of mutation resolution are generally known and can be
found among others in Miller (A Short Course in Bacterial Genetics,
A Laboratory Manual and Handbook for Escherichia coli and Related
Bacteria (Cold Spring Harbor Laboratory Press, 1992)) or the
Handbook "Manual of Methods for General Bacteriology" of the
American Society for Bacteriology (Washington D.C., USA,
1981)).
[0070] The present invention relates moreover to a method for the
microbial production of L-serine whereby the nucleic acids in the
microorganisms which code for the L-serine dehydratase in part or
completely are deleted or mutated or expressed to a lesser extent
or practically not at all by comparison with the naturally
available nucleic acids, using these genetically altered
microorganisms for the microbial production of L-serine, and
isolating the correspondingly formed L-serine from the culture
medium.
[0071] The genetically altered microorganisms produced in
accordance with the invention can be used for the purpose of
culturing L-serine in continuous cultures or discontinuously in
batch processes (set cultivation) or in a fed batch process or a
repeated fed batch process. A collection of known cultivation
methods can be found in the textbook of Chmiel (Bioprozesstechnik
1. Einfuhrung in die Bioverfahrensechnik (Gustav Fischer Verlag,
Stuttgart, 1991)) or in the Storhas (Bioreaktoren und periphere
Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
[0072] The culture medium used must be suitable to suitably satisfy
the requirements of the respective strain. Descriptions of culture
media for various microorganisms can be found in the handbook
"Manual of Methods for General Bacteriology" der American Society
for Bacteriology" der American Society for baceriology (Washington
D.C., USA, 1981) as carbon sources, sugars and carbohydrates like
for example glucose, saccharose, lactose, fructose, maltose,
molasses, starch and cellulose can be used, oils and fats like for
example soy oil, soy flour oil, peanut oil, cocoanut fats can be
used, fatty acids like for example palmitic acid, stearic acid and
linolaic acids can be used, alcohols like for example glycerine and
ethanol can be used and organic acids like for example acetic acid
can be used. These substances can be employed individually or as
mixtures. As nitrogen sources, organic nitrogen containing
compounds like peptones, yeast extract, meat extract, malt extract,
corn spring water, soybean meal and urea, or inorganic compounds
like ammonium sulfate, ammonium chloride ammonium phosphate,
ammonium carbonate and ammonium nitride are used. The nitrogen
sources can be used individually or as mixtures. As phoshorous
sources, phosphoric acid, potasiuim dihydrogen phosphate or
dipotassium phosphate or the corresponding sodium-containing salts
are used. The culture medium must contain further salts of metal
like for example magnesium sulfate or iron sulfate which are
required for growth. Finally essential nutrients like amino acids
and vitamins are added to the above-mentioned substances. The
culture medium can in addition have suitable precursors added to
it. The additives can be introduced into the culture in the form of
one time addition or can be fed to the culture suitably during
cultivation. For pH control of the culture basic compounds like
sodium hydroxide, potassium hydroxide, ammonia or acqueous ammonia
can be used or acid compounds like phosphoric acid or sulfuric acid
can be used in a suitable way. For control of foaming, antifoaming
agents like for example fatty acid polyglycol esters can be used.
To maintain the stability of plasmids suitable selectively
effective substances, for example antibiotics can be added to the
medium. To maintain the aerobic conditions, oxygen or
oxygen-containing mixtures like for example air are introduced into
the culture. The temperature of the culture is normally between
20.degree. C. and 45.degree. C. and preferably 25.degree. C. to
40.degree. C. The culture is maintained for a duration until
L-serine production is a maximum. This duration is normally from 10
hours to 160 hours.
[0073] The analysis of the L-serine formation can be carried out by
anion exchange chromatography with subsequent ninhydrin
derivatization as described by Spackman et al. (Analytical
Chemistry, 30 (1958), 1190) or the analysis can be effected by
reverse phase HPLC as described by Lindroth et al. (Analytical
Chemistry (1979) 51: 1167-1174.
[0074] The microorganisms which are the subject of the present
invention can produce L-serine from glucose, saccharose, lactose,
mannose, fructose, maltose, molasses, starch, cellulose or from
glycerine and ethanol. The method can use the coryneforme bacteria
representatives which have already been described in detail. A
selection of the results of the fermentation has been given in
Table 1. The genetically altered microorganisms of the invention
show a substantially improved L-serine production by comparison
with the corrsponding nontransformed microorganism (wild type) or
the micororganisms which contain only the vector without the gene
insert. In a special variation of the present invention it has been
shown that C. Glutamicum ATCC 13032.DELTA.panBC.DELTA.sdaA gives
rise to at least a 4-fold increase in the L-serine accomulation in
the medium by comparison with the control strain (Table 1). Through
the common overexpression of other genes, which act positively on
the L-serine biosynthesis pathway, a 16-fold increase in L-serine
production can be achieved.
[0075] Amino acid production strains, in accordance with the
present invention should be understood to be Corynebacterium
glutamicum strains or homologous microrganisms which are altered by
classical and/or molecular genetic methods so that metabolic flow
is amplified in the direction of the biosynthesis of amino acids or
their derivatives (metabolic engineering). For example, with these
amino acid production strains, one or more genes and/or the
corresponding enzyme have their regulation altered or are rendered
deregulated at different and correspondingly complex regulated key
positions in the metabolic pathway. The present invention includes
thereby all such already known amino acid production strains
preferably of the corynebacterium family or homologous
organisms.
[0076] Further, such production strains are encompassed within the
invention which the skilled worker in the art will recognize by
analogy with other microorganisms, for example, enterobacteria,
bacillaceen or yeast types can be produced by current methods.
[0077] The Figures show examples of plasmids which can be used as
well as experimental results with respect to nucleic acids or
microorganisms according to the invention.
[0078] It shows:
[0079] FIG. 1 The integration plasmid pK19mobsacB-DeltasdaA
Markings on the outer edge of the plasmid indicate the respective
restriction sites. The portion within the circle indicates the
following gene: TABLE-US-00001 kan canamycin resistance sacB
Sucrase OriT Transfer origin sdA' 5' end of the sdaA gene sda'' 3'
end of the sdaA gene
[0080] FIG. 2: A graph of the ratio between growth (square symbol
.quadrature.) and L-serine breakdown (circle symbol .largecircle.)
of C. glutamicum 13032.DELTA.panBC.DELTA.sdaA, clone 1
(.quadrature., .largecircle.) and C. glutamicum
13032.DELTA.panBC.DELTA.sdaA, clone 2 (.box-solid., .circle-solid.)
compared with C. glutamicum 13032.DELTA.panBC, clone 1
(.quadrature., .smallcircle.) and C. glutamicum
13032.DELTA.panBC,clone 2 (.box-solid., .circle-solid.). The
abscissa X represents the fermentation in hours (h). The ordinate
y.sub.1 is the growth of the microorganisms measured in terms of
optical density at 600 nm. The ordinate Y.sub.2 gives the L-serine
concentration in mM.
[0081] FIG. 3: The expression plasmid pEC-T18mob2-serA.sup.fbrCB.
The indicia on the outer edge of the plasmid show the resective
restriction sites. The indicia within the circle represent the
following genes: TABLE-US-00002 SerC Phosphoserine Transaminase
SerB Phsophoserine Phosphatase Rep Replication origin Per Partition
cell partition gene Tet Tetracycline resistance gene RP4-mob
Mobilizaiton origin OriV Source of DNA replication SerA-fbr
3-phosphoglycerate dehydrogenase
EXAMPLES
1. The Construction of sdaA-Deletion Mutant of C. glutamicum
ATCC13032 .DELTA.panBC.
[0082] The starting point was Corynebacterium glutamicum with a
nuclotide sequence (Genbank-Accession-Number BAB99038; SEQ-ID-No.
1) whose derivative polypeptide sequence showed 40% identity with
the described L-serine dehydratase of E.coli (NCBI-Accession-Number
P1095). By gene protected mutagenesis by the method of Link et al
(Link A J, Phillips D, Church G M, Methods for generating precise
deletions and insertions in the genome of wild-type Escherichia
coli: application to open reading frame characterization. J.
Bacteriol. 1997 October; 179(20):6228-37) and Schafer et al. (Gene
145: 69-73 (1994)) the sdaA-gene of C. glutamicum was deleted. The
following primers wre derived from the sdaA corynebacterial
sequence (NCBI Accession-Number AP005279): TABLE-US-00003 sdaA-1:
5'-TCGTGCAACTTCAGACTC-3' (AP005279 nucleotide 73635 - 73653);
sdaA-2: 5'-CCCATCCACTAAACTTAAACACGTCATAATGAACCCACC-3' (AP005279
complementary to nucleotide 74121 - 74139); sdaA-3:
5'-TGTTTAAGTTTAGTGGATGGGCCGACTAATGGTGCTGCG-3' (AP005279
complementary to nucleotide 74553 - 74571); sdaA-4:
5'-CGGGAAGCCCAAGGTGGT-3' (AP005279 nucleotide 75044 - 75062)
[0083] Primers sdaA-1 and sdaA-2 flank respectively the beginning
and the end of the sdaA-3 make available respective complementary
linker regions (see relevant text) which enable in a two-stage PCR
process (cross over PCR) a deletion of the sdaA gene in vitro. In a
first PCR reaction with the chromosomal DNA of C. glutamicum, the
primer combination sdaA-1 and sdaA-2 as well as sdaA-3 and sdaA-4
are used. The PCR reaction is carried out in 30 cycles in the
presence of 200 .mu.m deoxynucleotide triphosphates (dATP, dCTkP,
dGTP, dTTP), each with 600 nM of the corresponding oligonucleotide
sdaA-1 and sdaA-4 as well as 60 nm of oligonucleotide sdaA-2 and
sdaA-3, 100 ng of chromosomal DNA from Corynebacterium glutamicum
ATCC13032, 1/10 volumes 10-fold of reagion buffer and 2.6 units of
heat stabilized Taq-/Owi-DNA-Polymerase-Mischung mixture (Expand
High Fidelity PCR System of Firm of Roche Diagnostics, Mannheim,
Deutschland) in a Sthermocycler (PTC-100, MJ Research, Inc.,
Watertown, USA) under the following conditions: 94.degree. C. for
30 seconds, 50.degree. C. for 30 seconds and 72.degree. C. for 40
seconds. The elongation step at 42.degree. C. was extended after 10
cycles by about 5 seconds per cycle. After the PCR reaction, the
DNA fragments containing each having a length of 500 bp were
isolated with QIAExII Gelextraction kit (Qiagen) in accordance with
the requirements of the manufacturer on an 0.8% agarose gel and
both fragments were used as templates in the second PCR. As primers
the primers sdaA-1 and sdaA-4 were used. This time the reaction was
carried out in 35 cycles in the presence of 200 .mu.m
deoxynucleotide triphosphates, 600 nM each of the corresponding
olegonitrides, 2-mg each of the isolated template DNA from the
first PCR, 1/10 volume of 10 fold reaction buffer and 2.6 units of
Taq-/Pwo-DNA-Polymerase mixture under the following conditions:
94.degree. C. for 30 seconds, 50.degree. C. for 30 seconds and
72.degree. C. for 80 seconds. Again the elongation steps after 10
cycles were extended by 5 seconds each. After PCR reaction to 1000
bp long DNA fragments which contain the inactive sdaA gene with a
420 bp long central deletion was isolated on a 0.8% agarose gel and
cloned, blunt end with the aid of a Sure Clone Kit (Amersham
Pharmacia Biotech) in the SmaI-restriction site of the inactivation
vector pk19mobsacB (Schafer et al Gene 145: 69-73 (1994) which can
replicate only in an E.coli but not in C. glutamicum. The obtained
plasmid pK19mobsacB_.DELTA.sdaA (FIG. 1) is tested by restriction
mapping for correctness. The cloning was effected in the
Escherichia coli strain DH5.alpha.mcr (Grant et al., Proceedings of
the National Academy of Sciences of the United States of America
USA (1990) 87: 4645-4649).
[0084] Then the plasmid is incorporated by electroporation in C.
glutamicum 13032.DELTA.panBC (Radmacher E, Vaitsikova A, Burger U,
Krumbach K, Sahm H, Eggeling L. Linking central metabolism with
increased pathway flux: L-valine accumulated by Corynebacterium
glutamicum. Appl Environ Microbiol. 2002 68(5):2246-50) and subject
to selection with integration of the vector. This strain is
pantothenate auxotropic as a result of the deletion of the
pantothenate biosynthesis genes panB and panC and is characterized
in that it has an amplified accumulation of pyruvate about 50 mM
alanin and 8 mm valine because of the pantothenate limitation. In
addition the strain can form about 100 .mu.M L-serine and is
suitable as a starting strain for the construction of L-serine
producers. It contains Kanamycin resistant clones of C. Glutamicum
13032.DELTA.panBC by which inactivation vector is integrated in the
genome. To allow selection of the excision of the vector,
kanamycin-resistant clones are plated out on saccharose containing
LB medium (Sambrook et al., Molecular cloning. A laboratory manual
(1989) Cold Spring Harbour Laboratory (Press) with 15 g/l Agar, 2%
glucose/10% saccharose) and colonies are obtained in which the
vector has again been lost as a result of a second recombination
event. (Jager et al. 1992, Journal of Bacteriology 174: 5462-5465).
Two of these clones whose mucleotides have sdaA genes deleted from
positions 506 to 918 are designated and
13032.DELTA.panBC.DELTA.sdaA, clone 1 and
13032.DELTA.panBC.DELTA.sdaA, clone 2 and are used in the further
investigations.
2. The Influence of the sdaA Deletion Upon L-serine
Decomposition
[0085] In the following, a test was made whether the deleted sdaA
gene indeed participates in L-serine decomposition. For this
purpose a growth experiment was carried out with each of the two
clones of the strains C. glutamicum 13032.DELTA.panBC.DELTA.sdaA in
comparison with strain C. glutamicum 13032.DELTA.panBC on minimal
medium (Keilhauser et al., Journal of Bacteriology 175 (1993)
5595-5603) which additionally contains 2% glucose 1 .mu.M
pantothenate and 100 mM L-serine. The growth and consumption of
L-serine were followed. The results are given in FIG. 2.
[0086] The results in FIG. 2 show that the deletion of the sdaA
genes results in about 40% reduced decomposition of L-serine.
3. Influence of the Deletion of the sdaA Gene on L-serine
Formation
[0087] To test what the influence was of the deletion of the
L-serine dehydratase gene upon L-serine formation the strains
13032.DELTA.panBC.DELTA.sdaA (clone 1, clone 2) and
13032.DELTA.panBC (clone 1, clone 2) with the plasmid
pec-T18mob2-sera.sup.fbrserCserB the plasmid is formed (FIG. 3)
from the vector pEC-T18mob2 (Tauch, A., Kirchner, O., Loffler, B.,
Gotker, S., Puhler A., and Kalinowski J. Efficient
Electrotransformation of Corynebacterium diphtheria with a
MiniReplicon Derived from the Cornyebacterium glutamicum Plasmid
pGA1. Curr. Microbiol. 45(5), 362-367 (2002)), of the
corynebacterial gene serA.sup.fbr (Peters-Wendisch P., Netzer R,
Eggeling L. Sahm H. 3-Phosphoglycerate dehydrogenase from
Corynebacterium glutamicum: the C-terminal domain is not essential
for activity but is required for inhibition by L-serine. Appl
Microbiol Biotechnol. 2-2 December; 60(4); 437-41) as well as serC
and serB (German ptent application 100 44 831.3 of 11 Sep.
2000.
[0088] After electroporation, the strains
13032.DELTA.panBC.DELTA.sdaApSerA.sup.fbrCB and
13032.DELTA.panBCpSerA.sup.fbrCB were obtained.
[0089] For testing L-serine output the two strains
13032.DELTA.panBCpSerA.sup.fbrCB are cultivated in complex medium
(CgIII with 2% glucose and 5 .mu.g/l tetracycline) and the
fermentation medium CGXII (J Bacteriol (1993) 175: 5595-5603), each
seeded from the preculture to the medium contained in addition 50
.mu.g/l kanamycin nd 1 .mu.M pantothenate. As controls, the two
starting strains 13032.DELTA.panBC and 13032.DELTA.panBC.DELTA.sdaA
were cultured in the same manner although the medium did not
contain tetracycline. For each at least two independent
fermentations were carried out. After culturing for 30 hours at
30.degree. C. of a rotating shaker at 120 RPM, the L-serine
quantity accumulated in the medium was determined. The
determination of the amino acid concentration was carried out by
means of high presssure liquid chromatography (J Chromat (1983)
266: 471-482). The results of the fermentation are shown in Table 1
and indicate that the exclusion of L-serine dehydratase led to a
4-fold increase in the L-serine accumulation in the medium
independently of whether the L-serine biosynthesis genes
serA.sup.fbr, serC and serB were overexpressed. The overexpression
of the L-serine biosynthesis genes serA.sup.fbr, serC and serB
however resulted in 16 fold increase in L-serine accumulation in
the culture supernatent generally. Thus the use of the constructed
and described deletion mutant .DELTA.sdaA resulted in a method
which improved the L-serine formation decisively. TABLE-US-00004
TABLE 1 Accumulation of L-serine in the culture supernatent of
Corynebacterium glutamicum 13032.DELTA.panBC and
13032.DELTA.panBC.DELTA.sdaA after expression of the genes
serA.sup.fbr, serC and serB Strain OD.sub.600 L-Serine [mM]
13032.DELTA.panBC 40 0.1 13032.DELTA.panBC.DELTA.sdaA 42 0.4
13032.DELTA.panBCpserA.sup.fbrCB 30 1.6
13032.DELTA.panBC.DELTA.sdaApserA.sup.fbrCB 30 6.6
4. Determination of the L-serine Dehydratase Activity
[0090] For determining the L-serine dehydratase activity the wild
type strands WT pXMJ19 (Jacoby M., Burkovski A (1999) Construction
and application of new Corynebacterium glutamicum vectors.
Biotechnol. Technique 13:437-441), overexpression strand WT
pXMJ19_sdA and the deletion strains .DELTA.sdaA pXMJ19 were
cultured in CgXII minimal medium as in Keilhauer et al., (1993).
The medium contained 30 mg/l protocatechuic acid, 100 mM glucose
and 100 mM L-serine. The cells were cultivated in the presence of 1
mM isopropyl-beta-D-thiogalactopyranoside and in the exponential
growth phse at an optical density of 6-8, measured by a Pharmacia
Biotech ultrospec 3000 spectral photometer were harvested. They
were then centrifuged for 10 minutes at 4500 rpm and 4.degree. C.,
suspended in 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid buffer (pH 8.0) and centrifuged again. Thereafter the cells
were taken up in 50 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid buffer (pH 8.0),
1 mM FeSO.sub.4 and 10 mM dithiothreitol. The cell breakdown was
effected by means of ultrasonic treatment (Branson sonifier 250;
duty cycle 25%, output control 2.5, 10 minutes) on ice.
[0091] To determine the L-serine dehydratase activity the reaction
set contained 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid buffer (pH 8.0), 10 mM dithiothreitol and 10-100 .mu.l new
extract. The detection of the pyruvate formation from the serine
was effected as described (Ohmori et al., 1991). The reaction was
started by adding 50 mM L-serine and after 10 minutes was stopped
by the addition of 1,2-diamino-4,5-dimethoxybenzene reagent in a
ratio of 1:1. The reagent, as described in Ohmori et al 1991 was
comprised of 4 mg 1,2-diamino-4,5-dimethoxybenzol dissolved in 42.4
ml H.sub.2O, 3.5 ml .beta.-mercaptoethanol and 4.1 ml HCl (37% ig)
then incubation was carried out for 2 hours at 102.degree. dry
heat.
[0092] Detection and quantification of the
2-hydroxy-6,7-dimethoxy-3-methylquinoxaline derivative produced by
the pyruvate was carried out by means of high pressure liquid
chromatography also as described. (Ohmori et al., 1991). The
protein determination in the raw extract followed by means of the
Bradford method (Bradford 1976) using the protein assays (The firm
Bio-Rad). The specific L-serine dehydratase activity of the two
strands are given in Table 2. TABLE-US-00005 TABLE 2 Specific
Activity of the L-Serine Dehydratase in the Strains 13032 WT
pXMJ19_sdaA (Overexpressed), 13032 WT pXMJ19 (Wild type with empty
vectors) and 13032 .DELTA.sdaA pXMJ19. spec. Activity C. Glutamicum
Strain [nmol/min * mg] 13032 WT pXMJ19_sdaA 0.221 13032 WT pXMJ19
0.003 13032 .DELTA.sdaA pXMJ19 0
[0093]
Sequence CWU 1
1
2 1 1449 DNA Corynebacterium glutamicum 1 tcgtgcaact tcagactctt
acggaggcga tggaccaaaa acaactacaa tcaagcagat 60 caccttgtac
accaccatag aaaaggccca ccctcagcca tggctatcag tgttgttgat 120
ctatttagca tcggtatcgg accatcatcc tcacataccg tcggccccat gagagccgcc
180 ctcacgtata tctctgaatt tcccagctcg catgtcgata tcacgttgca
cggatccctt 240 gccgccaccg gtaaaggcca ctgcactgac cgggcggtat
tactgggtct ggtgggatgg 300 gaaccaacga tagttcccat tgatgctgca
ccctcacccg gcgcgccgat tcctgcgaaa 360 ggttctgtga acgggccaaa
gggaacggtg tcgtattccc tgacgtttga tcctcatcct 420 cttccagaac
accccaatgc cgttaccttt aaaggatcaa ccacaaggac ttatttgtcg 480
gtgggtggtg ggttcattat gacgttggag gatttccgga agctggacga tatcggatca
540 ggtgtgtcaa ccattcatcc agaggcagag gtgccttgtc cttttcagaa
gagttcccaa 600 ttactcgcat atggtcgcga ttttgcggag gtcatgaagg
ataatgagcg cttaatccac 660 ggggatcttg gcacagtgga tgcccatttg
gatcgagtgt ggcagattat gcaggagtgc 720 gtggcacaag gcatcgcaac
gccggggatt ttaccgggtg ggttgaatgt gcaacgtcgg 780 gcgccgcagg
tacacgcgct gattagcaac ggggatacgt gtgagctggg tgctgatctt 840
gatgctgtgg agtgggtgaa tctgtacgcc ttggcggtga atgaagaaaa cgccgctggt
900 ggtcgtgtgg ttactgctcc gactaatggt gctgcgggga ttattccggc
ggtgatgcac 960 tatgcgcggg attttttgac aggttttggg gcggagcagg
cgcggacgtt tttgtatacc 1020 gcgggtgcgg tgggcatcat cattaaggaa
aatgcctcga tctctggcgc ggaggtgggg 1080 tgtcagggtg aggttggttc
agcgtccgcg atggcggctg ccgggttgtg tgcagtctta 1140 ggtggttctc
cgcaacaggt ggaaaacgcc gcggagattg cgttggagca caatttggga 1200
ttgacgtgcg atccggtggg cgggttagtg cagattccgt gtattgaacg caacgctatt
1260 gctgccatga agtccatcaa tgcggcaagg cttgcccgga ttggtgatgg
caacaatcgc 1320 gtgagtttgg atgatgtggt ggtcacgatg gctgccaccg
gccgggacat gctgaccaaa 1380 tataaggaaa cgtcccttgg tggtttggca
accaccttgg gcttcccggt gtcgatgacg 1440 gagtgttag 1449 2 449 PRT
Corynebacterium glutamicum 2 Met Ala Ile Ser Val Val Asp Leu Phe
Ser Ile Gly Ile Gly Pro Ser 1 5 10 15 Ser Ser His Thr Val Gly Pro
Met Arg Ala Ala Leu Thr Tyr Ile Ser 20 25 30 Glu Phe Pro Ser Ser
His Val Asp Ile Thr Leu His Gly Ser Leu Ala 35 40 45 Ala Thr Gly
Lys Gly His Cys Thr Asp Arg Ala Val Leu Leu Gly Leu 50 55 60 Val
Gly Trp Glu Pro Thr Ile Val Pro Ile Asp Ala Ala Pro Ser Pro 65 70
75 80 Gly Ala Pro Ile Pro Ala Lys Gly Ser Val Asn Gly Pro Lys Gly
Thr 85 90 95 Val Ser Tyr Ser Leu Thr Phe Asp Pro His Pro Leu Pro
Glu His Pro 100 105 110 Asn Ala Val Thr Phe Lys Gly Ser Thr Thr Arg
Thr Tyr Leu Ser Val 115 120 125 Gly Gly Gly Phe Ile Met Thr Leu Glu
Asp Phe Arg Lys Leu Asp Asp 130 135 140 Ile Gly Ser Gly Val Ser Thr
Ile His Pro Glu Ala Glu Val Pro Cys 145 150 155 160 Pro Phe Gln Lys
Ser Ser Gln Leu Leu Ala Tyr Gly Arg Asp Phe Ala 165 170 175 Glu Val
Met Lys Asp Asn Glu Arg Leu Ile His Gly Asp Leu Gly Thr 180 185 190
Val Asp Ala His Leu Asp Arg Val Trp Gln Ile Met Gln Glu Cys Val 195
200 205 Ala Gln Gly Ile Ala Thr Pro Gly Ile Leu Pro Gly Gly Leu Asn
Val 210 215 220 Gln Arg Arg Ala Pro Gln Val His Ala Leu Ile Ser Asn
Gly Asp Thr 225 230 235 240 Cys Glu Leu Gly Ala Asp Leu Asp Ala Val
Glu Trp Val Asn Leu Tyr 245 250 255 Ala Leu Ala Val Asn Glu Glu Asn
Ala Ala Gly Gly Arg Val Val Thr 260 265 270 Ala Pro Thr Asn Gly Ala
Ala Gly Ile Ile Pro Ala Val Met His Tyr 275 280 285 Ala Arg Asp Phe
Leu Thr Gly Phe Gly Ala Glu Gln Ala Arg Thr Phe 290 295 300 Leu Tyr
Thr Ala Gly Ala Val Gly Ile Ile Ile Lys Glu Asn Ala Ser 305 310 315
320 Ile Ser Gly Ala Glu Val Gly Cys Gln Gly Glu Val Gly Ser Ala Ser
325 330 335 Ala Met Ala Ala Ala Gly Leu Cys Ala Val Leu Gly Gly Ser
Pro Gln 340 345 350 Gln Val Glu Asn Ala Ala Glu Ile Ala Leu Glu His
Asn Leu Gly Leu 355 360 365 Thr Cys Asp Pro Val Gly Gly Leu Val Gln
Ile Pro Cys Ile Glu Arg 370 375 380 Asn Ala Ile Ala Ala Met Lys Ser
Ile Asn Ala Ala Arg Leu Ala Arg 385 390 395 400 Ile Gly Asp Gly Asn
Asn Arg Val Ser Leu Asp Asp Val Val Val Thr 405 410 415 Met Ala Ala
Thr Gly Arg Asp Met Leu Thr Lys Tyr Lys Glu Thr Ser 420 425 430 Leu
Gly Gly Leu Ala Thr Thr Leu Gly Phe Pro Val Ser Met Thr Glu 435 440
445 Cys
* * * * *