# Patent

## Title
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.

## Background
This application is a divisional under 35 U.S.C. §120 of Ser. No. 10/375,039, filed Feb. 28, 2003, the entirety of which is incorporated by reference.


BACKGROUND OF THE INVENTION
1. Field of the Invention
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.
2. Discussion of the Background
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.
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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
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.
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.
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).
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.
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.
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.
“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.
“Isolated” as used herein means separated out of its natural environment.
“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.
“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.
“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.
“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.
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.
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.
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)).
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 2nd Edition (Springer Verlag, New York, 1997).
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).
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:

    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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);
    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).


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).
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° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° 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° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.
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° 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° 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° C.
Generally, stringent conditions are selected to be about 5° 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° 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° 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° 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° C. (aqueous solution) or 32° 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 1, 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).
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.
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.
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.
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.
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.
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.
“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.
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.
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.
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.
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, N.Y. (2001) and Current Protocols in Molecular Biology, Ausebel et al, (Eds.), John Wiley and Sons, Inc., New York, 2000.
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, N.Y. (2001) and Current Protocols in Molecular Biology, Ausebel et al, (Eds.), John Wiley and Sons, Inc., New York (2000).
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. 
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, N.Y. (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).
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, N.Y. (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.
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, N.Y. (1988).
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
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 
M. methylotrophus AS1 was cultured at 30° C. in the 121 medium described in the Catalogue of Strains (The National Collections of Industrial and Marine Bacteria Ltd., 1994).
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 KK, Tokyo, Japan). The recovered fragments were ligated to the high-copy number vector pUC118 treated by HincII and bacterial alkaline phosphatase (Takara Shuzo, Kyoto, Japan), and this was designated pUC118 library.
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 (Bemaidi, A. and Bernardi, F. (1984) Nucleic Acids Res. 12, 9415–9426). This library composed of large DNA fragments was designated pMW118 library.
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
The pUC118 library were transformed into Escherichia coli DH5α and plated on Luria-Bertani medium containing 100 μg/ml ampicillin and 40 μg/ml 5-bromo-4-chloro-3-indolyl-α-D-galactoside (X-Gal). The white colonies were picked up and cultured in Luria-Bertani medium containing 100 μg/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, MA, USA). This assembly treatment yielded 60 contigs with more than 5 kb in size.
As for pMW118 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
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.
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.
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.

## Claims

1. An isolated polynucleotide, which encodes a protein comprising the amino acid sequence of SEQ ID NO:4.

2. A vector comprising the isolated polynucleotide of claim 1.

3. An isolated host cell transformed with 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. An isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:3.

7. A vector comprising the isolated polynucleotide of claim 6.

8. An isolated host cell transformed with the isolated polynucleotide of claim 6.

9. The host cell of claim 8, which is a Methylophilus bacterium.

10. The host cell of claim 8, which is a Methylophilus methylotrophus bacterium.

11. An isolated polynucleotide, which hybridizes under stringent conditions to the isolated polynucleotide of claim 10, wherein the stringent conditions comprise hybridization in 50% formamide 1M NaCl and 1% SDS at 37° C.and a wash in 0.1×SSC at 65° C.

12. A vector comprising the isolated polynucleotide of claim 11.

13. An isolated host cell tranformed with the isolated polynucleotide of claim 11.

14. An isolated polynucleotide, which is at least 95% identical to the polynucleotide of claim 6, and wherein said polynucleotide encodes a polypeptide having 3-dehydroquinate synthase activity.

15. A vector comprising the isolated polynucleotide of claim 14.

16. An isolated host cell transformed with the isolated polynucleotide of claim 14.