U.S. patent application number 11/690917 was filed with the patent office on 2007-10-25 for polynucleotides encoding polypeptides involved in amino acid biosynthesis in methylophilus methylotrophus.
Invention is credited to Yousuke Nishio, Shinichi Sugimoto, Yoshihiro Usuda, Hisashi Yasueda.
Application Number | 20070249017 11/690917 |
Document ID | / |
Family ID | 32907753 |
Filed Date | 2007-10-25 |
United States Patent
Application |
20070249017 |
Kind Code |
A1 |
Usuda; Yoshihiro ; et
al. |
October 25, 2007 |
Polynucleotides Encoding Polypeptides Involved in Amino Acid
Biosynthesis in Methylophilus Methylotrophus
Abstract
The present invention provides polypeptides and polynucleotides
involved in amino acid biosynthesis in Methylophilus methylotrophus
and methods of producing amino acids in microorganisms having
enhanced or attenuated expression of these polypeptides and/or
polynucleotides.
Inventors: |
Usuda; Yoshihiro;
(Kawasaki-shi, JP) ; Nishio; Yousuke;
(Kawasaki-shi, JP) ; Yasueda; Hisashi;
(Kawasaki-shi, JP) ; Sugimoto; Shinichi;
(Kawasaki-shi, JP) |
Correspondence
Address: |
CERMAK & KENEALY LLP;ACS LLC
515 EAST BRADDOCK ROAD
SUITE B
ALEXANDRIA
VA
22314
US
|
Family ID: |
32907753 |
Appl. No.: |
11/690917 |
Filed: |
March 26, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10375039 |
Feb 28, 2003 |
7029893 |
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11690917 |
Mar 26, 2007 |
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11232405 |
Sep 22, 2005 |
7220570 |
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11690917 |
Mar 26, 2007 |
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Current U.S.
Class: |
435/69.1 ;
435/252.3; 435/320.1; 536/23.1 |
Current CPC
Class: |
C12P 13/04 20130101;
C12N 9/1205 20130101; C07K 14/195 20130101; C12N 15/52 20130101;
C12N 9/93 20130101 |
Class at
Publication: |
435/069.1 ;
435/252.3; 435/320.1; 536/023.1 |
International
Class: |
C12P 1/04 20060101
C12P001/04; C07H 21/04 20060101 C07H021/04; C12N 1/21 20060101
C12N001/21; C12N 15/00 20060101 C12N015/00 |
Claims
1. An isolated polynucleotide, which encodes a protein comprising
the amino acid sequences of SEQ ID NO:30 and SEQ ID NO:32.
2. A vector comprising the isolated polynucleotide of claim 1.
3. An isolated host cell comprising the isolated polynucleotide of
claim 1.
4. The host cell of claim 3, which is a Methylophilus
bacterium.
5. The host cell of claim 4, which is a Methylophilus
methylotrophus bacterium.
6. A method of producing at least one L-amino acid comprising
culturing the host cell of claim 5 for a time and under conditions
suitable for producing the amino acid; and collecting the amino
acid produced.
7. An isolated polynucleotide comprising the nucleotide sequences
of SEQ ID NO:29 and SEQ ID NO:31.
8. A vector comprising the isolated polynucleotide of claim 7.
9. An isolated host cell comprising the isolated polynucleotide of
claim 7.
10. The host cell of claim 9, which is a Methylophilus
bacterium.
11. The host cell of claim 10, which is a Methylophilus
methylotrophus bacterium.
12. A method of producing at least one L-amino acid comprising
culturing the host cell of claim 11 for a time and under conditions
suitable for producing the amino acid; and collecting the amino
acid produced.
13. An isolated polynucleotide, which hybridizes under stringent
conditions to the isolated polynucleotide of claim 7, wherein the
stringent conditions comprise hybridization in 50% formamide, 1M
NaCl, and 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at
65.degree. C., and wherein said polynucleotide encodes a
polypeptide having pyruvate carboxylase activity.
14. A vector comprising the isolated polynucleotide of claim
13.
15. An isolated host cell comprising the isolated polynucleotide of
claim 13.
16. The method of claim 15, wherein said host cell is a
Methylophilus bacterium.
17. The method of claim 16, wherein said host cell is a
Methylophilus methylotrophus bacterium.
18. A method of producing at least one L-amino acid comprising
culturing the host cell of claim 17 for a time and under conditions
suitable for producing the amino acid; and collecting the amino
acid produced.
19. An isolated polynucleotide, which is at least 95% identical to
the polynucleotide of claim 7, and wherein said polynucleotide
encodes a polypeptide having pyruvate carboxylase activity.
20. A vector comprising the isolated polynucleotide of claim
19.
21. An isolated host cell comprising the isolated polynucleotide of
claim 19.
22. The host cell of claim 21, wherein said host cell is a
Methylophilus bacterium.
23. The host cell of claim 22, wherein said host cell is a
Methylophilus methylotrophus bacterium.
24. A method of producing at least one L-amino acid comprising
culturing the host cell of claim 23 for a time and under conditions
suitable for producing the amino acid; and collecting the amino
acid produced.
Description
[0001] This application claims priority as a divisional under 35
U.S.C. .sctn.120 to U.S. patent application Ser. No. 10/375,039,
filed Feb. 28, 2003, which issued as U.S. Pat. No. 7,029,893, and
is a divisional under 35 U.S.C. .sctn.120 of U.S. patent
application Ser. No. 11/232,405, filed Sep. 22, 2005, the
entireties of which are incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to novel polynucleotides
encoding proteins involved in biosynthesis of amino acids including
phenylalanine, tryptophan, tyrosine, aspartate, lysine, methionine,
or threonine, derived from microorganisms belonging to
methylotrophic bacteria and fragments thereof, polypeptides encoded
by the polynucleotides and fragments thereof, polynucleotide arrays
comprising the polynucleotides and fragments thereof.
[0004] 2. Discussion of the Background
[0005] Amino acids such as L-lysine, L-glutamic acid, L-threonine,
L-leucine, L-isoleucine, L-valine and L-phenylalanine are
industrially produced by fermentation by using microorganisms that
belong to the genus Brevibacterium, Corynebacterium, Bacillus,
Escherichia, Streptomyces, Pseudomonas, Arthrobacter, Serratia,
Penicillium, Candida or the like. In order to improve the
productivity of amino acids, strains of the aforementioned
microorganisms that have been isolated from nature or artificial
mutants thereof have been used. Various techniques have also been
disclosed for enhancing activities of L-amino acid biosynthetic
enzymes by using recombinant DNA techniques to increase the L-amino
acid-producing ability.
[0006] L-amino acid production has been increased considerably by
breeding of microorganisms such as those mentioned above and by
improvements in production methods. However, in order to meet a
future increase in the demand for L-amino acids, development of
methods for more efficiently producing L-amino acids at lower cost
are still desired.
[0007] Conventional methods for producing amino acids by
fermentation using methanol, which is a raw fermentation material
available in large quantities at a low cost, employ Achromobacter
or Pseudomonas microorganisms (Japanese Patent Publication (Kokoku)
No. 45-25273/1970), Protaminobacter microorganisms (Japanese Patent
Application Laid-open (Kokai) No. 49-125590/1974), Protaminobacter
or Methanomonas microorganisms (Japanese Patent Application
Laid-open (Kokai) No. 50-25790/1975), Microcyclus microorganisms
(Japanese Patent Application Laid-open (Kokai) No. 52-18886/1977),
Methylobacillus microorganisms (Japanese Patent Application
Laid-open (Kokai) No. 4-91793/1992), Bacillus microorganisms
(Japanese Patent Application Laid-open (Kokai) No. 3-505284/1991)
and others.
[0008] However, only a few methods have been described for
producing L-amino acids using Methylophilus bacteria in conjunction
with recombinant DNA technology. Although methods described in EP 0
035 831 A, EP 0 037 273 A and EP 0 066 994 A have been described as
methods for transforming Methylophilus bacteria using recombinant
DNA, applying recombinant DNA techniques to improvement of amino
acid productivity of Methylophilus bacteria has not been described.
Only WO-00/61723 and WO-02/38777 disclose the improved production
of lysine and phenylalanine, respectively, using genes involved in
biosynthesis of each respective amino acid. In particular,
WO-00/61723 discloses the ask gene, the dapA gene, the dapB gene,
and the lysA gene, which encode aspartkinase, dihydrodipicolinate
synthase, dihydrodipicolinate reductase, and diaminopimelinate
decarboxylase, respectively. WO-02/38777 discloses the aroG gene
and the pheA gene, which encode 3-deoxy-D-arabino-heptulosonate
7-phosphate synthase and bi-functional enzyme of chorismate mutase
and prephenate dehydratase, respectively.
[0009] Therefore, prior to the present invention, only the ask
gene, the dapA gene, the dapB gene, the lysA gene, the aroG gene
and the pheA gene have been disclosed. Other genes isolated from
Methylophilus bacteria that are involved in amino acid biosynthesis
and which can be used to improve the yield of amino acids in
cultured microorganisms remain elusive and undisclosed.
SUMMARY OF THE INVENTION
[0010] An object of the present invention is to provide novel
measures for the improved production of amino acids or an amino
acid, where these amino acids include asparagine, threonine,
serine, glutamate, glycine, alanine, cysteine, valine, methionine,
isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine,
tryptophan, arginine and the salts thereof. In a preferred
embodiment the amino acids are L-amino acids.
[0011] Such a process includes bacteria, which express a protein
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID
NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ
ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36,
SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID
NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, and SEQ ID
NO:54.
[0012] In one embodiment the polypeptides are encoded by a
polynucleotide selected from the group consisting of SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,
SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID
NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ
ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39,
SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID
NO:49, SEQ ID NO:51, and SEQ ID NO:53. In another embodiment the
polypeptides are encoded by other polynucleotides which have
substantial identity to the herein described polynucleotides or
those which hybridize under stringent conditions.
[0013] Another object of the invention is to provide polynucleotide
sequences selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID
NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ
ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31,
SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID
NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ
ID NO:51, and SEQ ID NO:53; as well as those polynucleotides that
have substantial identity to these nucleotide sequences, preferably
at least 95% identity.
[0014] Another object of the invention is to provide isolated
polypeptides having a sequence selected from the group consisting
of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ
ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28,
SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID
NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ
ID NO:48, SEQ ID NO:50, SEQ ID NO:52, and SEQ ID NO:54; as well as
those polypeptides that have substantial identity to these amino
acid sequences, preferably at least 95% identity.
[0015] A further object of the invention is a method for producing
a protein or proteins by culturing host cells containing the herein
described polynucleotides under conditions and for a time suitable
for expression of the protein and collecting the protein produced
thereby.
[0016] Another object is the use of host cells having the
polynucleotides described herein to produce amino acids, as well as
the use of such isolated polypeptides in the production of amino
acids.
[0017] Other objects of the invention include methods of detecting
nucleic acid sequences homologous to at least one of: SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,
SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID
NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ
ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39,
SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID
NO:49, SEQ ID NO:51, and SEQ ID NO:53, particularly nucleic acid
sequences encoding polypeptides that herein described proteins or
polypeptides and methods of making nucleic acids encoding such
polypeptides.
[0018] The above objects highlight certain aspects of the
invention. Additional objects, aspects and embodiments of the
invention are found in the following detailed description of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0019] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art of molecular biology. Although methods
and materials similar or equivalent to those described herein can
be used in the practice or testing of the present invention,
suitable methods and materials are described herein. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In addition, the materials, methods, and examples are illustrative
only and are not intended to be limiting.
[0020] Reference is made to standard textbooks of molecular biology
that contain definitions and methods and means for carrying out
basic techniques, encompassed by the present invention. See, for
example, Sambrook et al., Molecular Cloning: A Laboratory Manual,
Third Edition, Cold Spring Harbor Laboratory Press, New York
(2001), Current Protocols in Molecular Biology, Ausebel et al
(eds.), John Wiley & Sons, New York (2001) and the various
references cited therein.
[0021] Methylophilus methylotrophus (M. methylotrophus) is a gram
negative ribulose monophosphate cycle methanol-utilizer, which can
be used for the large-scale production of a variety of fine
chemicals including amino acids, nucleic acids, vitamins,
saccharides, and so on. The polynucleotides of this invention,
therefore, can be used to identify microorganisms, which can be
used to produce fine chemicals, for example, by fermentative
processes. Modulation of the expression of the polynucleotides
encoding enzymes in the amino acid biosynthesis of the present
invention, can be used to modulate the production of one or more
fine chemicals from a microorganism (e.g., to improve the yield of
production of one or more fine chemicals from Methylophilus or
Methylbacillus species).
[0022] The proteins encoded by the polynucleotides of the present
invention are capable of, for example, performing a function
involved in the metabolism of intermediates in M. methylotrophus,
such as, oxaloacetate, pyruvate, phosphoenolpyruvate, L-aspartate,
L-homoserine, O-succinyl homoserine, homoserine phosphate, O-acetyl
homoserine, homocysteine, tetrahydrodipicolinate,
N-succinyl-alpha-amino-epsilon-keto-pimelate, N-succinyl
diaminopimelate, LL-diaminopimelate, Meso-diaminopimelate,
3-deoxy-D-arabino-heptulosonate 7-phosphate, 3-dehydroquinate,
3-dehydroshikimate, shikimate, shikimate 3-phosphate,
5-enolpyruvoylshikimate-3-phosphate, chorismate, prephenate,
4-hydroxyphenylpyruvate, anthranilate, phosphoribosyl anthranilate,
1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate,
indoleglycerol phosphate, L-lysine, L-phenylalnine, L-tryptophan,
L-tyrosine, L-methionine, and L-threonine.
[0023] Given the availability of cloning vectors used in M.
methylotrophus, such as those disclosed in Methane and Methanol
Utilizers, Plenum Press, New York (1992) edited by J. Colin Murrell
and Howard Dalton, the nucleic acid molecules of the present
invention may be used in the genetic engineering of this organism
to make it better or more efficient producer of one or more fine
chemicals.
[0024] There are a number of mechanisms by which the alteration of
a protein of the present invention may affect the yield,
production, and/or efficiency of production of a fine chemical from
M. methylotrophus bacteria, which have the altered protein
incorporated. Improving the ability of the cell to utilize pyruvate
or phosphoenolpyruvate (e.g., by manipulating the genes encoding
enzymes involved in the conversion of each compound into
oxaloacetate), one may increase the yield or productivity of
desired fine chemicals derived from oxaloacetate. Furthermore, by
suppressing the activity of enzymes involved in the wasteful
pathway such as the conversion of chorismate to
4-hydroxyphenylpyruvate, one may also increase the yield or
productivity of desired phenylalanine.
[0025] "L-amino acids" or "amino acids" as used herein means one or
more amino acids, including their salts, preferably chosen from the
following: L-asparagine, L-threonine, L-serine, L-glutamate,
L-glycine, L-alanine, L-cysteine, L-valine, L-methionine,
L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine,
L-lysine, L-tryptophan and L-arginine.
[0026] "Isolated" as used herein means separated out of its natural
environment.
[0027] "Substantial identity" as used herein refers to
polynucleotides and polypeptides which are at least 70%, preferably
at least 80% and more preferably at least 90% to 95% identical to
the polynucleotides and polypeptides, respectively, according to
the present invention.
[0028] "Polynucleotide" as used herein relates to
polyribonucleotides and polydeoxyribonucleotides, it being possible
for these to be non-modified RNA or DNA or modified RNA or DNA.
[0029] "Polypeptides" as used herein are understood to mean
peptides or proteins which comprise two or more amino acids bonded
via peptide bonds. In particular, the term refers to polypeptides
which are at least 70%, preferably at least 80% and more preferably
at least 90% to 95% identical to the polypeptides according to the
present invention. Included within the scope of the present
invention are polypeptide fragments of the polypeptides having a
sequence selected from the group consisting of SEQ ID NO:2, SEQ ID
NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ
ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,
SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID
NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ
ID NO:52, and SEQ ID NO:54 or those which are identical to those
described herein.
[0030] "Polynucleotides which encode the polypeptide" of the
invention as used herein is understood to mean the sequences
exemplified in this application as well as those sequences which
have substantial identity to the nucleic acid sequences at least
one of: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID
NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ
ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27,
SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID
NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ
ID NO:47, SEQ ID NO:49, SEQ ID NO:51, and SEQ ID NO:53 and which
encode a molecule having one or more of the bioactivities of the
associated gene products. Preferably, such polynucleotides are
those which are at least 70%, preferably at least 80% and more
preferably at least 90% to 95% identical to the nucleic acid
sequences at least one of: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15,
SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID
NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ
ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43,
SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, and SEQ ID
NO:53.
[0031] Polynucleotides according to the invention may be employed
as probes to isolate and/or identify RNA, cDNA and DNA molecules,
e.g., full-length genes or polynucleotides which code for the
polypeptides described herein. Likewise, the probes can be employed
to isolate nucleic acids, polynucleotides or genes which have a
high sequence similarity or identity with the polynucleotides of
the invention.
[0032] Polynucleotides of the invention may also be used to design
primers useful for the polymerase chain reaction to amplify,
identify and/or isolate full-length DNA, RNA or other
polynucleotides with high sequence homology or identity to the
polynucleotides of the invention, as well as, polynucleotides that
encode the polypeptides of the invention. Preferably, probes or
primers are at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29 or 30 nucleotides in length. Oligonucleotides with a
length of at least 35, 40, 45, 50, 100, 150, 200, 250 or 300
nucleotides may also be used.
[0033] Methods of DNA sequencing are described inter alia by Sanger
et al. (Proceedings of the National Academy of Sciences of the
United States of America USA, 74:5463-5467, (1977)).
[0034] A person skilled in the art will find instructions for
amplification of DNA sequences with the aid of the polymerase chain
reaction (PCR) inter alia in the handbook by Gait: Oligonucleotide
Synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and
in Newton and Graham: PCR 2.sup.nd Edition (Springer Verlag, New
York, 1997).
[0035] Additionally, methods employing DNA chips, microarrays or
similar recombinant DNA technology that enables high throughput
screening of DNA and polynucleotides that encode the herein
described proteins or polynucleotides with high sequence homology
or identity to the polynucleotides described herein. Such methods
are known in the art and are described, for example, in Current
Protocols in Molecular Biology, Ausebel et al (eds), John Wiley and
Sons, Inc. New York (2000).
[0036] The polynucleotides and polypeptides of the present
invention are involved in amino acids biosynthesis in M.
methylotrophus. By way of example, the present inventors provide
the following cited references (each of which are incorporated
herein by reference) demonstrating that assays to assess the
enzymatic activity of the polypeptides of the present invention are
known and, as such, determination of whether a sequence falls
within the scope of the present claims may be readily ascertained.
These polynucleotides and polypeptides include: [0037] 1. Shikimate
kinase enzyme comprises the amino acid sequence of SEQ ID NO:2 and
is encoded by the aroK gene which comprises the polynucleotide SEQ
ID NO:1 (Huang, L., Montoya, A. L. and Nester, E. W., J. Biol.
Chem. (1975) 250:7675-7681); [0038] 2. 3-dehydroquinate synthase
enzyme comprises the amino acid sequence of SEQ ID NO:4 and is
encoded by a aroB gene which comprises the polynucleotide SEQ ID
NO:3 (Frost, J. W., Bender, J. L. Kadonaga, J. T. and Knowles, J.
R., Biochemistry (1984) 23:4470-4475); [0039] 3. 3-dehydroquinate
dehydratase enzyme comprises the amino acid sequence of SEQ ID NO:6
and is encoded by a aroQ gene which comprises the polynucleotide
SEQ ID NO:5 (Chaudhuri, S., Duncan, K. and Coggins, J. R., Methods
Enzymol. (1987) 142:320-324); [0040] 4. Shikimate dehydrogenase
enzyme comprises the amino acid sequence of SEQ ID NO:8 and is
encoded by a aroE gene which comprises the polynucleotide SEQ ID
NO:7 (Chaudhuri, S., Anton, I. A. and Coggins, J. R., Methods
Enzymol. (1987) 142:315-320); [0041] 5. 5-enolpyruvyl shikimate
3-phosphate synthase enzyme comprises the amino acid sequence of
SEQ ID NO:10 and is encoded by a aroA gene comprising SEQ ID NO:9
(Lewendon, A. and Coggins, J. R., Biochem. J. (1983) 213:187-191);
[0042] 6. Chorismate synthase enzyme comprises the amino acid
sequence of SEQ ID NO:12 and is encoded by a aroC gene comprising
SEQ ID NO:11 (White, P. J., Millar, G. and Coggins, J. R., Biochem.
J. (1988) 251:313-322); [0043] 7. Chorismate mutase--prephenate
dehydrogenase enzyme comprises the amino acid sequence of SEQ ID
NO:14 and is encoded by a tyrA gene comprising SEQ ID NO:13
(Davidson, B. E. and Hudson, G. S., Methods Enzymol. (1987)
142:440-450); [0044] 8. Anthranilate synthase, component I enzyme
comprises the amino acid sequence of SEQ ID NO:16 and is encoded by
a trpE gene comprising SEQ ID NO:15 (Bauerle, R., Hess, J. and
French, S., Methods Enzymol. (1987) 142:366-386); [0045] 9.
Anthranilate synthase, component II enzyme comprises the amino acid
sequence of SEQ ID NO:18 and is encoded by a trpG gene comprising
SEQ ID NO:17 (Bauerle, R., Hess, J. and French, S., Methods
Enzymol. (1987) 142:366-386); [0046] 10. anthranilate
phosphoribosyl transferase enzyme comprises the amino acid sequence
of SEQ ID NO:20 and is encoded by a trpD gene comprising SEQ ID
NO:19 (Hommel, U., Lustig, A. and Kirschner, K., Eur. J. Biochem.
(1989) 180:33-40); [0047] 11. Phosphoribosyl anthranilate isomerase
enzyme comprises the amino acid sequence of SEQ ID NO:22 and is
encoded by a trpF gene comprising SEQ ID NO:21 (Hoch, S. O., J.
Bacteriol (1979) 139:362-368); [0048] 12. Indole-3-glycerol
phosphate synthase enzyme comprises the amino acid sequence of SEQ
ID NO:24 and is encoded by a trpc gene comprising SEQ ID NO:23
(Hoch, S. O., J. Bacteriol (1979) 139:362-368); [0049] 13.
Tryptophan synthase, B protein comprises the amino acid sequence of
SEQ ID NO:26 and is encoded by a trpB gene comprising SEQ ID NO:25
(Miles, E. W., Bauerle, R. and Ahmed, S. A., Methods Enzymol 1987
(142) 398-414); [0050] 14. Tryptophan synthase, A protein comprises
the amino acid sequence of SEQ ID NO:28 and is encoded by a trpA
gene comprising SEQ ID NO:27 (Miles, E. W., Bauerle, R. and Ahmed,
S. A., Methods Enzymol 1987 (142) 398-414); [0051] 15. Pyruvate
carboxylase A-subunit comprises the amino acid sequence of SEQ ID
NO:30 and is encoded by a pycA gene comprising SEQ ID NO:29
(Mukhopadhyay, B. et. al., Arch. Microbiol. (2000) 174:406-414);
[0052] 16. Pyruvate carboxylase B-subunit enzyme comprises the
amino acid sequence of SEQ ID NO:32 and is encoded by a pycB gene
comprising SEQ ID NO:31 (Mukhopadhyay, B. et. al., Arch. Microbiol.
(2000) 174:406-414); [0053] 17. Phosphoenolpyruvate carboxylase
enzyme comprises the amino acid sequence of SEQ ID NO:34 and is
encoded by a ppc gene comprising SEQ ID NO:33 (O'Regan, M. et. al.
Gene (1989) 77:237-251); [0054] 18. Aspartate aminotransferase 1
enzyme comprises the amino acid sequence of SEQ ID NO:36 and is
encoded by a aat1 gene comprising SEQ ID NO:35 (Sung, M. H. et. al.
J. Bacteriol. (1990) 172:1345-1351); [0055] 19. Aspartate
aminotransferase 2 enzyme comprises the amino acid sequence of SEQ
ID NO:38 and is encoded by a aat2 gene comprising SEQ ID NO:37
(Sung, M. H. et. al. J. Bacteriol. (1990) 172:1345-1351); [0056]
20. Tetrahydrodipicolinate succinylase enzyme comprises the amino
acid sequence of SEQ ID NO:40 and is encoded by a dapD gene
comprising SEQ ID NO:39 (Richaud, C. et. al., J. Biol. Chem. (1984)
259:14824-14828); [0057] 21. Succinyl diaminopimelate
aminotransferase enzyme comprises the amino acid sequence of SEQ ID
NO:42 and is encoded by a dapC gene comprising SEQ ID NO:41 (Fuchs,
T. M. et. al., J. Bacteriol. (2000) 182:3626-3631); [0058] 22.
Succinyldiaminopimelate desuccinylase enzyme comprises the amino
acid sequence of SEQ ID NO:44 and is encoded by a dapE gene
comprising SEQ ID NO:43 (Bouvier, J. et. al., J. Bacteriol. (1992)
174:5265-71); [0059] 23. Diaminopimelate epimerase enzyme comprises
the amino acid sequence of SEQ ID NO:46 and is encoded by a dapF
gene comprising SEQ ID NO:45 (Richaud, C., Higgins, W.,
Mengin-Lecreulx, D. and Stragier, P., J. Bacteriol. (1987)
169:1454-1459); [0060] 24. Homoserine O-acetyltransferase enzyme
comprises the amino acid sequence of SEQ ID NO:48 and is encoded by
a metX gene comprising SEQ ID NO:47 (Yamagata, S., J. Bacteriol
(1987) 169:3458-3463); [0061] 25. O-acetylhomoserine sulfhydrylase
enzyme comprises the amino acid sequence of SEQ ID NO:50 and is
encoded by a metY gene comprising SEQ ID NO:49 (Ozaki, H. and
Shiio, I., J. Biochem. (1982) 91:1163-1171); [0062] 26. Homoserine
kinase enzyme comprises the amino acid sequence of SEQ ID NO:52 and
is encoded by a thrB gene comprising SEQ ID NO:51 (Huo, X. and
Viola, R. E., Arch. Biochem. Biophys. (1996) 330:373-379); [0063]
27. Threonine synthase enzyme comprises the amino acid sequence of
SEQ ID NO:54 and is encoded by a thrc gene comprising SEQ ID NO:53
(Malumbres, M., Mateos, L. M., Lumbreras, M. A., Guerrero, C. and
Martin, J. F., Appl. Environ. Microbiol. (1994) 60:2209-2219).
[0064] The terms "stringent conditions" or "stringent hybridization
conditions" includes reference to conditions under which a
polynucleotide will hybridize to its target sequence, to a
detectably greater degree than other sequences (e.g., at least
2-fold over background). Stringent conditions are
sequence-dependent and will be different in different
circumstances. By controlling the stringency of the hybridization
and/or washing conditions, target sequences can be identified which
are 100% complementary to the probe (homologous probing).
Alternatively, stringency conditions can be adjusted to allow some
mismatching in sequences so that lower degrees of similarity are
detected (heterologous probing).
[0065] Typically, stringent conditions will be those in which the
salt concentration is less than approximately 1.5 M Na ion,
typically about 0.01 to 1.0 M Na ion (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions also may be achieved with the addition of destabilizing
agents such as formamide. Exemplary low stringency conditions
include hybridization with a buffer solution of 30 to 35%
formamide, 1 M NaCl, 1% SDS (w/v; sodium dodecyl sulphate) at
37.degree. C., and a wash in 1.times. to 2.times.SSC
(20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to
55.degree. C. Exemplary moderate stringency conditions include
hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at
37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to
60.degree. C. Exemplary high stringency conditions include
hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C.,
and a wash in 0.1.times.SSC at 60 to 65.degree. C.
[0066] Specificity is typically the function of post-hybridization
washes, the critical factors being the ionic strength and
temperature of the final wash solution. For DNA--DNA hybrids, the
Tm can be approximated from the equation of Meinkoth and Wahl
(Anal. Biochem., 138:267-284, 1984): Tm=81.5.degree. C.+16.6 (log
M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of
monovalent cations, % GC is the percentage of guanosine and
cytosine nucleotides in the DNA, % form is the percentage of
formamide in the hybridization solution, and L is the length of the
hybrid in base pairs. The Tm is the temperature (under defined
ionic strength and pH) at which 50% of a complementary target
sequence hybridizes to a perfectly matched probe. Tm is reduced by
about 1.degree. C. for each 1% of mismatching; thus, Tm,
hybridization and/or wash conditions can be adjusted to hybridize
to sequences of the desired identity. For example, if sequences
with approximately 90% identity are sought, the Tm can be decreased
10.degree. C.
[0067] Generally, stringent conditions are selected to be about
5.degree. C. lower than the thermal melting point (Tm) for the
specific sequence and its complement at a defined ionic strength
and pH. However, severely stringent conditions can utilize
hybridization and/or wash at 1, 2, 3, or 4.degree. C. lower than
the thermal melting point (Tm); moderately stringent conditions can
utilize a hybridization and/or wash at 6, 7, 8, 9, or 10.degree. C.
lower than the thermal melting point (Tm); low stringency
conditions can utilize a hybridization and/or wash at 11, 12, 13,
14, 15, or 20.degree. C. lower than the thermal melting point (Tm).
Using the equation, hybridization and wash compositions, and
desired Tm, those of ordinary skill will understand that variations
in the stringency of hybridization and/or wash solutions are
inherently described. If the desired degree of mismatching results
in a Tm of less than 45.degree. C. (aqueous solution) or 32.degree.
C. (formamide solution), it is preferred to increase the SSC
concentration so that a higher temperature can be used. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen, Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays", Elsevier, New York (1993); and Current
Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds.,
Greene Publishing and Wiley-Interscience, New York (1995).
[0068] Stringent hybridization conditions are understood to mean
those conditions where hybridization, either in solution or on a
solid support, occur between two polynucleotide molecules which are
70% to 100% homologous in nucleotide sequence which include 75%,
80%, 85%, 90%, 95%, 98% and all values and subranges
therebetween.
[0069] Homology, sequence similarity or sequence identity of
nucleotide or amino acid sequences may be determined conventionally
by using known software or computer programs. To find the best
segment of identity or similarity of sequences, BLAST (Altschul et
al (1990) J. Mol. Biol. 215:403-410 and Lipman et al (1990) J. Mol.
Biol. 215:403-410), FASTA (Lipman et al (1985) Science
227:1435-1441), or Smith and Waterman (Smith and Waterman (1981) J.
Mol. Biol. 147:195-197) homology search programs can be used. To
perform global alignments, sequence alignment programs such as the
CLUSTAL W (Thompson et al (1994) Nucleic Acids Research
22:4673-4680) can be used.
[0070] The present invention also provides processes for preparing
amino acids using bacteria that comprise at least one
polynucleotide whose expression is enhanced or attenuated.
Likewise, the invention also provides processes for preparing amino
acids using bacteria that comprise at least on polypeptide whose
activity is enhanced or attenuated. Preferably, a bacterial cell
with enhanced or attenuated expression of one or more of the
polypeptides and/or polynucleotides described herein will improve
amino acid yield at least 1% compared to a bacterial strain not
having the enhanced or attenuated expression. For the production of
amino acids the M. methylotrophus polynucleotides described herein
may be used to target expression, either by disruption to turn off
or increase or enhance the expression or relative activity of the
polypeptide enzymes encoded therein.
[0071] The term "enhancement" as used herein means increasing
intracellular activity of one or more polypeptides in the bacterial
cell, which in turn are encoded by the corresponding
polynucleotides described herein. To facilitate such an increase,
the copy number of the genes corresponding to the polynucleotides
described herein may be increased. Alternatively, a strong and/or
inducible promoter may be used to direct the expression of the
polynucleotide, the polynucleotide being expressed either as a
transient expression vehicle or homologously or heterologously
incorporated into the bacterial genome. In another embodiment, the
promoter, regulatory region and/or the ribosome binding site
upstream of the gene can be altered to achieve the over-expression.
The expression may also be enhanced by increasing the relative
half-life of the messenger RNA.
[0072] In another embodiment, the enzymatic activity of the
polypeptide itself may be increased by employing one or more
mutations in the polypeptide amino acid sequence, which increases
the activity. For example, altering the relative Km of the
polypeptide with its corresponding substrate will result in
enhanced activity. Likewise, the relative half-life of the
polypeptide may be increased.
[0073] In either scenario, that being enhanced gene expression or
enhanced enzymatic activity, the enhancement may be achieved by
altering the composition of the cell culture media and/or methods
used for culturing.
[0074] "Enhanced expression" or "enhanced activity" as used herein
means an increase of at least 10%, 25%, 50%, 75%, 100%, 150%, 200%,
300%, 400% or 500% compared to a wild-type protein, polynucleotide,
gene; or the activity and/or the concentration of the protein
present before the polynucleotides or polypeptides are
enhanced.
[0075] The term "attenuation" as used herein means a reduction or
elimination of the intracellular activity of the polypeptides in a
bacterial cell that are encoded by the corresponding
polynucleotide. To facilitate such a reduction or elimination, the
copy number of the genes corresponding to the polynucleotides
described herein may be decreased or removed. Alternatively, a weak
and/or inducible promoter may used to direct the expression of the
polynucleotide, the polynucleotide being expressed either as a
transient expression vehicle or homologously or heterologously
incorporated into the bacterial genome. For example, the endogenous
promoter or regulatory region of the gene corresponding to the
isolated polynucleotides described herein may be replaced with the
aforementioned weak and/or inducible promoter. Alternatively, the
promoter or regulatory region may be removed. The expression may
also be attenuated by decreasing the relative half-life of the
messenger RNA.
[0076] In another embodiment, the enzymatic activity of the
polypeptide itself may be decreased or deleted by employing one or
more mutations in the polypeptide amino acid sequence, which
decreases the activity or removes any detectable activity. For
example, altering the relative Kd of the polypeptide with its
corresponding substrate will result in attenuated activity.
Likewise, a decrease in the relative half-life of the polypeptide
will result in attenuated activity.
[0077] By attenuation measures, the activity or concentration of
the corresponding protein is in general reduced to 0 to 75%, 0 to
50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration
of the wild-type protein or of the activity or concentration of the
protein in the starting microorganism.
[0078] Suitable vectors for carrying M. methylotrophus
polynucleotides include those vectors which can direct expression
of the gene in bacterial cells as known in the art. One embodiment
of the present invention is whereby the vectors contain an
inducible or otherwise regulated expression system whereby the M.
methylotrophus polynucleotides may be expressed under certain
conditions and not expressed under other conditions. Furthermore,
in another embodiment of the invention, the M. methylotrophus
polynucleotides can be constitutively expressed. Examples of such
vectors and suitable cells in which they can be introduced are
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY (2001) and Current Protocols in Molecular Biology,
Ausebel et al, (Eds.), John Wiley and Sons, Inc., New York,
2000.
[0079] Methods of introducing M. methylotrophus polynucleotides or
vectors containing the M. methylotrophus polynucleotides include
electroporation, conjugation, calcium-mediated transfection,
infection with bacteriophage and other methods known in the art.
These and other methods are described in Sambrook et al., Molecular
Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY (2001) and Current
Protocols in Molecular Biology, Ausebel et al, (Eds.), John Wiley
and Sons, Inc., New York (2000).
[0080] The microorganisms that can be used in the present invention
should have the ability to produce amino acids, preferably L-amino
acids, from a suitable carbon source, preferably carbon sources
such as methanol, glucose, sucrose, lactose, fructose, maltose,
molasses, starch, cellulose glycerol or ethanol. The microorganisms
can be Methylophilus bacteria, preferably Methylophilus
methylotrophus.
[0081] Suitable culture conditions for the growth and/or production
of M. methylotrophus polynucleotides are dependent on the cell type
used. Likewise, culturing cells that contain attenuated or enhanced
expression of the M. methylotrophus polynucleotides or
polypeptides, as described herein, may be cultured in accordance
with methods known in the art. Examples of culture conditions for
various cells is described in Sambrook et al., Molecular Cloning: A
Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY (2001); Current Protocols in Molecular
Biology, Ausebel et al, (Eds.), John Wiley and Sons, Inc., 2000;
and Cells: A Laboratory Manual (Vols. 1-3), Spector et al, (Eds.),
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
(1988).
[0082] Following culturing the polypeptide or protein products,
which are encoded by the M. methylotrophus polynucleotides, may be
purified using known methods of protein purification. These methods
include high performance liquid chromatography (HPLC), ion-exchange
chromatography, size exclusion chromatography; affinity separations
using materials such as beads with exposed heparin, metals, or
lipids; or other approaches known to those skilled in the art.
These and other methods of protein purification are disclosed in
Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001);
Current Protocols in Molecular Biology, Ausebel et al, eds., John
Wiley and Sons, Inc., 2000 and Protein Purification, Scopes and
Cantor, (Eds.), Springer-Verlag, (1994). Likewise, the amino acids
produced may be purified by methods known in the art using similar
chromatography devices.
[0083] The invention also provides antibodies that bind to the
polypeptides of the present invention. Antibodies binding to the
polypeptides can be either monoclonal or polyclonal, preferably the
antibodies are monoclonal. Methods for obtaining antibodies that
bind to the polypeptides are known in the art and are described,
for example, in Harlow and Lane, Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
(1988).
[0084] Having generally described this invention, a further
understanding can be obtained by reference to certain specific
examples which are provided herein for purposes of illustration
only, and are not intended to be limiting unless otherwise
specified.
EXAMPLES
[0085] Whole genome sequencing using random shotgun method is
described in Fleischman R. D. et. al. (1995) Science, 269:
496-512.
Example 1
Construction of Genomic Libraries of Methylophilus
methylotrophus
[0086] M. methylotrophus AS1 was cultured at 30.degree. C. in the
121 medium described in the Catalogue of Strains (The National
Collections of Industrial and Marine Bacteria Ltd., 1994).
[0087] Cells were collected by centrifugation. Genomic DNA was
isolated using the Genome-tip system (Qiagen K. K., Tokyo, Japan).
The genomic DNA was sheared and fragmentized by sonication. The
resultant fragments in the 1- to 2-kb size range were purified by
gel electrophoresis through 1% low-melting agarose gel, followed by
recovery using the Wizard DNA purification kit (Promega K K, Tokyo,
Japan). The recovered fragments were ligated to the high-copy
number vector pUC 118 treated by HincII and bacterial alkaline
phosphatase (Takara Shuzo, Kyoto, Japan), and this was designated
pUC 118 library.
[0088] For larger fragments (9- to 11-kb in size), the genomic DNA
was partially digested by restriction endonuclease Sau3AI, followed
by 0.6% agarose gel electrophoresis. The DNA fragments
corresponding 9-kb to 11-kb in size were excised from gel and were
recovered using the DNACELL (Daiichi Pure Chemicals, Tokyo, Japan).
The recovered fragments were ligated into the low-coy number vector
pMW118 (Nippon Gene, Toyama, Japan), which is a derivative of the
pSC101 (Bernaidi, A. and Bernardi, F. (1984) Nucleic Acids Res. 12,
9415-9426). This library composed of large DNA fragments was
designated pMW118 library.
[0089] General DNA manipulation was performed according to
previously described methods (Sambrook et. al. (1989) "Molecular
Cloning: A Laboratory Manual/Second Edition", Cold Spring Harbor
Laboratory Press).
Example 2
DNA sequencing and Sequence Assembly
[0090] The pUC118 library were transformed into Escherichia coli
DH5.alpha. and plated on Luria-Bertani medium containing 100
.mu.g/ml ampicillin and 40 .mu.g/ml
5-bromo-4-chloro-3-indolyl-.alpha.-D-galactoside (X-Gal). The white
colonies were picked up and cultured in Luria-Bertani medium
containing 100 .mu.g/ml ampicillin. The individual colony was
cultured in the well of the 96 deep-well plates, and the plasmids
were isolated using QIAprep Turbo Kit (Qiagen). The DNA fragments
inserted into pUC118 were sequenced using a M13 reverse primer. The
shotgun sequencing was performed with the BigDye terminators and
3700 DNA analyzer (Applied Biosystems Japan, Tokyo, Japan).
Approximately 50,000 samples from pUC118 library corresponding to
coverage of approximately 8-fold to the genome size were analyzed
and the sequences were assembled by Phred/Phrap software
(CodonCode, Mass., USA). This assembly treatment yielded 60 contigs
with more than 5 kb in size.
[0091] As for pMW 18 library, 2,000 clones corresponding to
coverage of approximately 5-fold were sequenced using both M13
forward and reverse primers. The end-sequence data were analyzed
and the linking clones between contigs were selected from pMW118
library. The inserted fragments of selected clones were amplified
by the polymerase chain reaction (PCR) using LA Taq polymerase
(Takara Shuzo) and M. methylotrophus genomic DNA as a template.
These products of PCR were entirely sequenced as described in
Example 1, and the gap DNA sequences between contigs were
determined. By the additional sequence information, the Phrap
assembly software reduced the number of contigs with more than 5 kb
in size to 24. Then the 48 DNA primers with sequences complementary
to the end-sequences of the 24 contigs were prepared. All possible
pairwise combination of the primers were tested by PCR to amplify
the DNA fragments of M. methylotrophus genomic DNA. The amplified
products were sequenced directly. In several cases, the additional
primers complementary to different sequences at the end of the
contig were used. This strategy could close all of the remaining
physical gaps and resulted in a single circular contig. Several
regions that had been sequenced in only one direction and had
postulated secondary structure were confirmed. By this research,
the genome of M. methylotrophus was found to be a single circular
with the size of 2,869,603 bases and GC content of 49.6%.
Example 3
Sequence Analysis and Annotation
[0092] Sequence analysis and annotation was managed using the
Genome Gambler software (Sakiyama, T. et. al. (2000) Biosci.
Biotechnol. Biochem. 64: 670-673). All open reading frames of more
than 150 bp in length were extracted and the translated amino acid
sequences were searched against non-redundant protein sequences in
GenBank using the BLAST program (Altschul, S. F. et. al. (1990) J.
Mol. Biol. 215, 403-410). Of putative polynucleotide encoding
sequences with significant similarities to the sequences in public
databases (BLASTP scores of more than 100), the genes involved in
biosynthesis of amino acids were selected. Start codons (AUG or
GUG) were putatively identified by similarity of the genes and
their proximity to the ribosome binding sequences (Shine, J. and
Dalgarno, L. (1975) Eur. J. Biochem. 57: 221-230). Careful
assignment of gene function resulted in the identification of
aromatic amino acid biosynthetic genes, the shikimate kinase gene
(aroK), the 3-dehydroquinate synthase gene (aroB), the
3-dehydroquinate dehydratase gene (aroQ), the shikimate
dehydrogenase gene (aroE), the 5-enolpyruvyl shikimate 3-phosphate
synthase gene (aroA), the chorismate synthase gene (aroC), the
chorismate mutase--prephenate dehydrogenase gene (tyrA), the
anthranilate synthase, component I gene (trpE), the anthranilate
synthase, component II gene (trpG), the anthranilate phosphoribosyl
transferase gene (trpD), the phosphoribosyl anthranilate isomerase
gene (trpF), the indole-3-glycerol phosphate synthase gene (trpC),
the tryptophan synthase, B protein gene (trpB), and the tryptophan
synthase, A protein gene (trpA). The pyruvate carboxylase A-subunit
gene (pycA) and the pyruvate carboxylase B-subunit gene (pycB) were
found probably in operon. The phosphoenolpyruvate carboxylase gene
(ppc) and the aspaatate amino acid biosynthetic genes, the two
aspartate aminotransferase genes (aat1 and aat2), the
tetrahydrodipicolinate succinylase gene (dapD), the succinyl
diaminopimelate aminotransferase gene (dapC), the
succinyldiaminopimelate desuccinylase gene (dapE), the
diaminopimelate epimerase gene (dapF), the homoserine
O-acetyltransferase gene (metX), the O-acetylhomoserine
sulfhydrylase gene (metY), the homoserine kinase gene (thrB), and
the threonine synthase gene (thrC) were identified.
[0093] Obviously, numerous modifications and variations on the
present invention are possible in light of the above teachings. It
is therefore to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described herein.
[0094] While the invention has been described in detail with
reference to preferred embodiments thereof, it will be apparent to
one skilled in the art that various changes can be made, and
equivalents employed, without departing from the scope of the
invention. Each of the aforementioned documents is incorporated by
reference herein in its entirety.
* * * * *