KR100906179B1 - Polypeptides and biosynthetic pathways - Google Patents

Abstract

The present invention can be used to prepare monatin from glucose, tryptophan, indole-3-lactic acid, indole-3-pyruvate and 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid. It relates to methods and compositions. The present invention also provides a process for preparing indole-3-pyruvate and 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid intermediate. The compositions provided herein comprise nucleic acid molecules, polypeptides, chemical structures and cells. The methods of the present invention include both in vitro and in vivo methods and in vitro methods include chemical reactions.

Description

POLYPEPTIDES AND BIOSYNTHETIC PATHWAYS

Related Application Information

This application claims priority to US patent application 60 / 374,831, filed April 23, 2002.

The present invention relates to polypeptides and biosynthetic pathways useful for the production of indole-3-pyruvate, 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid (MP) and / or monatin.

Indole Pyruvate

Indole-3-pyruvate is a potent antioxidant that is thought to counteract oxidative stress in high oxygen tissues (Politi et al. "Recent advances in Tryptophan Research", edited by GAFilippini et al., Plenum Press, New York, 1996, pp 291-8). Indole pyruvate is also an intermediate in the pathway that produces indole-acetic acid (IAA), a major plant growth hormone auxin (diffuse growth promoter). IAA exhibits activity in sub-microgram amounts in a range of physiological processes, such as spermatologic dominance, flexibility, vein elongation, induction of mesenchymal cell division and initiation of rooting. Synthetic auxin is used in horticulture to induce rooting and to promote fruit deletion and growth. High concentrations of synthetic auxin are effective as herbicides on broadleaf plants. Natural auxin produced by fermentation can be seen as environmentally friendly compared to synthetic herbicides. Growth regulators sold £ 400 million ($ 1.4 billion U.S.) worldwide in 1999.

Some patents for indole acetic acid and derivatives thereof include US Pat. No. 5,843,782 (Micropropagation of rose plants, auxin used in culture medium) and US Pat. No. 5,952,231 (Micropropagation of rose plants).

In addition to plant related utility, indole acetic acid is also useful for pharmaceutical use. U.S. Patent 5,173,497 ("Method of preparing alpha-oxopyrrolo [2,3-B] indole acetic acids and derivatives") proposes the use of this compound useful for treating memory impairments, such as those associated with Alzheimer's disease and senile dementia. have. The mechanism proposed in US Pat. No. 5,173,497 is that the compound inhibits polypeptide acetylcholinesterase and increases the concentration of acetylcholine in the brain.

Indole-3-carbinol is produced from indole-3-acetic acid via peroxadase catalytic oxidation and can be readily converted to diindolinylmethane. Both compounds have been reported to eliminate toxins and promote hormone production that is beneficial to women's health.

Tryptophan derivatives

Chlorinated D-tryptophan has been identified as a nutritive sweetener and there is increasing interest in other derivatives that follow. Monatin is a natural sweetener similar in composition to the amino acid tryptophan. Monatin can be extracted from the root bark of Sclerochiton ilicofolius , a South African shrub, and has been promising in the food and beverage industry as a high intensity sweetener. Some patents for monatin include US Patent 5994559 (Synthesis of monatin-A high intensity natural sweetner), US Patent 4975298 (3- (1-amino-1,3-dicarboxy-3-hydroxy-but-4-yl) -indole compounds), US Patent 5128164 (Composition for human consumption containing 3- (1-amino-1,3-dicarboxy-3-hydroxy-but-4-yl) -indole compounds; and US Patent 5128482 (Process for the production of 3-1 (1-amino-1,3-dicarboxy-3-hydroxy-but-4-yl) indole).

Some of the monatin precursors described herein may also be usefully used as synthetic sweeteners or as intermediates in the synthesis of monatin derivatives.

The present invention is derived from glucose, tryptophan, indole-3-lactic acid and / or via monatin precursors such as indole-3-pyruvate and 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid. Several biosynthetic pathways for making monatin are provided. The invention also discloses polypeptide and nucleic acid sequences that can be used to prepare monatin, indole-3-pyruvate and 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid.

Monatin is indole-3-pyruvate, 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid (monatin precursor, MP, alpha-keto form of monatin), indole-3 -Via lactic acid, tryptophan and / or glucose (FIG. 1). Methods of producing or preparing monatin or intermediates thereof are illustrated in FIGS. 1-3 and 11-13, which convert the substrate into a first product and then convert the first product into a second product. Reactions that last until the desired end product is produced.

1-3 and 11-13 show potential intermediates and final products in boxes. For example, conversion from one product to another, such as glucose to tryptophan, tryptophan to indole-3-pyruvate, indole-3-pyruvate to MP, MP to monatin, or indole-3-lactic acid (indol- Lactate) to indole-3-pyruvate can be expressed in this manner. This conversion can be promoted chemically or biologically. The term “converts” means that chemical means or polypeptides are used in the reaction that changes from the first intermediate to the second intermediate. The term "chemical conversion" refers to a reaction that is not actively promoted by a polypeptide. The term "biological conversion" means a reaction that is actively promoted by a polypeptide. The conversion can occur in vivo or in vitro. If a biological transformation is used, the polypeptide and / or cells can be immobilized on the support, such as by chemically attaching on the polymer support. This conversion can be carried out using all reactors known to those skilled in the art, such as batch reaction or continuous reactors.

In addition, the present invention provides a method of preparing a first product by contacting a substrate with a first polypeptide, preparing a second product by contacting the formed first product with a second polypeptide, and then forming the second product with the third polypeptide. Also provided is a method comprising the step of contacting with a third product to produce a third product, such as monatin. The polypeptides used and the resulting products are shown in FIGS. 1-3 and 11-13.

The present invention also discloses polypeptides and coding sequences thereof that can be used in the conversion reactions shown in FIGS. 1-3 and 11-13. In some instances, polypeptides having one or more point mutations that modify the substrate specificity and / or activity of the polypeptide are also used for monatin production.

The present invention also discloses isolated recombinant cells that produce monatin. This cell can be any cell, such as a plant, animal, bacterium, yeast, algae, embryonic or fungal cell.

In certain embodiments, the disclosed cells are cells that possess one or more of the following activities, such as tryptophan aminotransferase (EC 2.6.1.27), tyrosine (aromatic) aminotransferase (EC 2.6.1.5), multiple substrate amino Transferase (EC 2.6.1.-), aspartate aminotransferase (EC 2.6.1.1), tryptophan dehydrogenase (EC 1.4.1.19), tryptophan-phenylpyruvate aminotransferase (EC 2.6.1.28), L- Amino acid oxidase (EC 1.4.3.2), tryptophan oxidase (without EC number, Hadar et al., J. Bacterial 125: 1096-1104, 1976 and Furuya et al., Biosci Biotechnol Biochem 64: 1486-93, 2000) , D-amino acid dehydrogenase (EC 1.4.99.1), D-amino acid oxidase (EC 1.4.3.3), D-alanine aminotransferase (EC 2.6.1.21), synthetase / degrading enzyme (EC 4.1.3.- ), Such as 4-hydroxy-4-methyl-2-oxoglutarate aldolase (EC 4.1.3.17) or 4-hydroxy-2-oxoglutarate aldolase (EC 4.1.3.16), synthetic Enzyme / min Enzyme (4.1.2.-), D- tryptophan aminotransferase (Kohiba and Mito, Proceedings of the 8th international Symposium on Vitamin B 6 and Carbonyl Catalysis, Osaka, Japan 1990), phenylalanine dehydrogenase (EC 1.4.1.20) and And / or cells that have two or more activities, such as glutamate dehydrogenase (EC 1.4.1.2, 1.4.1.3, 1.4.1.4).

In another embodiment, the cell is a cell having one or more of the following activities, such as indole lactate dehydrogenase (EC 1.1.1.110), R-4-hydroxyphenyl lactate dehydrogenase (EC 1.1.1.222), 3- (4) -hydroxyphenylpyruvate reductase (EC 1.1.1.237), lactate dehydrogenase (EC 1.1.1.27, 1.1.1.28, 1.1.2.3), (3-imidazol-5-yl) lactate dehydrogenase ( EC 1.1.1.111), lactate oxidase (EC 1.1.3.-), synthetases / degrading enzymes (4.1.3.-) such as 4-hydroxy-4-methyl-2-oxoglutarate aldolase (EC 4.1.3.17) or 4-hydroxy-2-oxoglutarate aldolase (EC 4.1.3.16), synthetase / degrading enzyme (4.1.2.-), tryptophan dehydrogenase (EC 1.4.1.19) , Tryptophan-phenylpyruvate aminotransferase (EC 2.6.1.28), tryptophan aminotransferase (EC 2.6.1.27), tryptophan-phenylpyruvate aminotransferase (EC 2.6.1.5), multi-substrate aminotransferase (EC 2.6 .1.-), Aspartate Army Transcriptase (EC 2.6.1.1), phenylalanine dehydrogenase (EC 1.4.1.20), glutamate dehydrogenase (EC 1.4.1.2, 1.4.1.3, 1.4.1.4), D-amino acid dehydrogenase (EC 1.4.99.1) Cells containing at least two or at least three of the following activities: D-tryptophan aminotransferase and / or D-alanine aminotransferase (EC 2.6.1.21).

In addition, the disclosed cells may be cells which possess one or more of the following activities, such as tryptophan aminotransferase (EC 2.6.1.27), tyrosine (aromatic) aminotransferase (EC 2.6.1.5), multiple substrate aminotransferases (EC) 2.6.1.-), aspartate aminotransferase (EC 2.6.1.1), tryptophan dehydrogenase (EC 1.4.1.19), tryptophan-phenylpyruvate aminotransferase (EC 2.6.1.28), L-amino acid oxidase (EC 1.4.3.2), tryptophan oxidase (no EC number), D-amino acid dehydrogenase (EC 1.4.99.1), D-amino acid oxidase (EC 1.4.3.3), D-alanine aminotransferase (EC 2.6. 1.21), indole lactate dehydrogenase (EC 1.1.1.110), R-4-hydroxyphenyl lactate dehydrogenase (EC 1.1.1.222), 3- (4) -hydroxyphenylpyruvate reductase (EC 1.1.1.237) , Lactate dehydrogenase (EC 1.1.1.27, 1.1.1.28, 1.1.2.3), (3-imidazol-5-yl) lactate dehydrogenase (EC 1.1.1.111), lactate oxidase (EC 1.1.3.-) , synthesis Bovine / degrading enzyme (4.1.3.-) such as 4-hydroxy-4-methyl-2-oxoglutarate aldolase (EC 4.1.3.17) or 4-hydroxy-2-oxoglutarate egg Dolase (EC 4.1.3.16), synthetase / degrading enzyme (4.1.2.-), glutamate dehydrogenase (EC 1.4.1.2, 1.4.1.3, 1.4.1.4), phenylalanine dehydrogenase (EC 1.4.1.20) And / or cells that have two or more or three or more activities, such as D-tryptophan aminotransferase.

Monatin is the first polypeptide that converts tryptophan and / or indole-3-lactic acid to indole-3-pyruvate and tryptophan or indole-3-lactic acid (both indole- form D or L of tryptophan or indole-3-lactic acid). Contacting with a substrate converted to 3-pyruvate; those skilled in the art will understand that the polypeptide selected in this step would ideally exhibit suitable specificity); Contacting the indole-3-pyruvate with a second polypeptide that converts the resulting indole-3-pyruvate to 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid (MP) step; And contacting the MP with a third polypeptide that converts the MP to monatin. Examples of polypeptides that can be used for this conversion are illustrated in FIGS. 2 and 3.

The present invention also provides, in another aspect, a cell containing a composition such as MP, two or more, or sometimes three or more, or four or more polypeptides encoded in at least one exogenous nucleic acid sequence.

These and other aspects of the invention will be apparent from the following detailed description and illustrative examples.

1 depicts the biosynthetic pathway used to prepare monatin and / or indole-3-pyruvate. One route produces indole-3-pyruvate through tryptophan and the other route through 3-lactic acid. Monatin is then produced via 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid (MP) intermediate.

The compounds shown in the boxes are the products produced by the substrate and the biosynthetic pathway.

The composition shown next to the arrow is a cofactor or a reactant that can be used while the substrate is converted to the product. The cofactors or reactants used will vary with the polypeptides used in the specific steps of the biosynthetic pathway. Cofactor PLP (pyridoxal 5′-phosphate) can catalyze the reaction regardless of polypeptide, so simply providing PLP allows the substrate to proceed to the product.

2 is a more detailed schematic of the biosynthetic pathway using MP intermediates. Substrates used in each step of this route are boxed. Polypeptides that convert each substrate are listed next to the arrows located between each substrate. Each polypeptide is listed by its generic name and enzyme classification (EC) number.

3 is a more detailed schematic of the biosynthetic pathway for converting indole-3-lactic acid to indole-3-pyruvate. Substrates are marked with boxes, and the polypeptides that convert each substrate are listed next to the arrows located between each substrate. Each polypeptide is listed by its generic name and enzyme classification (EC) number.

4 illustrates one possible reaction that can produce MP via chemical means.

5A and 5B are chromatograms showing the results obtained by LC / MS for enzymatically produced monatin.

6 is an electrospray mass spectrum of enzymatically synthesized monatin.

7A and 7B are chromatograms showing LC / MS / MS daughter ion analysis of monatin produced in an enzymatic mixture.

8 is a chromatogram showing the results of high resolution mass spectrometry of enzymatically produced monatin.

9A-9C are chromatograms showing chiral separation of (A) R-tryptophan, (B) S-tryptophan and (C) enzymatically produced monatin.

10 is a bar graph showing the relative amount of monatin produced in bacterial cells after IPTG induction. (-) Indicates no substrate (tryptophan or pyruvate).                 

11 and 12 are schematic diagrams showing the route used to increase the yield of monatin produced from tryptophan or indole-3-pyruvate.

FIG. 13 is a schematic diagram illustrating pathways that can be used to increase the yield of monatin produced from tryptophan or indole-3-pyruvate.

Sequence list

Nucleic acid sequences and amino acid sequences listed in the appended sequence listings are presented using standard letter abbreviations for nucleotide bases and three letter codes for amino acids. Each nucleic acid sequence has shown only one strand, but it should be understood to include complementary strands which may be known by reference to the strands presented as such.

SEQ ID NOs: 1 and 2 are Sinorhizobium The nucleic acid sequence and amino acid sequence of the aminotransferase derived from meliloti ) are shown respectively (tatA gene, called a tyrosine or aromatic aminotransferase in literature).

SEQ ID NOs: 3 and 4 are Rhodobacter Nucleic acid and amino acid sequences of tyrosine aminotransferases derived from sphaeroides ) (2.4.1) are shown respectively (homologous aminotransferases in the genomics software due to homology with tatA Estimated).

SEQ ID NOs: 5 and 6 show nucleic acid sequences and amino acid sequences of aminotransferases derived from Rhodobacter spheroid (35053), respectively (2.4.1 sequences, novel cloned based on SEQ ID NOs: 3 and 4).

SEQ ID NOs: 7 and 8 show the nucleic acid and amino acid sequences of the broad substrate aminotransferase (bsat) derived from Leishmania major , respectively.

SEQ ID NOs: 9 and 10 show nucleic acid sequences and amino acid sequences of aromatic aminotransferases (ara T) derived from Bacillus subtilis , respectively.

SEQ ID NOs: 11 and 12 are Lactobacillus amyloborus amylovorus ) shows the nucleic acid sequence and amino acid sequence of the aromatic aminotransferase derived from (identified by the homology to the aromatic aminotransferase).

SEQ ID NOs: 13 and 14 show nucleic acid sequences and amino acid sequences of multiple substrate aminotransferases (msa) derived from R. spheroid (35053), respectively (Accession No. AAAE01000093.1 (bp 14743-16155) and Accession No. ZP00005082). Identified as multiple substrate aminotransferases by homology with .1).

SEQ ID NOs: 15 and 16 show the primers used to clone the B. subtilis D-alanine aminotransferase (dat) sequence.

SEQ ID NOs: 17 and 18 show the primers used to clone the S. melototti tatA sequence.

SEQ ID NOs: 19 and 20 show the primers used to clone the B. subtilis araT aminotransferase sequence.

SEQ ID NOs: 21 and 22 show the primers used to clone the multiple substrate aminotransferase sequences of Rhodobacter spheroids (2.4.1 and 35053).                 

SEQ ID NOs: 23 and 24 show the primers used to clone the bsat sequence of Laishmania major.

SEQ ID NOs: 25 and 26 show the primers used to clone the araT sequence of Lactobacillus amyloborus.

SEQ ID NOs: 27 and 28 show the primers used to clone the R. spheroid tatA sequences (2.4.1 and 35053).

SEQ ID NOs: 29 and 30 show the primers used to clone the E. coli aspC sequence (gene sequence Genbank Accession No. AE000195.1, protein sequence Genbank Accession No. AAC74014.1).

SEQ ID NOs: 31 and 32 show amino acid sequences and nucleic acid sequences of aromatic aminotransferases (tyrB) derived from E. coli, respectively.

SEQ ID NOs: 33 and 34 show the primers used to clone the E. coli tyrB sequence.

SEQ ID NOs: 35 to 40 show primers used to clone a polypeptide having 4-hydroxy-2-oxoglutarate aldolase (KHG) (EC 4.1.3.16) activity.

SEQ ID NOs: 41 and 42 are tyrosine phenols derived from tryptophanase (tna) and Citrobacter freundii derived from E. coli encoding proteins P00913 (GI: 401195) and P31013 (GI: 401201), respectively. The nucleic acid sequence of the degrading enzyme (tpl) is shown, respectively.                 

SEQ ID NOs: 43-46 show the primers used to clone the tryptophanase polypeptide and β-tyrosinase (tyrosine phenolase) polypeptide.

SEQ ID NOs: 47-54 show primers used to mutate the tryptopase polypeptide and the β-tyrosinase polypeptide.

SEQ ID NOs: 55-64 show the primers used to clone a polypeptide having 4-hydroxy-4-methyl-2-oxoglutarate aldolase (EC 4.1.3.17) activity.

SEQ ID NOs: 65 and 66 show the nucleic acid and amino acid sequences of 4-hydroxy-4-methyl-2-oxoglutarate aldolase (proA) derived from C.testosteroni, respectively. .

SEQ ID NOs 67 and 68 show the primers used to clone C.testosterone 4-hydroxy-4-methyl-2-oxoglutarate aldolase (proA) in an operon carrying E. coli aspC in pET30 Xa / LIC will be.

SEQ ID NOs: 69-72 show the primers used to clone E. coli aspC and C. testosterone proA in pESC-his.

SEQ ID NOs: 73 and 74 show sequences added to the 5 'end of the primers used for cloning the genes disclosed herein.

Detailed Description of the Preferred Embodiments

Acronyms and Terms

The following description of terms and methods is intended to describe the invention in more detail and to be an indicator when those skilled in the art practice the invention. As used herein, the term "containing" means "including" and the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the expression "containing a protein" includes one such protein or a plurality of proteins, and the expression "containing a cell" includes one or more kinds of cells and their equivalents known to those skilled in the art. And the like.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those disclosed herein can be used in the practice or testing of the present invention, but suitable methods and materials are set forth below. The materials, methods, and examples presented are illustrative and should not be considered as limiting. The following detailed description and claims will also reveal other features and advantages of the present invention.

cDNA (complementary DNA ): DNA fragments that determine transcription and lack internal, non-coding segments (introns) and regulatory sequences. cDNA can be synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.

Conservative substitutions: substitution of one or more amino acids with an amino acid residue having similar biochemical properties (eg, two, five or ten residues). In general, conservative substitutions have little or no effect on the activity of the resulting polypeptide. For example, an tryptophan aminotransferase polypeptide that ideally contains one or more conservative substitutions will retain tryptophan aminotransferase activity. For example, standard procedures such as site directed mutagenesis or PCR can be used to prepare polypeptides comprising one or more conservative substitutions through manipulation of the nucleotide sequence encoding the polypeptide.

Substitutable variants are those wherein at least one residue in the amino acid sequence is removed and another residue is inserted at that position. Examples of amino acids that may be substituted for the original amino acid present in a protein and that are considered conservative substitutions include: ala-ser or thr; arg-substitution by gln, his or lys; asn-substitution with glu, gln, lys, his, asp; asp-substitution with asn, glu or gln; cys = substituted with ser or ala; gln-substitution with asn, glu, lys, his, asp or arg; glu-asn, gln, lys or asp; gly-substituted with pro; his-asn, lys, gln, arg, tyr; ile-substitution with leu, met, val, phe; leu-substituted with ile, met, val, phe; lys-asn, glu, gln, his, arg substitution; substitution with met-ile, leu, val, phe; phe-substituted with trp, tyr, met, ile, or leu; ser-thr, substituted by ala; thr-substitution by ser or ala; trp-phe, substituted by tyr; tyr-substitution by his, phe or trp; And val-met, ile, leu.

Further information on conservative substitutions can be found in other literature, in particular in Ben-Bassat et al. (J. Bacteril. 169: 751-7, 1987), O'Regan et al., (Gene 77: 237-51, 1989). ), Sahin-Toth et al., (Protein Sci. 3: 240-7, 1994), Hochuli et al., (Bio / Technology 6: 1321-5, 1988), WO 00/67796 (Curd et al.) And standard books on genetics and molecular biology.

Exogenous : The term "exogenous" as used herein with reference to nucleic acids and specific cells refers to any nucleic acid from which the particular cell found in nature is not of origin. Thus, non-naturally occurring nucleic acids are considered exogenous to the cell when introduced into the cell. Naturally occurring nucleic acids may also be exogenous for certain cells. For example, a complete chromosome isolated from cells of human X is a nucleic acid that is exogenous to cells of human Y when it is introduced into Y cells.

Functional equivalence: to have equivalent functions. In the case of enzymes, functionally equivalent molecules include other molecules that retain the function of the enzyme. For example, functional equivalents can be provided by sequence changes in the enzyme sequence, where a peptide with one or more sequence changes retains the function of the unmodified peptide to exhibit its enzymatic activity. In certain embodiments, the functional equivalent of tryptophan aminotransferase possesses the property of converting tryptophan to indole-3-pyruvate.

Examples of sequence modifications include, but are not limited to, conservative substitutions, deletions, mutations, structural shifts and insertions. As an example, where a given polypeptide binds an antibody, its functional equivalent is a polypeptide that binds the same antibody. That is, functional equivalents include peptides that have the same binding specificity as the polypeptide and can be used as reagents in place of the polypeptide. As an example, functional equivalents include polypeptides where the binding sequence is discontinuous and the antibody binds to a linear epitope. That is, if the peptide sequence is MPELANDLGL (amino acids 1 to 10 of SEQ ID NO: 12), the functional equivalent includes a discontinuous epitope which can be represented as: NH2-**-M ** P ** E ** L * * A ** B ** D ** L ** G ** L-COOH. In this example, if the conformation of this polypeptide can bind to a monoclonal antibody that binds amino acids 1 to 10 of SEQ ID NO: 12, then the polypeptide is a functional equivalent to amino acids 1 to 10 of SEQ ID NO: 12.

Hybridization: As used herein, the term "hybridization" refers to a method of testing complementarity of nucleotide sequences of two nucleic acid molecules, tested based on the properties of complementary single-stranded DNA and / or RNA to form double-stranded molecules. do. Nucleic acid hybridization techniques can be used to obtain isolated nucleic acids that fall within the scope of the invention. Briefly, all nucleic acids having some homology with the sequences set forth in SEQ ID NO: 11 can be used as probes that can identify similar nucleic acids through hybridization under moderate to high stringency conditions. Once identified, the nucleic acid can then be purified, sequenced and analyzed to determine whether it is within the scope of the present invention.

Hybridization can be performed through Southern analysis or Northern analysis to identify DNA sequences or RNA sequences that hybridize to the probe, respectively. This probe can be labeled with a radioisotope such as biotin, digoxigenin, polypeptide or ³² P. The analyte DNA or RNA is electrophoretically separated on agarose or polyacrylamide gels and then transferred to nitrocellulose, nylon or other suitable membranes, followed by standard techniques known in the art (eg Sambrook et al., (1989) the method described in sections 7.39-7.52 of Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, NY). Generally, probes are nucleotides of at least about 20 lengths. For example, probes corresponding to 20 consecutive nucleotide sequences set forth in SEQ ID NO: 11 can be used to identify identical or similar nucleic acids. In addition, probes of nucleotides longer or shorter than 20 may be used.

The invention also provides for at least about 12 bases in length (eg, at least about 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50). , 60, 100, 250, 500, 750, 1000, 1500, 2000, 3000, 4000 or 5000 bases) and having the sequence set forth in SEQ ID NO: 11 under hybridization conditions An isolated nucleic acid sequence is provided that hybridizes to either the sense or antisense strand of the nucleic acid. Here, the hybrid condition may be a hybrid condition of medium or high stringency.

In describing the present invention, medium stringency hybrid conditions are 25 mM KPO ₄ (pH 7.4), 5X SSC, 5X Denhardt's solution, 50 μg / ml denatured and sonicated sperm DNA, 50% formamide, 10% Hybridization was performed at about 42 ° C. in a hybrid solution containing dextran sulfate and 1-15 ng / ml probe (about 5 × 10 ⁷ cpm / μg), and a wash solution containing 2 × SSC and 0.1% sodium dodecyl sulfate was added. Use to perform the wash at about 50 ° C.

High stringency hybrid conditions include 25 mM KPO ₄ (pH 7.4), 5X SSC, 5X Denhardt's solution, 50 μg / ml denatured and sonicated salmon sperm DNA, 50% formamide, 10% dextran sulfate and 1-15ng Hybridization was performed at about 42 ° C. in a hybrid solution containing / ml of probe (about 5 × 10 ⁷ cpm / μg) and at about 65 ° C. using a wash solution containing 0.2 × SSC and 0.1% sodium dodecyl sulfate. Means to carry out a wash.

Isolated: The term "isolated" as used herein in reference to a nucleic acid refers directly to the naturally occurring genome of the organism from which it is derived (one at the 5 'end and the other at the 3' end. A naturally occurring nucleic acid that does not directly contact two sequences. For example, an isolated nucleic acid is a recombinant DNA molecule of any length, provided that one of the nucleic acid sequences that are observed to be in direct contact with the recombinant DNA molecule in the naturally occurring genome is either free or absent. It is not limited. That is, the isolated nucleic acid may be a vector, autonomous replicating plasmid, a virus (e.g., a recombinant DNA (eg, cDNA or genomic DNA fragments generated by PCR or restriction enzyme processing) that exists as a separate molecule regardless of other sequences). Retrovirus adenovirus, herpes virus), or recombinant DNA incorporated into genomic DNA of prokaryotic or eukaryotic cells. The isolated nucleic acid may also include recombinant DNA molecules that are part of a hybrid or fusion nucleic acid sequence.

The term "isolated" as used herein in reference to nucleic acids also includes any non-naturally occurring nucleic acid because there is no sequence directly contacting in the naturally occurring genome and no non-naturally occurring nucleic acid sequence is observed in nature. Sometimes. For example, non-naturally occurring nucleic acids, such as engineered nucleic acids, are considered isolated nucleic acids. The engineered nucleic acid can be prepared using conventional molecular cloning or chemical nucleic acid synthesis techniques. The isolated non-naturally occurring nucleic acid is present independently of other sequences or integrated into the vector, autonomous replicating plasmid, virus (eg retrovirus, adenovirus or herpes virus), or genomic DNA of prokaryotic or eukaryotic cells. May exist. In addition, non-naturally occurring nucleic acids may include nucleic acid molecules that are part of a hybrid or fusion nucleic acid sequence.

It will be appreciated by those skilled in the art that gel fragments containing nucleic acid or genomic DNA restriction digests present among hundreds to millions of other nucleic acid molecules, such as in cDNA or genomic libraries, are not considered isolated nucleic acids.

Nucleic Acid: As used herein, the term “nucleic acid” is intended to include both RNA and DNA, including but not limited to cDNA, genomic DNA and synthetic (eg, chemically synthesized) DNA. The nucleic acid may be double stranded or single stranded. If single stranded, the nucleic acid may be the sense strand or the antisense strand. In addition, nucleic acids may be circular or linear.

Operationally Linked: If the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence, the first nucleic acid sequence is "operably linked" with the second nucleic acid sequence. For example, if a promoter affects the transcription of a coding sequence, that promoter is operably linked to the coding sequence. In general, operably linked DNA sequences are contiguous and are located in the same reading frame as needed for binding of two polypeptide coding regions.

Peptide Modifications: The invention also includes enzymes and their combinations. In addition, analogs (non-peptide organic molecules), derivatives (peptide molecules with chemical functional groups derived from the starting peptide sequences) and variants (homologs) can also be used in the methods described herein if the desired enzymatic activity is present. have. Peptides disclosed herein comprise sequences of amino acids that may be naturally occurring and non-naturally occurring L-amino acids and / or D-amino acids.

Peptides may be modified by a variety of chemical techniques to produce derivatives that have substantially the same activity as unmodified peptides and, in some cases, other desirable properties. For example, the carboxylic acid group of a protein may be provided or esterified in the form of a salt of a pharmaceutically acceptable cation whether or not at the carboxy terminus or side chain to form a C1 to C16 ester, or the formulas NR1R2 where R1 and R2 are each Can be converted to or combined with an amide represented by H or independently C1 to C16 alkyl to form a heterocyclic ring such as a five or six membered ring. The amino group of the peptide, whether amino or terminal or side chain, is in the form of a pharmaceutically acceptable acid addition salt, such as HCl salt, HBr salt, acetate, benzoate, toluene sulfonate, maleate, tartarate and other organic acid salts or It may be modified with C1 to C16 alkyl or dialkyl amino or further converted to amide.

The hydroxyl groups of the peptide side chains can be converted to C1 to C16 alkoxy or C1 to C16 esters using known techniques. The phenyl and phenol rings of the peptide side chains are substituted with one or more halogen atoms, such as F, Cl, Br or I or substituted with C1 to C16 alkyl, C1 to C16 alkoxy, carboxylic acids and esters thereof or amides of these carboxylic acids. Can be. The methylene groups of the peptide side chains may extend to homogeneous C2 to C4 alkylenes. Thiols may be protected with any of a number of known protecting groups, such as acetamide groups. In addition, those skilled in the art will be well aware of how to introduce ring structures into the peptides of the present invention in order to limit their conformation by selecting structures that enhance stability. For example, addition of C- or N-terminal cysteines to a peptide results in the formation of disulfide bonds upon oxidation of the peptide, resulting in cyclic peptides. Other peptide cyclization methods include the formation of thioethers and carboxy and amino termini amides and esters.

Peptide mimetics and organomimetics also fall within the scope of the present invention, wherein the three-dimensional arrangement of the chemical mimetics of the peptidomimetic and organic mimetics mimics the three-dimensional arrangement of the peptide backbone and the amino acid side chains constructed. Such peptidomimetics and organomimetics of the proteins disclosed herein have detectable enzymatic activity. When used through computer modeling, the drug action cluster is an ideal three-dimensional definition with the structural conditions necessary for biological activity. Peptide mimetics and organomimetics can be designed for each drug action stage using current computer modeling software (computer-aided drug design or CADD). Techniques used in CADD are described by Walters, "Computer-Assisted Modeling of Drugs", in Klegerman & Groves (eds.), Pharmaceutical Biotechnology, 1993, Interpharm Press: Buffalo Grove, IL, pp. 165-74 and Ch. 102 in Munsosn (ed.). Principles of Pharmacology, 1995, Chapman & Hall. Also included in the scope of the present invention are mimetics produced using such techniques. As an example, mimetics are those that mimic the enzymatic activity exhibited by enzymes, variants, fragments or fusions thereof.

Probes and Primers: Nucleic acid probes and primers can be readily prepared based on the amino acid sequences and nucleic acid sequences disclosed herein. A "probe" includes an isolated nucleic acid containing a detectable label or reporter molecule. Examples of labels include, but are not limited to, radioactive isotopes, ligands, chemiluminescent agents, and polypeptides. Guidance on labeling methods and label selection suitable for various purposes is described, for example, in Sambrook et al. (Ed), Molecular Cloning: A Laboratory Manual 2nd ed., Vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al. (Ed.) Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York (with periodic updates), 1987]. Is discussed.

A “primer” is generally a nucleic acid molecule having 10 or more nucleotides (eg, a nucleic acid molecule having about 10 to about 100 nucleotides). Primers can be annealed to complementary target nucleic acid strands through nucleic acid hybridization to form a hybrid between the primer and the target nucleic acid strands, and then extended along the target nucleic acid strands using, for example, DNA polymerase polypeptides. Primer pairs can be used for amplification of nucleic acid sequences, such as through polymerase chain reaction (PCR) or other nucleic acid amplification methods known in the art.

Methods of making and using probes and primers are described, for example, in []. PCR primer pairs can be prepared from known sequences using, for example, computer programs for such purposes as Primer (Version 0.5, © 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). Those skilled in the art will appreciate that although the specificity of a particular probe or primer increases with length, the probe or primer can vary in size, ranging from full length sequences to sequences consisting of as few as five short nucleotides. Thus, for example, a primer consisting of 20 consecutive nucleotides can be annealed if it is desired to target with a primer with higher specificity than a primer consisting only of 15 nucleotides. That is, to obtain higher specificity, for example, 10, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 , 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750 , 1800, 1850, 1900, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050 , 3100, 3150, 3200, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400 , Including 4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 5000, 5050, 5100, 5150, 5200, 5250, 5300, 5350, 5400, 5450 or more consecutive nucleotides You can choose.

Promoter: An array of nucleic acid regulatory sequences that induce transcription of a nucleic acid. The promoter includes a TATA factor in the case of an essential nucleic acid sequence, such as a polymerase type II promoter, present near the transcription initiation site. Promoters may include distant enhancers (enhancers) or suppressors (repressors) that may be present at positions thousands of base pairs away from the transcription initiation site.

Purified: The term "purified" as used herein does not require absolute purity and should be understood as somewhat relative. For example, the purified polypeptide or nucleic acid agent may be at a concentration higher than the concentration of polypeptide or nucleic acid that may be present in the natural environment within the organism. For example, if the polypeptide content in the preparation is at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% of the total protein content of the preparation, it is considered purified. can do.

Recombinant : A “recombinant” nucleic acid is a sequence created by artificially combining (1) a sequence that does not originally exist in the organism to be expressed or (2) two different distinct short sequences. Such artificial combinations are often done by chemical synthesis or, more generally, by artificially manipulating the fragments of an isolated nucleic acid, for example using genetic engineering. "Recombinant" also refers to nucleic acid molecules that have been artificially engineered but contain regulatory sequences and coding regions such as those found in isolated organisms.

Sequence identity: Similarity between amino acid sequences is referred to as similarity between sequences, also called sequence identity. Sequence identity is often measured through a ratio of identity (or similarity or homology), with higher ratios indicating that the two sequences are more similar. Peptide homologs or variants such as SEQ ID NO: 12 show relatively high sequence identity when aligned by standard methods.

Methods for aligning sequences for comparison purposes are known in the art. Various programs and alignment algorithms have been described in numerous literature [Smith and Waterman, Adv. Appl. Math. 2: 482, 1981; Needleman and Wunsch, J. Mol. Biol. 48: 443-53, 1970: Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444-8, 1988; Higgins and Sharp, Gene 73: 237-44, 1988; Higgins and Sharp, CABIOS 5: 151-3, 1989; Corpet et al., Nucleic Acids Research 16: 10881-90, 1988; And Altschul et al., Nature Genet. 6: 119-29, 1994].

The NCBI Basic Local Alignment Search Tool (BLAST ™) (Altschul et al., J. Mol. Biol. 215: 403-10, 1990) used in conjunction with sequencing programs blastp, blastn, blastx, tblastn and tblastx Available from the National Center for Biotechnology Information (NCBI, Bethesda, MD) and the Internet.

Peptide variants are generally characterized by using NCBI Blast 2.0 and forming gaps with blasp set for the difold parameters and having at least 50% or more sequence identity counted at full length alignment with amino acid sequences. When comparing amino acid sequences consisting of more than about 30 amino acids, use the Blast 2 sequence function using the default BLOSUM62 matrix set for the default variables (gap entity cost 11, gap cost 1 for each residue). When aligning short peptides (peptides less than about 30 amino acids), alignment is performed via the Blast 2 sequence function using the PAM30 matrix set for the default parameters (open gap 9, extension gap 1 penalty). Proteins with significantly greater similarity to control sequences show increased identity ratios, such as at least 80%, at least 90%, at least 95%, at least 98%, even at least 99% sequence identity as assessed by this method. Where fewer than the entire sequence is compared for sequence identity, homologues and variants generally exhibit at least 80% or more sequence identity in a short window of 10 to 20 amino acids and at least 85% depending on similarity to the control sequence. , At least 90%, at least 95% or 98% sequence identity. Methods for measuring sequence identity in such a short window are described on a website maintained by NCBI (Bedesda, MD). Those skilled in the art will appreciate that such ranges of sequence identity serve only as a guiding role, and there is absolutely the likelihood that strong significant analogues outside the ranges indicated are obtained.

Similar methods may be used to determine the sequence identity ratio of nucleic acid sequences. As a specific example, homologous sequences are aligned with native sequences and the number of matches is determined by counting the number of positions at which the same nucleotide or amino acid residue is present in both sequences. The sequence identity ratio is determined by dividing the number of matches by the length of the sequence described in the identified sequence (e.g., SEQ ID NO: 11) or the segmented length (e.g., 100 contiguous nucleotides or amino acid residues from the sequence described in the identified sequence) Measure by multiplying the value by 100. For example, when aligned with the sequence set forth in SEQ ID NO: 11, the nucleic acid sequence with 1166 matches is 75.0% identical to the sequence set forth in SEQ ID NO: 11 (ie 1166 ÷ 1554 * 100 = 75.0). Sequence identity ratios round to the nearest 1/10 unit. For example, 75.11, 75.12, 75.13 and 75.14 round off to 75.1 and 75.15, 75.16, 75.17, 75.18 and 75.19 round off to 75.2. Also note that in particular, the length of a target sequence containing 20 nucleotide sequences aligned with 20 contiguous nucleotides from a sequence identified as follows, in particular, that the length is always an integer, is 75% sequence identity with the sequence identified above. Contains regions that share (ie, 15 ÷ 20 * 100 = 75).

Specific Binders: Agents that can specifically bind any polypeptide disclosed herein. Examples include polyclonal antibodies, monoclonal antibodies (including humanized monoclonal antibodies) and fragments of monoclonal antibodies such as Fab, F (ab ') ₂ , Fv fragments and epitopes of such polypeptides. Other agents, including but not limited to.

Antibodies to a polypeptide (or fragment, variant or fusion thereof) provided herein can be used to purify or identify the polypeptide. The amino acid sequences and nucleic acid sequences provided herein can be used to prepare specific antibody based binders that recognize the polypeptides described herein.

Monoclonal antibodies or polyclonal antibodies can be generated against the polypeptides of the invention, portions thereof or variants thereof. Most preferably, the polypeptide is specifically detected using antibodies raised against one or more epitopes present on the polypeptide antigen. That is, an antibody raised against one or more specific polypeptides will recognize and bind that particular polypeptide, while the other polypeptide will not substantially recognize or bind. Whether the antibody specifically binds to a particular polypeptide can be determined by one of a number of standard immunoassays, such as Western blotting (eg, Sambrook et al. (Ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

Western blotting allows certain antibody preparations (e.g., those produced in mice to polypeptides having the amino acid sequence set forth in SEQ ID NO: 12) to suitable polypeptides (e.g., polypeptides having the amino acid sequence set forth in SEQ ID NO: 12). To determine if specifically detected, total cell proteins can be first extracted from the cells and then separated via SDS-polyacrylamide gel electrophoresis.

The isolated total cellular protein is then transferred to a membrane (eg nitrocellulose) and incubated with the antibody preparation. After washing the membrane to remove nonspecifically bound antibody, the presence of the specifically bound antibody was determined by addition of a polypeptide such as alkaline phosphatase (5-bromo-4-chloro-3-indoleyl phosphate / nitro blue tetrazolium). Measurements can be made using a suitable second antibody (eg, an anti-mouse antibody) that binds to a dark blue compound by time immunolocalized alkaline phosphatase.

Substantially pure polypeptides suitable for use as immunogens can be obtained from transfected cells, transformed cells or wild type cells. The polypeptide concentration in the final product can be adjusted to a concentration of several μg / ml, for example, by concentration using an Amicon filter. In addition, the size of the polypeptide can be used as an immunogen in a variety of sizes ranging from full length polypeptide to polypeptide having about 9 amino acid residues. Such polypeptides can be obtained in cell culture or chemically synthesized by standard methods or by cleavage into smaller polypeptides that can purify larger polypeptides. Polypeptides with as little as 9 amino acid residue lengths may be immunogenic when given to the immune system associated with major histocompatibility complex (MHC) molecules, such as MHC class I or MHC class II molecules. Thus, at least 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100 of all amino acid sequences disclosed herein , 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350 or more Contiguous amino acid residues can be used as immunogens to produce antibodies.

Monoclonal antibodies against any of the polypeptides disclosed herein can be prepared from murine hybridomas according to the classical methods of Koehler and Milstein (Nature 256: 495-7, 1975) or methods derived therefrom.

Polyclonal antisera containing antibodies against heterologous epitopes of any of the polypeptides disclosed herein can be prepared by immunizing a suitable animal with the polypeptide (or fragment thereof), which polypeptide is modified to enhance immunity. Or may not be modified. Effective immune protocols for rabbits can be found in Vaitukaitis et al. J. Clin. Endocrinol. Metab. 33: 988-91, 1971.

Instead of whole antibodies, antibody fragments that can be readily expressed in prokaryotic hosts can also be used. Methods of making and using immunologically effective portions of monoclonal antibodies, also referred to as "antibody fragments", are known and include those known in the literature, such as: Better & Horowitz (Methods Enzymol. 178: 476-96, 1989) ), Glockshuber et al. (Biochemistry 29: 1362-7, 1990), US Pat. No. 5,648,237 ("Expression of Functional Antibody Fragments"), US Pat. No. 4,946,778 ("Single Polypeptide Chain Binding Molecules", US Pat. 5,455,030 ("Immunotherapy Using Single Chain Polypeptide Binding Molecules") and references cited therein.

Transformed : A "transformed" cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques or the like. Transformation includes any technique that can introduce nucleic acid molecules into the cells described above, such as transfection with viral vectors, conjugation, transformation with plasmid vectors and electroporation, lipofection, particle total acceleration. Introduction of naked-type DNA by, but is not limited to such.

Variants, Fragments or Fusion Proteins: The proteins disclosed herein also include variants, fragments and fusions thereof. A protein (eg SEQ ID NO: 12), a fusion protein, or a DNA sequence encoding a fragment or variant of the protein (eg SEQ ID NO: 11) is a gene that allows the protein to be expressed in eukaryotic cells, bacteria, insects and / or plants. Can be manipulated. DNA sequences can be modified and operably linked to other regulatory sequences to effect expression. The final product containing regulatory sequences and proteins is called a vector. This vector can be introduced into eukaryotic cells, bacteria, insects and / or plant cells. Once introduced into the cell, the vector drives protein production.

The fusion protein is a protein such as tryptophan aminotransferase (or a variant, polymorph, mutant or fragment thereof) such as SEQ ID NO: 12, which has a desired activity such as converting tryptophan to indole-3-pyruvate, etc. It is included in a state bound to another amino acid sequence that does not inhibit. In one example, the other amino acid sequence is about 10 or less, 12 or less, 15 or less, 20 or less, 25 or less, 30 or less or 50 amino acids in length.

It will be appreciated by those skilled in the art that modifications to the DNA sequence can be made in a variety of ways without affecting the biological activity of the encoded protein. For example, PCR can be used to make modifications to DNA sequences encoding proteins. Such a variant may be a variant optimized for codons used to express the protein as preferred in the host cell, or a variant optimized for other sequence changes that promote expression.

Vector: A nucleic acid molecule that is introduced into a cell to produce a transformed cell. The vector may comprise a nucleic acid sequence such as origin of replication that allows replication within the cell. Vectors may also include genetic components other than one or more selectable marker genes known in the art.

Biosynthesis  Overview of the route

As shown in FIGS. 1-3 and 11-13, a number of biosynthetic pathways can be used for the purpose of producing monatin or its intermediates, such as indole-3-pyruvate or MP. There are several different polypeptides for the purpose of converting each substrate (glucose, tryptophan, indole-3-lactic acid, indole-3-pyruvate and MP) to each product (tryptophan, indole-3-pyruvate, MP and monatin). Can be used. Moreover, such reactions can be carried out in vivo or in vitro or through a combination of in vivo and in vitro reactions, such as non-enzymatic chemical reactions. Accordingly, FIGS. 1-3 and 11-13 are illustrative and illustrate a number of different routes that can be used to obtain the desired product.

In glucose To tryptophan  transform

Many organisms can synthesize tryptophan from glucose. Thus, it is possible to clone constructs containing the genes necessary for producing monatin, MP and / or indole-3-pyruvate from glucose and / or tryptophan in such organisms. It is shown herein that tryptophan can be converted to monatin.

In other instances, organisms may be genetically engineered using known polypeptides that produce tryptophan or overproduce tryptophan. For example, US Pat. No. 4,371,614, which is incorporated herein by reference, describes an E. coli strain transformed with a plasmid containing wild-type tryptophan operon.

The maximum titer of tryptophan disclosed in US Pat. No. 4,371,614 is about 230 ppm. Similarly, WO8701130 (incorporated herein) describes E. coli strains genetically engineered to produce tryptophan and discusses the increased fermentation production of L-tryptophan. Those skilled in the art will appreciate that organisms capable of producing tryptophan from glucose can produce similar results using other carbon and energy sources that can be converted to glucose or fructose-6-phosphate. Examples of such carbon and energy sources include, but are not limited to, sucrose, fructose, starch, cellulose or glycerol.

From tryptophan  Indole-3- To pyruvate  transform

Several polypeptides can be used to convert tryptophan to indole-3-pyruvate. Examples of such polypeptides include enzyme classes (EC) 2.6.1.27, 1.4.1.19, 1.4.99.1, 2.6.1.28, 1.4.3.2, 1.4.3.3, 2.6.1.5, 2.6.1.-, 2.6.1.1 and 2.6. Contains ingredients of 1.21. This class includes tryptophan aminotransferases that convert L-tryptophan and 2-oxoglutarate to indole-3-pyruvate and L-glutamate (alternatively, L-phenylalanine-2-oxoglutarate aminotransferase, tryptophan Aminotransferase, 5-hydroxytryptophan-ketoglutarate aminotransferase, hydroxytryptophan aminotransferase, L-tryptophan aminotransferase, L-tryptophan transaminase (collectively referred to as L-tryptophan aminotransferase), and L-tryptophan: also called 2-oxoglutarate aminotransferase); D-tryptophan aminotransferase (alternatively, NAD (P) -L-tryptophan dehydrogenase, L-tryptophan dehydrogenase, L-Trp-, which converts D-tryptophan and 2-oxo acid to indole-3-pyruvate and amino acids) Dehydrogenase, TDH and L-tryptophan: NAD (P) oxidoreductase (also called deaminoation); D-amino acid dehydrogenases, which convert D-amino acids and FAD to indole-3-pyruvate and NH ₃ and FADH ₂ ; Tryptophan-phenylpyruvate aminotransferase (alternately, L-tryptophan-α-ketoisocaproate aminotransferase and L-tryptophan which converts L-tryptophan and phenylpyruvate to indole-3-pyruvate and L-phenylalanine : Also called phenylpyruvate aminotransferase); L-amino acid oxidase (also opio-amino acid oxidase and L-amino acid: oxygen oxidoreductase (deami), which converts L-amino acid and H ₂ O and O ₂ to 2-oxo acid and NH ₃ and H ₂ O ₂ Also called aging); D-amino acid oxidase (also called opio-amino acid oxidase and D-amino acid: oxygen oxidoreductase (deaminoation)); And tryptophan oxidase which converts L-tryptophan and H ₂ O and O ₂ to indole-3-pyruvate and NH ₃ and H ₂ O ₂ . This class also exhibits tyrosine (aromatic) aminotransferases, aspartate aminotransferases, D-amino acids (or D-alanine) aminotransferases, and a plurality of aminotransferase activities, some of which include tryptophan and 2-oxo acid. Also included are indole-3-pyruvate and a broad (multiple substrate) aminotransferase that can be converted to amino acids.

Eleven components of the class of aminotransferases exhibiting this activity are described in Example 1, including the novel aminotransferases set forth in SEQ ID NOs: 11 and 12. Thus, the present invention provides an isolated nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or even at least 99% sequence identity with the sequences set forth in SEQ ID NOs: 11 and 12 and Provide protein sequences. The present invention also provides fragments and fusions of SEQ ID NOs: 11 and 12 that encode proteins that possess or exhibit aminotransferase activity. Examples of fragments include fragments of at least 10, 12, 15, 20, 25, 50, 100, 200, 500, or 1000 contiguous nucleotides of SEQ ID NO: 11 or at least 6, 10, 15 of SEQ ID NO: 12 Fragments consisting of, but not limited to, 20, 25, 50, 75, 100, 200, 300 or 350 contiguous amino acids. The disclosed sequences (and variants, fragments and fusions thereof) can be part of a vector. Such vectors can be used for transformation of host cells to produce recombinant cells capable of producing indole-3-pyruvate from tryptophan and in some cases additionally MP and / or monatin.

L-amino acid oxidase (1.4.3.2) is known and its sequence can be obtained from several different sources, such as Vipera levetin ( Vipera). lebetine sp P81375), Ophiophagus hannah sp P81383, Agkistrodon rhojdostoma spP81382), Crotalus atrox sp P56742, Burkholderia cepacia , Arabidopsis thaliana , Caulobacter cresentus , Chlamidomonas reinhardt ( Chlamydomonas reinhardtii ), Mus Musculus musculus ), Pseudomonas syringae and Rhodococcus strains. Tryptophan oxidase has also been described in the literature and can be isolated from, for example, Coprinus sp. SF-1, Chinese cabbage with clubbing root disease, Arabidopsis thaliana, and mammalian liver. One component of the L-amino acid oxidase capable of converting tryptophan to indole-3-pyruvate was discussed in Example 3 below along with other sources for molecular cloning. Many D-amino acid oxidase genes are readily available in databases for molecular cloning.

Tryptophan dehydrogenases are well known, for example, spinach, Pisum sativum , Prosopis Juliflora, Pears, Mesquite, Wheat, Corn, Tomato, Tobacco, Chromobacte It can be separated from such as Chromobacterium violaceum and Lactobacilli. Many D-amino acid dehydrogenase gene sequences are known.

As shown in Figures 11-13, catalase can be added to reduce or even eliminate the presence of hydrogen peroxide if an amino acid oxidase such as tryptophan oxidase is used to convert tryptophan to indole-3-pyruvate.

Indole-3- From lactate  Indole-3- To pyruvate  transform

The reaction for converting indole-3-lactate to indole-3-pyruvate can be catalyzed by various polypeptides, such as 1.1.1.110, 1.1.1.27, 1.1.1.28, 1.1.2.3, 1.1.1.222, 1.1 There are .1.237, 1.1.3.- or 1.1.1.111 components of the polypeptide class. 1.1.1.110 The polypeptide class includes indole lactate dehydrogenase (also called indole lactate: NAD ⁺ oxidoreductase). The classes 1.1.1.27, 1.1.1.28 and 1.1.2.3 include lactate dehydrogenases (also called lactate dehydrogenases, also called lactate: NAD ⁺ redox enzymes). The classes 1.1.1.222 include (R) -4-hydroxyphenyl lactate dehydrogenase (D-aromatic lactate dehydrogenase, R-aromatic lactate dehydrogenase and R-3- (4-hydroxyphenyl) lactate: NAD (P) ⁺ (Also referred to as 2-oxidoreductase), and the class 1.1.1.237 includes 3- (4-hydroxyphenylpyruvate) reductases (hydroxyphenylpyruvate reductase and 4-hydroxyphenyl lactate: NAD ⁺ redox Also called enzymes). 1.1.3.- Class includes lactate oxidase and 1.1.1.111 class includes (3-imidazol-5-yl) lactate dehydrogenase ((S) -3- (imidazol-5-yl) lactate: NAD (P) ⁺ also called oxidoreductase). Several polypeptides belonging to this class will be able to produce indole-3-pyruvate from indole-3-lactic acid. An example of such a conversion is discussed in Example 2.

Chemical reactions may also be used to convert indole-3-lactic acid to indole-3-pyruvate. These chemical reactions include B2 catalysts (China Chemical Reporter, v 13, n 28, p 18 (1), 2002), atmospheric oxidation with diluted permanganate and perchlorate, or hydrogen peroxide in the presence of metal catalysts. It includes an oxidation step that can be achieved by using a variety of methods, including.

2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid in indole-3-pyruvate Switch to (MP)

Various known polypeptides can be used for the conversion of indole-3-pyruvate to MP. Examples of such polypeptide classes are 4.1.3.-, 4.1.3.16, 4.1.3.17 and 4.1.2-. This class includes carbon-carbon synthetases / degrading enzymes such as aldolases that catalyze the condensation of two carboxylic acid substrates. Peptide class EC 4.1.3.- is a synthetase / degrading enzyme that forms a carbon-carbon bond using an oxo acid substrate (eg indole-3-pyruvate) as an electrophile, while EC 4.1.2.- It is a synthetase / degrading enzyme that forms a carbon-carbon bond using an aldehyde substrate (eg benzaldehyde) as an electrophile.

For example, the polypeptides described in EP 1045-029 (EC 4.1.3.16, 4-hydroxy-2-oxoglutarate aldolase, 2-oxo-4-hydroxyglutarate aldolase or KHG aldolase 4-hydroxy-2-oxoglutarate glyoxylate degrading enzyme), also called glyoxylic acid and pyruvate, converts 4-hydroxy-2-ketoglutaric acid, polypeptide 4-hydroxy-4-methyl 2-oxoglutarate aldolase (EC 4.1.3.17, otherwise referred to as 4-hydroxy-4-methyl-2-oxoglutarate pyruvate degrading enzyme or ProA aldolase) is characterized by two pyruvates and The same two keto acids are condensed with 4-hydroxy-4-methyl-2-oxoglutarate. Reactions using such degrading enzymes are described herein.

1 and 2 and FIGS. 11 to 13 are schematic diagrams of reactions in which 3-carbon (C3) molecules bind to indole-3-pyruvate. Polypeptides using multiple components of EC 4.1.2.- and 4.1.3.-, in particular PLP, can utilize C3 molecules which are amino acids or derivatives thereof such as serine, cysteine and alanine. Aldol condensation catalyzed by representative components of EC 4.1.2.- and 4.1.3.- requires that the C3 molecules in this pathway are pyruvate or derivatives thereof. However, other compounds can also act as C3 carbon sources and can be converted to pyruvate. Pyruvates and ammonia are L-serine, L-cysteine and derivatives of serine and cysteine having sufficient leaving groups such as O-methyl-L-serine, O-benzyl-L-serine, S-methylcysteine, S-benzylcysteine Obtained by beta elimination reaction of S-alkyl-L-cysteine, O-acyl-L-serine and 3-chloro-L-alanine (e.g., catalyzed by tryptophanase or β-tyrosinase) Can be. Aspartate is catalyzed by PLP mediated beta-lyase enzymes such as tryptophanase (EC 4.1.99.1) and / or β-tyrosinase (EC 4.1.99.2, also called tyrosine-phenol degrading enzymes). Can act as a source of pyruvate. The rate of beta-lyase enzyme reaction is described by Muratou et al. (J. Biol. Chem 274: 1320-5, 1999) and shown in Example 8. Increase by performing site directed mutagenesis on the polypeptide (4.1.99.1-2). Such modifications allow the polypeptide to accommodate the dicarboxylic acid amino acid substrate. Lactate can also serve as a source of pyruvate, which is oxidized to pyruvate by addition of lactate dehydrogenase and oxidized cofactors or lactate oxidase and oxygen. Examples of such reactions are described below. For example, ProA aldolase can be contacted with indole-3-pyruvate when pyruvate is used as the C3 molecule, as shown in FIGS. 2 and 11-13.

MP can also be prepared using chemical reactions, for example the aldol condensation set forth in Example 5.                 

MP in With monatin  transform

The MP to monatin conversion can be catalyzed by one or more of the following polypeptides: tryptophan aminotransferase (2.6.1.27), tryptophan dehydrogenase (1.4.1.19), D-amino acid dehydrogenase (1.4.99.1 ), Glutamate dehydrogenase (1.4.1.2-4), phenylalanine dehydrogenase (EC 1.4.1.20), tryptophan-phenylpyruvate aminotransferase (2.6.1.28) or more generally aminotransferase family (2.6.1. Components such as aspartate aminotransferase (EC 2.6.1.1), tyrosine (aromatic) aminotransferase (2.6.1.5), D-tryptophan aminotransferase or D-alanine (2.6.1.21) aminotransferase (FIG. 2). The eleven components of such aminotransferases are described in Example 1 below with novel components of this class illustrated in SEQ ID NOs: 11 and 12. The reaction demonstrating the activity of these aminotransferases and dehydrogenases is described in Example 7. Presented.

This reaction may also be carried out through a chemical reaction. Amination of keto acid (MP) can be carried out through reductive amination with ammonia and sodium cyanoborohydride.

11-13 show another polypeptide that can be used to convert MP to monatin and can be used to increase the yield of monatin from indole-3-pyruvate or tryptophan. For example, where aspartate is an amino feeder, aspartate aminotransferase can be used to convert aspartate to oxaloacetate (FIG. 11). This oxaloacetate is converted into pyruvate and carbon dioxide by dehydrogenase, such as oxaloacetate dehydrogenase (FIG. 11). In addition, when lysine is used as the amino donor, lysine ε-aminotransferase can be used to convert lysine to allicin (FIG. 12). Allicin is spontaneously converted to 1-piperidine-6-carboxylate (FIG. 12). Polypeptides capable of catalyzing reductive amination reactions (eg, glutamate dehydrogenases) are used to convert MPs to monatin, which can recycle NAD (P) H and / or produce volatile products (FIG. 13) can be used, for example formate dehydrogenase.

Biosynthesis  Additional considerations when designing a path

Depending on the polypeptides used to produce indole-3-pyruvate, MP and / or monatin, cofactors, substrates and / or additional polypeptides may be provided to the producing cells for the purpose of enhancing product formation.

Removal of hydrogen peroxide

Hydrogen peroxide (HO) is a product that, if produced, is toxic to production cells and can be harmful to the resulting polypeptide or intermediate. The aforementioned L-amino acid oxidase produces H ₂ O ₂ as a product. Thus, when L-amino acid oxidase is used, the resulting H ₂ O ₂ must be removed or its concentration reduced to reduce potential damage to cells or products.

(스타필로코커스 크실로서스),

(바실러스 할로듀란스),

(칸디다 알비칸스),

(스트렙토마이세스 코엘리컬러),

(크산토모나스 캄페스트리스)(SwissProt 수탁번호)가 있으며, 이에 국한되는 것은 아니다. L-아미노산 산화효소, D-아미노산 산화효소 또는 트립토판 산화효소를 이용하는 생물촉매 반응기에도 역시 카탈라제 폴리펩티드를 첨가할 수 있다.Catalase can be used to reduce the concentration of H ₂ O ₂ in cells (FIGS. 11-13). The producing cell may express a gene or cDNA sequence encoding catalase (EC 1.11.1.6) that catalyzes the breakdown of hydrogen peroxide into water and oxygen gas. For example, catalase can be expressed from a vector transfected into production cells. Examples of catalase that can be used include

(Staphylococcus xylose),

(Bacillus halodurance),

(Candida Albicans),

(Streptomyces coelicolor),

(Swiss Prot Accession No.), but is not limited thereto. Catalase polypeptides can also be added to biocatalyst reactors using L-amino acid oxidase, D-amino acid oxidase or tryptophan oxidase.

PLP (Pyridoxal-5'- phosphate ) Usability control

As shown in FIG. 1, PLP may be used in one or more biosynthesis steps described herein. PLPs can be supplemented with PLP concentrations that do not limit the total efficiency of the reaction.

Biosynthetic pathways of vitamin B ₆ (PLP precursors) have been thoroughly studied in E. coli and some proteins have been crystallized (Laber et al., FEBS Letters, 449: 45-8, 1999). Pyridoxal phosphate biosynthesis requires three unique genes (pdxA, pdxB and pdxJ), while other metabolic pathways require two genes (epd or gapB and serC). One starter observed in the E. coli pathway is 1-deoxy-D-xylulose-5-phosphate (DXP). The synthesis of this precursor from common C2 and C3 carbon core metabolites is catalyzed by polypeptide 1-deoxy-D-xylulose-5-phosphate synthase (DSX). Another precursor is a threonine derivative formed from D-erythrose 4-phosphate, a C4 sugar. Genes required for conversion to phospho-4-hydroxyl-L threonine (HTP) are epd, pdxB and secC. The final reaction of PLP formation is the complex intramolecular condensation and ring closure reaction between DXP and HTP, catalyzed by the gene products of pdxA and pdxJ.

If PLP becomes the limiting nutrient during fermentation for monatin production, increasing the expression of one or more of the genes of this pathway in production host cells may also increase the yield of monatin. The host organism may comprise genes of its natural pathway in a plurality of copies, or may inject copies of genes of the non-natural pathway into the organism genome. In addition, multiple copies of the gene of the recovery pathway can be cloned into the host organism.

One recovery pathway, preserved in all organisms, recycles various derivatives of vitamin B ₆ into active PLP form. Polypeptides included in this pathway include pdxK kinase, pdxH oxidase and pdxY kinase. Overexpression of such one or more genes may increase PLP utilization.

The concentration of vitamin B ₆ may be raised through the elimination or inhibition of metabolic regulation of the natural biosynthetic pathway genes present in the host organism. PLP inhibits polypeptides involved in the biosynthesis of precursor threonine derivatives in strain 238-7 of the bacterial Flavobacterium species. The metabolism-free bacterial strain can overproduce pyridoxal derivatives to secrete up to 20 mg / L PLP. Genetic engineering of monatin producing host organisms in a similar manner may increase PLP production without overexpression of the biosynthetic pathway genes.

ammonium Usability

Tryptopase reactions can be directed in the synthesis direction (production of tryptophan from indole) by making ammonia more available or by removing water. Reductive amination reactions, such as those catalyzed by glutamate dehydrogenase, can also be induced in the forward direction by excess ammonium.

Ammonia can be made available as ammonium carbonate or ammonium phosphate salts in carbonate or phosphate buffer systems. Ammonia may also be provided as ammonium pyruvate or ammonium formate. Alternatively, ammonia may be supplied when the reaction is linked to a reaction that produces ammonia, such as a glutamate dehydrogenase or tryptophan dehydrogenase reaction. Ammonia can also be produced by adding natural substrates of EC 4.1.99 .- (tyrosine or tryptophan) which are hydrolyzed with phenol or indole, pyruvate and NH ₃ . This allows the enzyme to hydrolyze its desired substrate, resulting in an increased yield of synthetic product than the amount of normal equilibrium.

Removal of products and byproducts

The conversion of tryptophan to indole-3-pyruvate via tryptophan aminotransferase results in the production of glutamate through this reaction and requires 2-oxoglutarate (α-ketoglutarate) as an auxiliary substrate. May adversely affect the production rate of -3-pyruvate. This is because glutamate can inhibit the aminotransferase so that a large amount of auxiliary substrate can be consumed in the reaction. Moreover, high concentrations of glutamate adversely affect downstream separation processes.

Polypeptide glutamate dehydrogenase (GLDH) converts glutamate to 2-oxoglutarate to recycle auxiliary substrates in reactions catalyzed by tryptophan aminotransferase. GLDH also produces a reducing equivalent (NADH or NADPH) that can be used for the production of energy (ATP) of cells under aerobic conditions. In addition, the availability of glutamate by GLDH also reduces the formation of byproducts. This reaction also produces ammonia, which can act as a nitrogen source of the cell or be used as a substrate for reductive amination at the final stage of the route shown in FIG. Thus, production cells that overexpress GLDH polypeptide can be used to increase yield and reduce the cost of the medium and / or separation process.

In the route from tryptophan to monatin, if the appropriate enzyme class of aminotransferase is used, the amino supply of step 3 (e.g. glutamate or aspartate) is required for the amino receptor (e.g. 2-oxo-glutar) Acid salts or oxaloacetates). If two separate aminotransferases are used in this pathway, the efficiency of this pathway can be increased if the substrate of one aminotransferase does not competitively inhibit the activity of the other aminotransferase.

Many of the reactions in the pathways described herein are reversible, so that an equilibrium will form between the substrate and the product. Thus, successive removal of products from polypeptides can increase the yield of this pathway. For example, secretion of monatin into the fermentation broth using permease or other transport protein or selective crystallization of monatin from the biocatalyst reactor and at the same time recycling the substrate will increase the reaction yield.

Removal of by-products by further enzymatic reactions or substitution of amino feeder groups is another way to increase reaction yield. In Example 13 below, various examples are examined and illustrated in FIGS. 11 to 13. Ideally, a phase change (evaporation) or spontaneous conversion to an unreacted end product such as carbon dioxide or the like produces byproducts that are not available in the reverse reaction.

temperament Pool  control

Indole pools can be regulated through increased production of tryptophan precursors and / or through modification of catabolic pathways including indole-3-pyruvate and / or tryptophan. For example, functional deletion of a gene encoding EC 4.1.1.74 in a host cell can reduce or eliminate the production of indole-3-acetic acid from indole-3-pyruvate. Functional deletion of the gene encoding EC 4.1.99.1 in the host cell can reduce or eliminate the production of indole from tryptophan. Alternatively, excess indole may be used as a substrate in an in vitro or in vivo process that increases the amount of the gene encoding EC 4.1.99.1 (Kawasaki et al., J. Ferm. And Bioeng., 82: 604-). 6, 1996). In addition, genetic modifications can be made to increase the concentration of intermediates such as D-erythros-4-phosphate and corismate.

Tryptophan production is regulated in most organisms. One of the regulatory mechanisms is through the feedback inhibitory action of specific enzymes in this pathway, and as tryptophan concentration increases, the production rate of tryptophan decreases. In other words, when genetically engineered host cells are used to produce monatin via tryptophan intermediates, organisms that are not sensitive to tryptophan concentration may be used. For example, by repeated exposure to high concentrations of 5-methyl-tryptophan is a rose is a Kata Lantus mouse (Catharanthus roseus) resistant to growth inhibition by various tryptophan analogs strain starter bar (Schallenberg and Berlin, Z Naturforsch 34 : 541-5, 1979). Tryptophan synthase activity of the resulting strains was less affected by product inhibition reactions, presumably due to mutations in the genes. In a similar manner, host cells used for monatin production can also be optimized.

Tryptophan production can be optimized through the use of induction generation to produce polypeptides that are less sensitive to product inhibition. For example, selection may be performed on a plate that does not contain tryptophan in the medium and that contains a large amount of non-metabolic tryptophan analogues. US Patent No. 5,756,345; 4,742,007; 4,371,614 describes a method used to increase the tryptophan production rate of fermented organisms. The final step in tryptophan biosynthesis is the addition of serine to indole. Thus, increasing the usefulness of serine can increase the yield of tryptophan production.

The amount of monatin produced by the fermentation organism can be increased by increasing the amount of pyruvate produced by the host organism. Thus, some yeasts, such as Trichosporum cutaneum , Wang et al., Lett. Appl. Microbiol. 35: 338-42, 2002) and Torulopsis Glabrata glabrata , Li et al., Appl Microbiol. Biotechnol. 57: 451-91, 2001) can be used in the methods disclosed herein as overproducing pyruvate. Genetic modifications may also be performed in organisms to promote pyruvic acid production, for example, genetic modifications in E. coli strain W1485lip2 (Kawasaki et al., J. Ferm. And Bioeng. 82: 604-6, 1996). .

Chirality  control

The taste profile of monatin can be changed by controlling the stereochemistry (chirality) of the molecule. Thus, it may be desirable for different food systems to provide different monatin isomers in various concentration combinations. Chirality can be controlled through a combination of pH and polypeptide.

Racemization at the monatin C4 position (see molecule numbered above) can occur through deprotonation and reprotonation of alpha carbon, which can be done via pH shift or reaction with the cofactor PLP. Microorganisms are less likely to have a pH shift that is sufficient to cause racemization, but PLP is abundant. Methods of modulating chirality with polypeptides depend on the biosynthetic pathway used for monatin production.

When monatin is formed using the route shown in Figure 2, it should be noted that: In biocatalytic reactions, the chirality of C2 is determined by an enzyme that converts indole-3-pyruvate to MP. Since there are a plurality of enzymes (eg EC 4.1.2.-, 4.1.3.-) in the enzyme for converting indole-3-pyruvate to MP, it is possible to select an enzyme that forms the required isomer. Alternatively, the enantiospecificity of the enzyme that converts indole-3-pyruvate to MP may be changed through induction generation or the catalytic antibody may be engineered to catalyze the required reaction. Once MP is produced (via enzymatic or chemical condensation), amino groups can be added stereospecifically via aminotransferases as described herein. Depending on whether D-type or L-type aromatic acid aminotransferase is used, C4 of type R or S may be generated. Most aminotransferases are specific for L-isomers, but some plants also have D-tryptophan aminotransferases (Kohiba and Mito, Proceedings of the 8th International Symposium on Vitamin B ₆ and Carbonyl Catalysis, Osaka, Japan 1990 ). Moreover, D-alanine aminotransferase (2.6.1.21), D-methionine-pyruvate aminotransferase (2.6.1.41) and (R) -3-amino-2-methylpropanoate aminotransferase (2.6.1.61) And (S) -3-amino-2-methylpropanoate aminotransferase (2.6.1.22) were all identified. Some aminotransferases only allow substrates with a specific orientation to C2 carbon in this reaction. Thus, even if the conversion to MP is not stereospecific, the stereochemistry of the final product can be controlled through the appropriate choice of aminotransferases. Because this reaction is reversible, unreacted MP (unnecessary isomers) can be recycled to its constituents to modify the racemic mixture of MP.

Activation of substrate

Phosphorylated substrates such as phosphoenolpyruvate (PEP) and the like can be used in the reactions disclosed herein. Phosphorylated substrates are more advantageous in terms of energy and can be used to increase reaction rate and / or yield. In aldol condensation, the addition of phosphate groups stabilizes the enol tautomers of the nucleophilic substrate to further increase reactivity. As another reaction, phosphorylated substrates often provide better leaving groups. Similarly, the substrate may be activated through conversion to CoA derivatives or pyrophosphate derivatives.

Example  One

Tryptophan  Aminotransferase Cloning  And expression

This example relates to a method used for cloning tryptophan aminotransferase that can be used to convert tryptophan to indole-3-pyruvate.

Experiment overview

Eleven genes encoding aminotransferase were cloned into E. coli. This gene is composed of Bacillus subtilis D-alanine aminotransferase (dat, GenBank Accession No. Y14082.1 bp 28622-29470 and GenBank Accession No. NP_388848.1, nucleic acid sequence and amino acid sequence, respectively), and cynorazorbium melorirot ( Tyrosine aminotransferases (tatA, SEQ ID NOs: 1 and 2, respectively, nucleic acid sequences and amino acid sequences), Rhodobacter spheroid strain 2.4.1 tyrosine aminotransferases (tatA, sequences supported through homology) Nos. 3 and 4, nucleic acid sequence and amino acid sequence respectively, bsat supported by homology with Laishmania major broad substrate aminotransferase (bsat, L. Mexicana peptide fragment, SEQ ID NOs: 7 and 8, respectively, nucleic acid Sequence and amino acid sequence), Bacillus subtilis aromatic aminotransferase (araT, supported through homology, SEQ ID NOs: 9 and 10, nucleic acid sequence and amino acid sequence, respectively), Lactobacillus amylobo Rusu aromatic aminotransferase (araT supported by homology, SEQ ID NOs: 11 and 12, respectively, nucleic acid sequence and amino acid sequence), R. spheroids 35053 multiple substrate aminotransferase (supported through homology, SEQ ID NO: 13 and 14, nucleic acid sequence and amino acid sequence respectively, Rhodobacter spheroid strain 2.4.1 multi-substrate aminotransferase (msa supported through homology, Genbank Accession No. AAAE01000093.1, bp 14743-16155 and Genbank Accession No. ZP00005082 .1, nucleic acid sequence and amino acid sequence), Escherichia coli aspartate aminotransferase (aspC, Genbank Accession No. AE000195.1 bp 2755-1565 and Genbank Accession No. AAC74014.1, respectively, nucleic acid sequence and amino acid sequence) And E. coli tyrosine aminotransferases (tyrB, SEQ ID NOs: 31 and 32, nucleic acid sequences and amino acid sequences, respectively).

These genes were cloned, expressed and tested with commercial enzymes for activity at the conversion of tryptophan to indole-3-pyruvate. All 11 clones showed activity.

Identification of Bacterial Strains That May Contain Polypeptides Having the Desired Activity

The NCBI (National Center for Biotechnology Information) database does not have a gene designated as tryptophan aminotransferase, but organisms exhibiting this enzyme activity have been identified. L-Tryptophan aminotransferase (TAT) activity was measured from cell extracts or purified proteins from the following sources: Lysobacterial isolates from Festuca octoflora , mitochondria and cytosol of peas, sunflower crown humps Cells, Rhizobium leguminosarum biovar trifoli, Erwinia herbola pv Ziposophyll, Pseudomonas syringe pv. Savastannoy, Agrobacterium Tumation, Azospirilum Lipferum and Brasilens, Enterobacter Cloase, Enterobacter Aglomeranse, Bradyraizobium Elcanni, Candida Maltosa, Azotobacter Vinelandi, Rat Brain, Rat liver, cyno-razobium melorirot, Pseudomonas fluorescence CHA0, Lactococcus lactis, Lactobacillus casei, Lactobacillus helvetticus, wheat seed, barley, Peiceolus aureus (mung bean), Saccharomyces uba Rum (Carlsbergensis), Laishmania spp., Corn, tomato veins, pea plants, tobacco, pigs, Clostridium sporogenes and Streptomyces griesus.

Cloning  Genome DNA Separation of

S. melorirot (ATCC Accession No. 9930) was grown in TY medium (pH 7.2) at 25 ° C. Cells were grown until the optical density at 600 nm (OD ₆₀₀ ) became 1.85 and 2% inoculum was used for genomic DNA preparation. The Qiagen Genome Tip 20 / G Kit (Valencia, CA) was used for genomic DNA isolation.

Bacillus subtilis 6051 (ATCC) was grown at 30 ° C. in Bereto Nutrient Broth, Difco; Detroit, MI. Genomic DNA was isolated according to the Qiagen Genome Tip 20 / G protocol, except that the proteinase K and lysosome arbitrary concentrations were doubled and the inoculation time was increased 2-3 times.

Laishmania major ATCC 50122 genomic DNA was supplied at a concentration of 17 ng / μl in TE buffer (pH 8.0) in IDI, Inc. (Quebec, Canada).

Rhodobacter spheroids 2.4.1 (provided by Professor Sam Kaplan of Texas University, Houston), R. spheroids 35053 (ATCC number) and L. amyloborus genomic DNA were prepared by standard phenol extraction methods. Late logarithmic cells were harvested and resuspended in TEN buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl) and then lysed by adding 0.024 ml of sodium lauryl sarcosine per ml of cell suspension. After extracting the TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) three times or more with the same amount of saturated phenol, and then extracted once with 9: 1 chloroform: octanol, three times with chloroform to prepare a DNA solution. DNA was precipitated by adding 0.1 volume of 3M sodium acetate (pH 6.8) and 2 volumes of ethanol. The precipitate was collected by centrifugation and washed once with 70% ethanol. Finally, DNA was dissolved in 0.10 ml distilled water.

E. coli genomic DNA was isolated from strain DH10B (Invitrogen) and prepared using the Qiagen Genome-Tip ™ (500 / G) kit. 0.3 mg of purified DNA was obtained from 30 ml of the culture in which the strain was grown from LB to OD ₆₅₀ 1.87. Purified DNA was dissolved in Qiagen elution buffer (EB) at a concentration of 0.37 μg / μl.

Polymerase  Chain reaction protocol

Primers were prepared using a compatible overhang of the pET 30 Xa / LIC vector (Novagen, Madison, Wis.). The pET vector has 12 base single chain overhangs on the 5 'side of the Xa / LIC region, and 15 base single chain overhangs on the 3' side of the Xa / LIC region. This plasmid was designed for Ligation Independent Cloning, with His- and S-tags attached to the N-terminus and His-tag selectively attached to the C-terminus. An Xa protease recognition site (IEGR) was introduced immediately before the start codon of the gene so that the tag could be removed from the fusion protein.

(서열번호 73); 역방향 프라이머:

(서열번호 74).In the primer design, the following sequence was added at the 5 'end of the organism specific sequence: Forward primer:

(SEQ ID NO: 73); Reverse primer:

(SEQ ID NO: 74).

Bacillus subtilis dat primer:

(서열번호 15)N terminus:

(SEQ ID NO: 15)

(서열번호 16).C terminus:

(SEQ ID NO: 16).

Cynorazobium melorilot tatA primers:

(서열번호 17)N terminus:

(SEQ ID NO: 17)

(서열번호 18).C terminus:

(SEQ ID NO: 18).

Bacillus subtilis araT primer:

(서열번호 19)N terminus:

(SEQ ID NO: 19)

(서열번호 20).C terminus:

(SEQ ID NO: 20).

Rhodobacter spheroids msa (both 2.4.1 and 35053):                 

(서열번호 21)N terminus:

(SEQ ID NO: 21)

(서열번호 22).C terminus:

(SEQ ID NO: 22).

Laishmania major bast:

(서열번호 23)N terminus:

(SEQ ID NO: 23)

(서열번호 24).C terminus:

(SEQ ID NO: 24).

Lactobacillus amyloborus araT:

(서열번호 25)N terminus:

(SEQ ID NO: 25)

(서열번호 26).C terminus:

(SEQ ID NO 26).

Rhodobacter spheroids tatA (both 2.4.1 and 35053 strains):

(서열번호 27)N terminus:

(SEQ ID NO 27)

(서열번호 28).C terminus:

(SEQ ID NO 28).

Eserihia Collie aspC:

(서열번호 29)N terminus:

(SEQ ID NO 29)

(서열번호 30).C terminus:

(SEQ ID NO: 30).

Eserihia Collie tyrB:

(서열번호 33)N terminus:

(SEQ ID NO: 33)

(서열번호 34).C terminus:

(SEQ ID NO 34).

Genes derived from S. melototti (tatA) were amplified using the following PCR protocol. 50 μl of reaction was used with 0.1-0.5 μg template, 1.5 μM of each primer, 0.4 dM of each dNTP, 3.5 U of Expand High Fidelity Polymerase (Roche, Indianapolis, Indiana), and 1 × Expand ™ buffer (Mg addition). The thermocycler program used consisted of the following: 29 cycles consisting of hot start 96 ° C., 5 minutes, then 94 ° C. 30 seconds, 55 ° C. 2 minutes, 72 ° C. 2.5 minutes. After 29 repetitions, the samples were kept at 72 ° C. for 10 minutes and then stored at 4 ° C. This PCR protocol produced 1199 bp of product.

Gene sequences derived from R. spheroids (msa and tatA), L. amyloborus araT and Bacillus araT were amplified by the following PCR protocol. 50 μl reaction was used with 0.1-0.5 μg template, 1.5 μM of each primer, 0.4 mM of each dNTP, 3.5 U of Expand High Fidelity ™ Polymerase (Roche, Indianapolis, Indiana), and 1 × Expand ™ buffer (Mg addition). . The thermocycler program used consisted of: high temperature start 96 ° C., 5 minutes, then 94 ° C. 30 seconds, 40 to 60 ° C. 1 minute, 45 seconds (gradient thermocycler), 72 ° C. 2 minutes, 15 seconds. 29 cycles repeated. After 29 repetitions, the samples were kept at 72 ° C. for 10 minutes and then stored at 4 ° C.

In each R. spheroid msa gene, annealing temperatures of 42 ° C. and 48 ° C. produced several products, with characteristic bands at about 1464 bp. For L. amyloborus araT, 42 ° C., 48 ° C. and 56 ° C. annealing temperatures yielded a single product exhibiting a strong band at 1173 bp. For B. subtilis araT, 40 ° C., 45 ° C., 50 ° C., 55 ° C. annealing temperatures yielded one main product (1173 bp) in both genomic DNA and colonies. For L. major bsat, the purest product (1239 bp) was obtained at 55 ° C. annealing temperature. The Rhodobacter tatA gene provided a pure product of the correct size (1260 bp) with an annealing temperature of 50-55 ° C. In both E. coli and B. subtilis dat genes, annealing temperatures of 55 to 60 ° C. were used, and the annealing time was reduced to 45 seconds. As a result, a pure product of the correct size was obtained (approximately 1.3 kb for E. coli gene, 850 bp for dat gene).

Cloning

PCR products were gel purified from 0.8% or 1% TAE-agarose gel using the Qiagen gel extraction kit (Valencia, Calif.). This PCR product was quantified in comparison to a standard on an agarose gel and then treated with T4 DNA polymerase according to the manufacturer's recommended method for ligation independence cloning (Novagen, Madison, Wis.).

Briefly, about 0.2 pmol of the purified PCR product was treated with 1 U T4 DNA polymerase in the presence of dGTP for 30 minutes at 22 ° C. This polymerase continuously removes the base from the 3 'end of the PCR product. When the polymerase encounters a guanine residue, the polymerase activity in the 5 'to 3' direction of the enzyme inhibits exonuclease activity, effectively blocking further ablation. As a result, a Japanese chain overhang compatible with the pET Xa / LIC vector is created. The polymerase is inactivated by incubation at 75 ° C. for 20 minutes.

Vectors and treated inserts were annealed according to Novagogen's recommendations. Approximately 0.02 pmol of the treated insert and 0.01 pmol of the vector were incubated at 22 ° C. for 5 minutes, 6.25 mM EDTA (final concentration) was added and then incubated again at 22 ° C. Annealing reaction (1 μl) was added to NovaBlue ™ competent single cells (Novagen, Madison, Wis.) And incubated on ice for 5 minutes. After mixing, cells were transformed with heat shock at 42 ° C. for 30 seconds. The cells were left on ice for 2 minutes and recovered for 30 minutes at 37 ° C. under shaking at 225 rpm in 250 μl of SOC at room temperature. Cells were plated on LB plates containing kanamycin (25-50 μg / ml).

The plasmid DNA was purified using the Qiagen Spin Miniprep Kit and limited digestion with XhoI and XbaI to select the correct insert. Dideoxy chain termination DNA sequencing confirmed the plasmid sequences observed to have the correct inserts.

SEQ ID NOs: 1 to 14 and 31 and 32 are the nucleotide sequences of the recombinant aminotransferases and their corresponding amino acid sequences, with any change with the Genbank sequence resulting in silent mutations or conservative substitutions in the protein sequence. SEQ ID NOs: 11 and 12 are new sequences.

Gene Expression and Analysis

Plasmid DNA confirmed by sequencing was subcloned into E. coli expression host BLR (DE3) or BL21 (DE3) (Novagen, Madison, Wis.). After the culture was grown, plasmids were isolated using a Qiagen miniprep kit and analyzed by restriction enzyme digestion for identity.

First, expression was induced in BLR (DE3) and BL21 (DE3) cells using L. amyloborus araT, B. subtilis araT and S. melorirot tatA. In LB containing kanamycin (30 mg / L), OD ₆₀₀ was propagated to 0.5 to 0.8 and then cultured with 1 mM IPTG (isopropyl thiogalactoside). Samples were taken at 1, 2, and 4 hours. 2.0 ml of the sample was resuspended in 0.10 ml of 120 mM Tris-HCl (pH 6.8) containing 10% sodium dodecyl sulfate, 10% 2-mercaptoethanol and 20% glycerol and heated at 95 ° C. for 10 minutes. , Cooled and diluted with 0.10ml H ₂ O. An amount of this total cell protein sample was analyzed by SDS-PAGE using a 4-15% gradient gel. The amount of protein expression did not differ significantly between 2 and 4 hour induction, or between BLR (DE3) cells and BL21 (DE3) cells.

The cell extracts had a 4-hour sample and the cell pellets from 2 ml of culture were suspended in 0.25 ml Novagen BugBuster ™ reagent containing 0.25 μl Benzonase nuclease, then incubated at room temperature for 20 minutes under gentle shaking. , Was prepared by centrifugation at 16,000xg to remove cell debris. Supernatants (cell extracts) were loaded onto 4-15% gradient gels for analysis of soluble proteins of cells.

The three clones, L. amyloborus araT (SEQ ID NOs: 11 and 12), B. subtilis araT (SEQ ID NOs: 9 and 10), and S. melioti tatA (SEQ ID NOs: 1 and 2) are the correct size (approximately 45 kDa). Soluble protein). The B. subtilis araT gene product was overexpressed in the highest amount and / or more soluble than the other two gene products.

In subsequent expression, plasmid DNA from positive clones was subcloned into BL21 (DE3) because this host showed better proliferation. Cultures grown in LB containing 50 mg / L kanamycin were reinduced using 1 mM IPTG and induced until OD ₆₀₀ reached about 0.8. After 4 hours propagation at 37 ° C., centrifugation at 3000 rpm for 10 minutes (4 ° C.) was followed by TEGGP buffer (50 mM Tris-HCl, pH 7.0), 0.5 mM EDTA, 100 mg / L glutathione, 5% glycerol and Roche Complete Protease. Inhibitor mixture), followed by rapid freezing in -80 ° C ethanol.

Samples were weighed at 1 g of wet cell weight per 5 ml of BugBuster ™ (Novagen) reagent containing 5 μl / ml protease inhibitor mixture set # 3 (Calbiochem-Novabiochem Corp., San Diego, Calif.) And 1 μl / ml Benzonase nuclease. Cells were resuspended in proportions. Samples were incubated for 20 minutes at room temperature on an orbital shaker. Insoluble cell debris was removed by centrifugation at 16,000 g for 20 minutes at 4 ° C.

Cell extracts were analyzed by SDS-PAGE, and the activity of tryptophan aminotransferase was analyzed through subsequent production of indole-pyruvic acid according to the following method. 1 ml reaction was added to 50 mM sodium tetraborate (pH 8.5), 0.5 mM EDTA, 0.5 mM sodium arsenate, 50 μM pyridoxal phosphate, 5 mM α-ketoglutarate and 5 mM L-tryptophan. The reaction was initiated by the addition of cell-free extract or purified enzyme and incubated at 30 ° C. for 30 minutes. 20% TCA (200 μl) was added to stop the reaction and the precipitated protein was removed by centrifugation. Absorbance was measured at 327 nm and compared to a standard curve constructed with freshly prepared indole-3-pyruvate in assay buffer. A control reaction using no control substrate or a cell-free extract derived from clones transformed with pET30a alone was performed.

Because of the background value of the original E. coli aminotransferase present in the cell extract, the recombinant protein was purified using His-Bind cartridge according to the manufacturer's protocol (Novagen, Madison, WI). Elution fractions were desalted in PD-10 (Amersham Biosciences, Fitzkataway, NJ) and eluted with 50 mM Tris, pH 7.0. Purified proteins were analyzed by SDS-PAGE and aminotransferase activity.

The results obtained after induction (4 hours) at 37 ° C using 1 mM IPTG showed that L. major bsat, S. melototti tatA, Escherichia coli aspC and two R. spheroids tatA clones showed significant levels of tryptophan aminotransferase activity. Proved. The araT protein was overexpressed and soluble from B. subtilis but showed little enzymatic activity. The L. amyloborus araT gene product was found to be soluble in cell extracts, but only a small amount of protein with the correct molecular weight was identified after purification using His-Bind cartridge. The msa gene product was insoluble and further expression experiments were performed at 24 ° C. to minimize inclusion body formation. Various concentrations of IPTG between 10 μM and 1 mM were used to maximize the amount of soluble protein.

Table 1 below lists the inactivation (μg) of indole-3-pyruvate (I3P) formed per mg of protein per minute. In some cases, very low levels of recombinant protein showed high activity above the effective linear range of the assay. In this case, a '>' is shown before the inactive value.

Table 1

Inactivation of enzymes compatible with clones in the form of cell extracts (CE) and purified products (P)

enzyme Inactive (I3Pμg / proteinmg / min) exegesis L. Major bsat CE > 49.3 L. Major bsat P > 4280 S. melilotti tatA CE > 28.6 S. melilotti tatA P > 931 2.4.1 tatA CE > 41.2 2.4.1 tatA P 1086 35053 tatA CE > 62.3 35053 tatA P > 486 L. amyloborus araT CE 1.26 L. amyloborus araT P 0 Small amount of protein after His-Bind cartridge B. Subtilis araT CE 0 Undetectable B. Subtilis araT P 1.5-4.5 2.4.1 msa P 2.05 Extremely low soluble protein 2.4.1 msa P 0 Protein Not Detected After His-Band Cartridge 35053 msa CE 3.97 Extremely low soluble protein 35053 msa P 0 Protein Not Detected After His-Band Cartridge Escherichia coli aspC (P) 800 E. coli tyrB (P) One Poorly soluble B. subtilis D-aminotransferase (P) 2.7 Use of D-Tryptophan as a Substrate Widespread aminotransferase 22 Sigma cat # T7684 Pig type II-A 1.5 Sigma G7005 Pig Type I One Sigma G2751

As a result of sorting to compare all cloned recombinant proteins, it was found that there are not many highly conserved regions between the araT, tatA, bsat and msa sequences. The alignment of the recombinant protein, Rhodobacter tatA gene product homologue, L. major broad substrate aminotransferase and cynoraizobium melioroty tyrosine aminotransferase, which showed the highest activity, showed several conserved regions, but they were only weak at the protein level. There was an identity of 30-43%. The utility of a wide range of D-specific (D-alanine) aminotransferases can be used for the production of other stereoisomers of monatin.

Example  2

Indole-3- From lactate  Indole-3- To pyruvate  transform

As shown in Figures 1 and 3, indole-3-lactic acid can be used for the production of indole-3-pyruvate. The conversion between lactic acid and pyruvate is a reversible reaction, as is the conversion between indole-3-pyruvate and indole-3-lactate. The oxidation of indole-lactate was then performed due to the presence of a high background value at 340 nm with indole-3-pyruvate.

The standard assay mixture consisted of 0.1 mM 100 mM potassium phosphate (pH 8.0), 0.3 mM NAD ⁺ , lactate dehydrogenase (LDH) (Sigma-L2395, St. Louis, MO), 2 mM substrate. The analysis was performed in two replicates using a spectrometer (Molecular Devices SpectraMax Plus platereader) on a UV-permeable microtiter plate. The polypeptide and the buffer were mixed, injected into wells containing indole-3-lactic acid and NAD ⁺ , and the 340 nm absorbance of each well was read at intervals of 9 seconds after instant mixing. The reaction was kept at 25 ° C. for 5 minutes. The absorbance at 340 nm increased due to the production of NADH from NAD ⁺ . A negative control without NAD ⁺ and substrate added was also analyzed. It was observed that D-LDH (Sigma catalog number L2395) derived from Leukonostock Mesenteroids showed better activity on indole derivative substrates than L-LDH (Sigma catalog number L5275) derived from Bacillus stearotermophilus.

Similar methods were used for D-lactic acid and NAD + or NADH and pyruvate, which are natural substrates of D-LDH polypeptide. V _max of pyruvate reduction reaction were 100 to 1000 times higher than the V _max of lactate oxidation. The V _max of the indole-3-lactic acid oxidation by D-LDH was about 1/5 of the lactic acid V _max . In addition, the presence of indole-3-pyruvate was measured by using a 50 mM sodium borate buffer containing 0.5 mM EDT and 0.5 mM sodium arsenate and changing the absorbance at 327 nm (enol-borate derivative). Small absorbance changes were observed repeatedly compared to negative controls for both L-LDH and D-LDH polypeptides.

In addition, a broad specificity of lactate dehydrogenase (an enzyme with activity associated with EC 1.1.1.27, EC 1.1.1.28 and / or EC 1.1.2.3) can be cloned and used to indole-3 from indole-3-lactic acid. Pyruvate can be prepared. Sources of broad-specific dehydrogenase are Escherichia coli, Neisseria gonorrhoeae , and Lactobacillus plantarum ).

Or cell extracts of Clostridium sporogenes containing indole lactate dehydrogenase (EC 1.1.1.110); Indole-3-pyruvic acid is known to be active for the salts p- hydroxyphenyl lactate dehydrogenase (EC 1.1.1.222) trip Fano Soma crew if epi maseuti and test (Trypanosoma cruzi containing epimastigotes ) cell extracts; Pseudomonas acidovorans or E. coli cell extracts containing imidazol-5-yl lactate dehydrogenase (EC 1.1.1.111); Coleus blumei containing hydroxyphenylpyruvate reductase (EC 1.1.1.237); Alternatively, indole-3-pyruvate can be prepared by contacting indole-3-lactate with Candida maltosa cell extract containing D-aromatic lactate dehydrogenase (EC 1.1.1.222). References describing this activity include: Nowicki et al. (FEMS Microbiol Lett 71: 119-24, 1992), Jean and DeMoss (Canadian J. Microbiol. 14 1968, Coote and Hassall). Biochem. J. 111: 237-9, 1969), Cortese et al. (CRSeances Soc. Biol. Fil. 162 390-5, 1968), Petersen and Alfermann (Z. Naturalforsch. C: Biosci. 43 501-4, 1988), and Bhatnagar et al. (J. Gen Microbiol 135: 353-60, 1989). Also derived from Pseudomonas species (Gu et al., J. Mol. Catalysis B: Enzymatic: 18: 299-305, Lactate oxidase (2002) can be used to oxidize indole-3-lactic acid to indole-3-pyruvate.

Example  3

L- using L-amino acid oxidase Tryptophan  Indole-3- To pyruvate  transform

This example describes the method used to convert tryptophan to indole-3-pyruvate via oxidase (EC 1.4.3.2) as an alternative to tryptophan aminotransferase use described in Example 1. L-amino acid oxidase was purified from Crotalus durisus (Sigma, Catalog No. A-2805, St. Louis, MO). L-amino acid oxidase for molecular cloning includes polypeptides of the following accession numbers: CAD21325.1, AAL14831, NP_490275, BAB78253, A38314, CAB71136, JE0266, T08202, S48644, CAC00499, P56742, P81383, O93364, P81382 , P81375, S62692, P23623, AAD45200, AAC32267, CAA88452, AP003600 and Z48565.

The reaction mixture was prepared with a total volume of 1 ml in a microcentrifuge tube and incubated for 10 minutes with shaking at 37 ° C. The reaction mixture contains 5 mM L-tryptophan, 100 mM sodium phosphate buffer pH 6.6, 0.5 mM sodium arsenate, 0.5 mM EDTA, 25 mM sodium tetraborate, 0.016 mg catalase (83U, Sigma C-3515), 0.008 mg FAD (Sigma) and 0.005 to 0.125 unit of L-amino acid oxidase was added. All ingredients except tryptophan were added to the negative control, and all ingredients except oxidase were added to the control. Catalase was used to remove hydrogen peroxide formed during oxidative deamination. Sodium tetraborate and sodium arsenate were used to stabilize the enol-borate form of indole-3-pyruvate, showing a maximum absorbance at 327 nm. Indole-3-pyruvate standards were prepared in the reaction mixture at a concentration of 0.1 to 1 mM.

The purchased L-amino acid oxidase showed an inactivation of 540 μg of indole-3-pyruvate formed per minute per mg of protein. This was the same level of inactivation of tryptophan aminotransferase.

Example  4

Aldolase  Indole-3- Pyruvate  2- Hydroxy  2- (indole-3- Methyl ) -4-keto To glutaric acid  transform

This example converts indole-3-pyruvate to 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid monatin precursor (MP) using aldolase (degrading enzyme) It will be described a method that can be used to (Fig. 2). Aldol condensation is a reaction that forms a carbon-carbon bond between the β-carbon of one aldehyde or ketone and the carbonyl carbon of the other aldehyde or ketone. Carbonyl ions are formed in the carbon adjacent to the carbonyl group of one substrate, which acts as nucleophile attacking the carbonyl carbon (nucleophilic carbon) of the second substrate. Most commonly, the nucleophilic substrate is an aldehyde, with most aldolases belonging to the EC 4.1.2.- class. Often the nucleophilic substrate is pyruvate. It is not uncommon for aldolases to catalyze the condensation reaction between two keto acids or two aldehydes.

However, aldolases have been identified that catalyze the condensation of two carboxylic acids. For example, EP 1045-029 describes an example of the production of L-4-hydroxy-2-ketoglutaric acid from glyoxylic acid and pyruvate using Pseudomonas culture (EC 4.1.3.16). In addition, 4-hydroxy-4-methyl-2-oxoglutarate aldolase (4-hydroxy-4-methyl-2-oxoglutarate pyruvate degrading enzyme, EC 4.1.3.17) is known as both keto acids. It can catalyze the condensation reaction. Thus, similar aldolase polypeptides were used to catalyze the condensation of indole-3-pyruvate with pyruvate.

Cloning

4-hydroxy-4-methyl-2-oxoglutarate pyruvate degrading enzyme (ProA aldolase, EC 4.1.3.17) and 4-hydroxy-2-oxoglutarate glyoxylate degrading enzyme (KHG egg Dolase, EC 4.1.3.16) catalyzes a reaction very similar to the aldolase reaction of FIG. 2. Thus, primers were designed with overhangs compatible with the pET30 Xa / LIC vector (Novagen, Madson, WI). The design of such primers is as described above in Example 1.

The following primers were designed for pET30 Xa / LIC cloning:

1. Pseudomonas straminea proA gene (GenBank Accession No. 12964663 Version: 12964663) and Coomamonas testosterone proA gene (SEQ ID NOs: 65 and 66, respectively, nucleic acid sequence and amino acid sequence):

Forward (SEQ ID NO: 55)

.Reverse (SEQ ID NO: 56)

.

2. Cynorazorbium melorilot 1021 SMc00502 gene (homologous to proA, Genbank Accession Nos. 15074579 and CAC46344, nucleic acid sequence and amino acid sequence, respectively):

Forward (SEQ ID NO: 61)

.Reverse (SEQ ID NO: 62)

.

3. Sphingomonas spp. LB126 fldZ gene (Genbank Accession No. 7573247 Version: 7573247, putative acyl transferase encoding):

Forward (SEQ ID NO: 57)

.Reverse (SEQ ID NO: 58)

.

4. Atlobacter chiserie pcmE gene (Genbank Accession No. AF331043 Version: AF331043.1, oxalocitramalate aldolase coding):

Forward (SEQ ID NO: 59)

.Reverse (SEQ ID NO: 60)

.

5. Yersinian pestis strain CO92 YPO0082 gene (Genbank Accession No .: 15978115 Version: 15978115, possible transferase encoding):

Forward (SEQ ID NO: 63)

.Reverse (SEQ ID NO: 64)

.

6. Bacillus subtilis khg gene (Genbank Accession No. Z99115.1, GI: 2634478, 126711-127301 and CAB14127.1, respectively nucleic acid sequence and amino acid sequence):

Forward (SEQ ID NO: 35)

.Reverse (SEQ ID NO: 36)

.

7.E. coli khg gene (GenBank Accession No. AE000279.1 1331-1972 and AAC74920.1, nucleic acid sequence and amino acid sequence, respectively):

Forward (SEQ ID NO: 37)

Reverse (SEQ ID NO: 38) .

8. S. melilotti khg gene (Genbank Accession No. AL591792.1, GI: 15075850, 65353-64673 and CAC47463.1, respectively nucleic acid sequence and amino acid sequence):

Forward (SEQ ID NO: 39)

.Reverse (SEQ ID NO: 40)

.

Genomic DNA of the organisms described in 1 and 2, 6 to 8 above were purified according to the Qiagen genome-tip protocol. Similar techniques can be used to purify the genomic DNA of organisms described in 3-5.

Pseudomonas stramine (ATCC 33636) was grown at 30 ° C. in a medium containing nutrient liquid medium and hydroxybenzoate. Comamonas testosterone (ATCC 49249) was grown at 26 ° C. in a medium containing nutrient liquid medium and hydroxybenzoate. Sphingmonas spp. LB126 (Flemish Institute for Technological Research, VITO, B-2400 Mol, Belgium) is described by Wattiau et al. Research in Microbiol. 152: 861-72, 2001]. Atrocacter Kisseri (Gulf Ecology Division, National Health and Environmental Effects Research Laboratory, US Environmental Protectin Agency, Gulf Breeze, FL32561, USA), described by Eaton, J. Bacteriol. 183: 3689-3703, 2001. Proliferation was followed according to the protocol. Cynorazobium melorilot 1021 (ATCC 51124) was grown at 26 ° C. in ATCC TY medium and hydroxybenzoate medium. Ercinia pestis strain CO92 (ATCC) was grown at 26 ° C. in ATCC medium 739 horse blood agar. Bacillus subtilis 6051 (ATCC) was grown at 30 ° C. in Bereto nutritional liquid medium (Difco, Detroit, Mich.). E. coli genomic DNA was isolated from strain DH10B (Invitrogen) as described in Example 1.

The PCR, cloning and screening protocols described in Example 1 were used to clone the C. testosterone and S. melorirot proA sequences and the E. coli, B. subtilis and S. melorirot khg sequences. The same method can be used for cloning other sequences described above.

The sequencing of the positive clones used dideoxy chain termination sequencing using S-tags and T7 terminator primers (Novagen) and internal primers (Integrated DNA Technologies, Inc. (Coralville, IA)).                 

Expression and Activity Assay

Plasmid DNA (identified by sequencing) was subcloned into expression host BL21 (DE3) (Novagen). Cultures were grown in LB medium containing 50 mg / L kanamycin and plasmids were isolated using the Qiagen spin plasmid miniprep kit, followed by restriction enzyme digestion to identify identity. Induction experiments were performed using BL21 (DE3) constructs grown at 37 ° C. in LB medium containing 50 mg / L kanamycin. After OD ₆₀₀ reached about 0.6, protein expression was induced using 0.1 mM IPTG. Cells were grown for 4 hours at 30 ° C. and then harvested by centrifugation. The cells were then lysed using Bugbuster ™ reagent (Novagen) and His-tag recombinant protein was purified using the His-Bind cartridge (Example 1) described above. Purified protein was desalted using a PD-10 disposable column and eluted with 50 mM Tris-HCl buffer (pH 7.3) added ₂ mM MgCl ₂ .

Proteins were analyzed via SDS-PAGE of 4-15% gradient gels to detect the concentration of soluble protein at the estimated molecular weight of the recombinant fusion protein.

Protein activity was analyzed using indole-3-pyruvate and sodium pyruvate as substrates. The assay mixture contains 100 mM Tris-HCl (pH 7 to pH 8.9), 0 to 8 mM MgCl ₂ , 3 mM potassium phosphate (pH 8) and 6 mM of each substrate in 1 ml. The reaction was initiated by the addition of varying amounts of polypeptide (eg 10-100 μg) to the reaction and incubated at 25 ° C.-37 ° C. for 30 minutes, then filtered and frozen at −80 ° C.

proA  Activity result by gene product

The C. testosterone proA and S. melorirot SMc00502 gene constructs showed high expression rates upon IPTG induction. Recombinant protein was highly soluble in SDS-PAGE analysis of total protein and cell extract samples. The C. testosterone gene product was purified to> 95% purity. Since the yield of the S. meloriti gene product was extremely low after the affinity purification using the His-Bind cartridge, the cell extract was used for the enzyme analysis.

Both recombinant aldolases catalyzed the formation of MP from indole-3-pyruvate and pyruvate. Enzyme activity required the presence of divalent magnesium phosphate and potassium phosphate. In the absence of indole-3-pyruvate, pyruvate or potassium phosphate, no distinct product was observed. Small amounts of product were formed even in the absence of enzymes (generally lower concentrations than orders of magnitude).

The peak of the product eluted slightly later than indole-3-pyruvate from the reversed-phase C18 column, and the mass spectrum of this peak was the collision-inducing moiety ([M + H] ⁺ ) of 292.1, the expected ion for the product MP. Indicated. The major daughter fragments present in the mass spectrum are m / z = 158 (1H-indole-3-carbaldehyde carbonium ion) and 168 (3-buta-1,3-dienyl-1H-indole carbonium ion) , 274 (292-H ₂ O), 256 (292-2H ₂ O), 238 (292-3H ₂ O), 228 (292-CH ₄ O 3) and 204 (loss of pyruvate). The product also exhibited a UV spectrum characteristic of other indole containing compounds such as tryptophan, showing a lambda _max at 279 to 280 and a small shoulder at about 290 nm.

The amount of MP produced by C. testosterone aldolase increased as the reaction temperature was increased from room temperature to 37 ° C. and the substrate and magnesium amounts were increased. Synthetic activity of the enzyme decreased with increasing pH, and the desired pH for the maximum product was observed to be 7. Based on tryptophan standards, the amount of MP produced by standard assay using 20 μg purified protein was about 10-40 μg per 1 ml reactant.

Because the S. melototti and C. testosterone ProA aldolase coding sequences show a high degree of homology with the other genes described above, it was expected that both recombinant gene products could catalyze this reaction. Furthermore, aldolase having threonine (T) at positions 59 and 87, aldolase having arginine (R) at position 119, aldolase having aspartate (D) at position 120 and 31 Aldolases bearing histidine (H) at 71 (based on C. testosterone's numbering system) are expected to have similar activity.

khg  Activity result by gene product

B. subtilis and E. coli khg gene constructs express large amounts of protein when induced by IPTG, while S. meliothy khg has a lower expression rate. Recombinant proteins are highly soluble proteins, as determined by SDS-PAGE analysis of total protein and cell extracts. The B. subtilis and E. coli khg gene products were purified to> 95% purity, but the yield of S. memeliotti gene products did not increase as much after affinity purification using His-Bind cartridge.

There was no evidence that magnesium and phosphate were necessary for the activity of this enzyme. However, the literature reports that this assay is performed in sodium phosphate buffer, which reports that the enzyme is a bifunctional phosphorylated substrate such as 2-keto-3-deoxy-6-phosphogluconate (KDPG). It is showing activity. Enzyme analysis was performed as described above and in some cases phosphate was excluded. As a result, recombinant KHG aldolase produced MP but was not as active as ProA aldolase. In some cases the concentration of MP produced by KHG was about the same as that produced by magnesium and phosphate only. In other words, phosphate did not appear to increase KHG activity. Bacillus enzymes showed the highest activity and were about 20-25% higher than activity with magnesium and phosphate alone, as measured by SRM (see Example 10). Cynorizobium enzymes showed the lowest activity, possibly related to folding and solubility problems observed during expression. All three enzymes possessed lysine (position 130) for the formation of Schiff bases between active site glutamate (position 43 in the B. subtilis numbering system) and pyruvate, while B. subtilytm enzymes are active site residues. Phosphorus position 47 contained threonine but not arginine. B. subtilis KHG was smaller and appears to exist in clusters, unlike S. melorirot and E. coli enzymes and other enzymes with active site threonine. In other words, it is possible that the difference in the active sites is the cause of the increased activity of the B. subtilis enzyme.

Aldolase  Improvement of activity

Catalytic antibodies are as efficient as natural aldolase, allow a wide range of substrates and can be used to catalyze the reaction shown in FIG.

Aldolase can also be enhanced through directed evolution, e.g., through DNA shuffling and error-prone PCR to eliminate the need for phosphates and to conduct enantioselectivity. The KDPG aldolase (which is highly homologous to KHG described above) can be improved as previously described. KDPG aldolase polypeptides are useful for biochemical reactions because they have high specificity for the feed substrate but relatively fluid to the receptor substrate (ie, indole-3-pyruvate) (Koeller & Wong, Nature 409: 232-9, 2001). KHG aldolase is active in the condensation of many carboxylic acids with pyruvate. The mammalian form of KHG aldolase is believed to have a broader specificity than the bacterial form, such as higher activity against 4-hydroxy 4-methyl 2-oxoglutarate and 4-hydroxy-2-ketoglutar All of the stereoisomers of acid salts are allowed. The bacterial source appears to exhibit a 10-fold preference for the R ideal. Nearly 100 KHG homologs are available in the genomic database and have been demonstrated in activity Pseudomonas, Paracoccus, Providencia, cynorazobium, Morganella, Escherichia coli and mammalian tissue. This enzyme can be used as a starting point to achieve the desired enantioselectivity for monatin production.

Aldolases utilizing pyruvate and other substrates with bulky hydrophobic groups such as keto acids and / or indole can be "evolved" to match the specificity, rate and selectivity of this polypeptide. In addition to the KHG and ProA aldolases demonstrated herein, examples of such enzymes include, but are not limited to: KDPG aldolase and related polypeptides (KDPH); Transcarboxybenzalpyruvate hydratase-aldolase from nocardioid strains; 4- (2-carboxyphenyl) -2-oxobut-3-enoate aldolase (2'-carboxybenzalpyruvate aldolase) for condensing pyruvate and 2-carboxybenzaldehyde (aromatic ring-containing substrate) ; Trans-O-hydroxybenzylidenepyruvate hydratase-aldorase derived from Pseudomonas putida and sphingonamonas allomatisivoranes using pyruvate and an aromatic-containing aldehyde as a substrate; 3-hydroxyaspartate aldolase (erythro-3-hydroxy-L-aspartate glyoxylate degrading enzyme) which uses 2-oxo acid as a substrate and is believed to be present in the organism Micrococcus denitrippicans; Benzoin aldolase (benzaldehyde degrading enzyme) using a substrate containing a benzyl group; Dihydroneopterin aldolase; L-threo-3-phenylserine benzaldehyde-degrading enzyme (phenylserine aldolase) for condensing benzaldehyde and glycine; 4-hydroxy-2-oxovaleric acid aldolase; 1,2-dihydroxybenzylpyruvate aldolase; And 2-hydroxybenzalpyruvate aldolase.

Polypeptides with the required activity can be selected by selecting the clones using the following methods. Tryptophan mutant strains are transformed using vectors carrying the clones in the expression cassettes and propagated in medium containing small amounts of monatin or MP. Because aminotransferase and aldolase reactions are reversible, cells can produce tryptophan from racemic mixtures of monatin. In a similar manner to l, organisms (both recombinant and wild type) can be screened through the property of using MP or monatin as the carbon and energy source. One source of target aldolase is the expression library of various Pseudomonas and Lyzobacteria strains. Pseudomonads possess a number of unique catabolic pathways that degrade aromatic molecules, and also contain a number of aldolases, while lysobacteria contain aldolases and are known to proliferate in the plant root zone and the biosynthetic pathways of monatin Has a number of genes as described.

Example  5

Montan Precursor  Chemical synthesis

Example 4 describes a method of using aldolase to convert indole-3-pyruvate to 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid monatin precursor (MP) It was. This example describes an alternative method of chemically synthesizing MP.

MP is generally prepared via aldol-like condensation (FIG. 4). Briefly, typical aldol-type reactions include the generation of carboions of pyruvate esters using strong bases such as LDA (lithium diisopropylamide), lithium hexamethyldisilazane or butyl lithium. The resulting carboanions react with indole-pyruvate to form the binding product.

Protective groups that can be used to protect indole nitrogen include t-butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz), but are not limited thereto. Blocking groups of carboxylic acids include, but are not limited to, alkyl esters (eg, methyl, ethyl, benzyl esters). When such protecting groups are used, it is impossible to control the stereochemistry of the product formed. However, formation of one MP enantiomer may be advantageous if R 2 and / or R 3 is a chiral protecting group such as (S) -2-butanol, menthol or chiral amine (FIG. 4).

Example  6

Tryptophan  Or indole-3- From pyruvate With monatin  transform

In vitro methods using two enzymes, aminotransferase and aldolase, produced monatin from tryptophan and pyruvate. In the first step, alpha-ketoglutarate produces indole-3-pyruvate and glutamate as receptors to accept amino groups derived from tryptophan in transamination reactions. Aldolase catalyzes the second reaction and reacts pyruvate with indole-3-pyruvate in the presence of Mg ²⁺ and phosphate to give the alpha-keto derivative (MP), ie 2-hydroxy 2- (indole) of monatin. To yield 3--3-ylmethyl) -4-keto glutaric acid. The transfer of amino groups from glutamate formed in the first reaction produced the desired product monatin. Purification and characterization of the product confirmed that the formed isomer was S, S-monatin. The following describes the improvements carried out in this process along with alternative substrates, enzymes and conditions.

enzyme

4-hydroxy-4-methyl-2-oxoglutarate pyruvate degrading enzyme (ProA aldolase, proA gene) (EC4.1.3.17), which is an aldolase derived from Comamonas testosterone, was used in Example 4. Cloned, expressed and purified as described. 4-hydroxy-2-oxoglutarate glyoxylate degrading enzyme (KHG aldolase) from B. subtilis, Escherichia coli and S. melorirot (EC 4.1.3.16) is also described in Example 4. Cloned, expressed and purified as described.

The aminotransferases used with aldolase for monatin production include the L-aspartate aminotransferase encoded by the E. coli aspC gene, the tyrosine aminotransferase encoded by the E. coli tyrB gene, and the S. melotillo TatA enzyme. , Broad substrate aminotransferases encoded by the L. major bsat gene, glutamic acid-oxaloacetic acid aminotransferase (type IIa) derived from porcine heart. Cloning, expression and purification of non-mammalian proteins is described in Example 1. Glutamic acid-oxaloacetic acid aminotransferase (type IIa) from pig heart was obtained from Sigma (# G7005).

ProA Aldolase  And L- Aspartate  Method using aminotransferase

50 mM ammonium acetate, pH8.0, 4 mM MgCl ₂ , 3 mM potassium phosphate, 0.05 mM pyridoxal phosphate, 100 mM ammonium pyruvate, 50 mM tryptophan, 10 mM alpha-ketoglutarate, 160 mg of recombinant C. Testosterone ProA aldolase (Crude cell extract, about 30% aldolase), reaction mixture containing 233 mg of recombinant E. coli L-aspartate aminotransferase (crude cell extract, about 40% aminotransferase) in 1 liter was used. . All components except enzymes were mixed together and incubated at 30 ° C. until tryptophan was dissolved. Enzymes were then added and the reaction solution was incubated for 3.5 hours under gentle shaking (100 rpm). At 0.5 hours and 1 hour after the addition of the enzyme, a quantity of solid tryptophan (50 mmol each) was added to the reaction. All of the added tryptophan was not dissolved but the concentration was maintained at 50 mM or higher. After 3.5 hours the solid tryptophan was filtered off. The reaction mixture was analyzed by LC / MS with a standard amount of tryptophan as standard, and the concentration of tryptophan in the solution was 60.5 mM and the concentration of monatin was 5.81 mM (1.05 g).

The final product was purified by the following method. 90% clear solution was applied to the Biorad AG50W-X8 resin column (225 ml; binding capacity 1.7 meq / ml). The column was washed with water, collecting 300 ml fractions until the absorbance at 280 nm became <5% of the primary through the fractions. Then, 1M ammonium acetate pH 8.4 was eluted through the column to collect four 300 ml fractions. Four fractions containing monatin were evaporated to 105 ml using a rotary evaporator equipped with a lukewarm water bath. A precipitate formed upon volume reduction, which was filtered off during the evaporation process.

Analysis of column fractions by LC / MS confirmed that 99% of tryptophan and monatin were bound to the column. The precipitate formed during the evaporation process contained> 97% tryptophan and <2% monatin. The ratio of tryptophan: product in the supernatant was about 2: 1.

The supernatant (7 ml) was applied to a 100 ml Fast Flow DEAE Sepharose (Amersham Biosciences) column which was previously washed with 0.5 L 1 M NaOH, 0.2 L water, 1.0 L 1.0 M ammonium acetate pH 8.4 and 0.5 L water and converted to the acetate form. It was. The supernatant was loaded in an amount of <2 ml / min and washed with 3-4 ml / min water until the absorbance at 280 nm was about zero. Monatin was eluted with 100 mM ammonium acetate pH 8.4 and four 100 ml fractions were collected.

As a result of the fraction analysis, the ratio of tryptophan: monatin in the flow fraction was 85:15 and the ratio in the eluate fraction was 7:93. Assuming that the extinction coefficient of monatin at 280 nm is the same as tryptophan, the amount of product in the eluate fraction was 0.146 mmol. The total 1 L reactant produced about 2.4 mmol (about 710 mg) of monatin, yielding a recovery of 68%.

Eluate fractions from the DEAE Sepharose column were evaporated to <20 ml. For monatin characterization on the analytical scale, the product was further purified by applying a certain amount of product to a C ₈ preparative reversed phase column using chromatography conditions as described in Example 10. Waters Fractionlynx ™ software was used to automatically fractionate monatin based on the detection of m / z = 293 ions. Fractions from a C ₈ column with the corresponding protonated molecular ions of monatin were collected, evaporated to dryness and dissolved in a small amount of water. This fraction was used to characterize the product.

The resulting product was characterized in the following manner.

UV / Visible Spectroscopy. UV / Visible spectroscopic measurements of enzymatically produced monatin were performed using a Cary 100 Bio UV / Visible spectrophotometer. The purified product dissolved in water showed a maximum absorbance at 280 nm and a shoulder at 288 nm, which is typical of indole-containing compounds.

LC / MS analysis. Analysis of the mixture to detect monatin induced by in vitro biochemical reactions was performed as described in Example 10. Typical LC / MS analysis of monatin present in the in vitro enzymatic synthesis mixture is illustrated in FIG. 5. The bottom panel of FIG. 5 shows a selective ion chromatogram for protonated molecular ions of monatin at m / z = 293. Identification of monatin in such mixtures was confirmed through the mass spectrum shown in FIG. 6. Analysis of the purification product by LC / MS showed a single peak with absorbance at 280 nm and molecular ions of 293. The mass spectrum was the same as shown in FIG.

MS / MS analysis. LC / MS / MS daughter ion experiments as described in Example 10 were also performed for monatin. The daughter ion mass spectrum of monatin is shown in FIG. 7. All labeled fragment ions were virtually structured in FIG. 7. These include m / z = 275 (293-H ₂ O), 257 (293- (2xH ₂ O)), 230 (275-COOH), 212 (257-COOH), 168 (3-buta-1,3- Dienyl-1H-indole carbonium ion), 158 (1H-indole-3-carbaldehyde carbonium ion), 144 (3-ethyl-1H-indole carbonium ion), 130 (3-methylene-1H-indole Carbonium ions) and fragment ions of 118 (indole carbonium ions). Most of this was as obtained in MP (Example 4), which was expected if it was derived from the indole portion of the molecule. Some were about 1 mass unit higher than those observed for MP, presumably due to the presence of amino groups instead of ketones.

High resolution MS analysis. 8 shows the mass spectra obtained for purified monatin using an Applied Biosystems-Perkin Elmer Q-star hybrid quadrupole / flight time mass spectrometer. The mass of protonated monatin measured using tryptophan as internal mass control standard was 293.1144. The mass of protonated monatin calculated based on the C ₁₄ H ₁₇ N ₂ O ₅ element composition was 293.1137. This mass error of less than 2 ppm provides conclusive evidence that the enzymatically produced product is the elemental composition of monatin.

NMR spectroscopy. NMR experiments were performed using a Varian Inova 500 MHz device. The monatin sample (about 3 mg) was dissolved in 0.5 ml D ₂ O. First, solvent (D ₂ O) was used as an internal control as 4.78 ppm. Since the peak of water was large, ¹ H-NMR was performed while suppressing the peak of water. Due to the broadness of the water peak, the C-2 proton of monatin was then used as the control peak and added at a known value of 7.192 ppm.

Hundreds of primary scans for ¹³ C-NMR were found to be too dilute to provide adequate ¹³ C spectra within the allotted time. Thus, heteronuclear multiple quantum cohesion (HMQC) experiments were performed, which provide information on the correlation between hydrogen and attached carbon and the chemical shift of carbon.

Results for ¹ H and HMQC are summarized in Tables 2 and 3. As compared with known values, NMR data suggested that the enzymatically produced monatin was (S, S), (R, R) or mixtures thereof.

Chiral LC / MS Analysis. In order to confirm that the in vitro produced monatin is a single isomer rather than a mixture of (R, R) and (S, S) enantiomers, chiral LC / MS analysis was performed using the device shown in Example 10.

Chiral LC separations were performed using a Chirobiotic T (Advanced Separations Technology) chiral chromatography column at room temperature. Separation and detection based on the protocol provided by the supplier is optimized for the R- (D) and S- (L) isomers of tryptophan. The LC mobile phase was A) water containing 0.05% (v / v) trifluoroacetic acid; B) A mixture consisting of methanol containing 0.05% (v / v) trifluoroacetic acid was used. Elution was isocratic at 70% A and 30% B. The flow rate was 1.0 ml / min and PDA absorbance was monitored at 200 nm to 400 nm. The parameters of the instrument used in the chiral LC / MS analysis of monatin and tryptophan were used the same as described in Example 10 for LC / MS analysis. Mass spectra were collected in the region m / z 150-400. Ion chromatograms selected for protonated molecular ions ([M + H] ⁺ = 205 for R-tryptophan and S-tryptophan, [M + H] ⁺ = 293 for monatin), and the blood in the mixture The presence of the analytes was known directly.

Chromatograms for R- and S-tryptophan and monatin separated by chiral chromatography and monitored by MS are shown in FIG. 9. The single peak appearing in the monatin chromatogram suggested that this compound is a single isomer and the retention time is almost the same as S-tryptophan.

TABLE 2                 

¹ H NMR data

¹ Vleggaar et al. (JCS PerKin Trans. 1: 3095-8, 1992).

² Takeshi and Shusuke (JP2002060382, 2002-02-26).

TABLE 3

¹³ C NMR data (via HMQC spectrum)

¹ Vleggaar et al. (JCS PerKin Trans. 1: 3095-8, 1992).

Polarization measurement method . Oddness was measured with a Rudolph Autopol III polarimeter. Monatin was prepared in 14.6 mg / ml aqueous solution. The expected non-photoreactivity ([α] _D ²⁰ ) of S, S monatin (salt form) was -49.6 in 1 g / ml aqueous solution (Vleggaar et al.). The observed [α] _D ²⁰ was -28.1 for the enzymatically produced purified monatin, suggesting that it is an S, S isomer.

Improvement

Reaction conditions including reagent and enzyme concentrations were optimized and a yield of 10 mg / ml was obtained using the following reagent mixtures:

50 mM ammonium acetate pH 8.3, 2 mM MgCl ₂ , 200 mM pyruvate (sodium or ammonium salt), 5 mM alpha-ketoglutarate (sodium salt), 0.05 mM pyridoxal phosphate, degassing water to make final volume 1 ml after addition of enzyme, 3 mM potassium phosphate, 50 μg / ml recombinant ProA aldolase (cell extract; total protein concentration 167 μg / ml), L-aspartate aminotransferase (cell extract; total protein concentration 2500) encoded by E. coli aspC gene Μg / ml) and 1000 μg / ml, and solid tryptophan (saturated; some are undissolved during the reaction) to make concentrations> 60 mM. The mixture was incubated for 4 hours at 30 ° C. under gentle stirring or mixing.

substitution

The concentration of alpha-ketoglutarate can be reduced to 1 mM and 9 mM aspartate can be supplemented with the same amount of monatin. In the first step, alternative amino acid receptors such as oxaloacetate can be used.                 

Using mutant L. major broad substrate aminotransferase instead of E. coli L-aspartate aminotransferase, monatin was obtained in similar yields. However, a second unidentified product (3-10% of the main product) having a molecular weight of 292 was detected in the LC-MS analysis. When the aminotransferase was added to E. coli tyrB coding enzyme, S. meloriti tat A coding enzyme or glutamic acid-oxaloacetic acid aminotransferase (type IIa) derived from swine heart, the monatin concentration was 0.1 to 0.5 mg / ml. Obtained. When the reaction is initiated from indole-3-pyruvate, reductive amination can be carried out using glutamate dehydrogenase and NADH in the last step (as in Example 7).

KHG aldolase derived from B. subtilis, E. coli and S. melorirot was used with E. coli L-aspartate aminotransferase to produce monatin enzymatically. The following conditions were used for the reaction: 50 mM ammonium acetate pH 8.3, 2 mM MgCl ₂ , 200 mM pyruvate, 0.05 mM pyridoxal phosphate, degassed water to bring the final volume to 0.5 ml after enzyme addition, 3 mM potassium phosphate, 20 Μg / ml of recombinant B. subtilis KHG aldolase (purified), approximately 400 μg / ml crude E. coli L-aspartate aminotransferase (AspC) from cell extract, and 12 mM indole-3- Pyruvate. The reaction was incubated at 30 ° C. for 30 minutes under shaking. The amount of monatin produced using B. subtilis enzyme was 80 ng / ml and was increased by increasing amounts of aldolase. Substitution of saturated tryptophan and 5 mM alpha-ketoglutarate instead of indole-3-pyruvate and glutamate increased the production of monatin to 360 ng / ml. 30 μg / ml of these three KHG enzymes, each dissolved in 50 mM Tris pH 8.3, were used in combination with a saturated amount of tryptophan to repeat the reaction and run for 1 hour to improve detection rate. The Bacillus enzyme showed the highest activity as in Example 4, producing about 4000 ng / ml of monatin. Escherichia coli KHG produced 3000 ng / ml monatin and S. melorirot enzyme produced 2300 ng / ml.

Example  7

With MP With monatin  Interchange

Amination of MP to form monatin can be catalyzed by an aminotransferase as identified in Examples 1 and 6 or a dehydrogenase that requires a reducing cofactor such as NADH or NADPH. This reaction is reversible and can be measured in both directions. When using dehydrogenases, the aromaticity can be controlled primarily by the concentration of the ammonium salt.

Dehydrogenase activity. Oxidative deamination of monatin was monitored as NAD (P) ⁺ showed an increase in absorbance at 340 nm when converted to NAD (P) H, which is more chromogenic. Monatin was produced and purified enzymatically as described in Example 6.

The assay mixture used a typical mixture made of 50 mM Tris-HCl, pH 8.0-8.9, 0.33 mM NAD ⁺ or NADP ⁺ , 2-22 units of glutamate dehydrogenase (Sigma), and 0.2 mL of 10-15 mM substrate. Analysis was performed in duplicate on a UV-permeable microtiter plate using a Molecular Devices SpectraMax Plus platereader. Mixtures containing enzyme, buffer and NAD (P) ⁺ were injected into the wells containing the substrate and mixed briefly and then monitored for an increase in absorbance at 340 nm at 10 second intervals. The reaction solution was incubated at 25 ° C. for 10 minutes. Negative control was performed without substrate addition and glutamate was used as positive control. Type III glutamate dehydrogenase obtained from bovine liver (Sigma # G-7882) was used for the conversion of monatin to monatin precursor at a conversion rate of about 1/100 of the conversion rate of glutamic acid to alpha-ketoglutarate. Catalyzed.

Aminotransferase activity. The monatin aminotransferase assay was described in Aspartate aminotransferase (AspC) derived from E. coli, Tyrosine aminotransferase (TyrB) derived from E. coli, Broad substrate aminotransferase (BSAT) derived from L. major, and described in Example 1. Two commercialized porcine glutamate-oxaloacetate aminotransferases were performed. Oxaloacetate and alpha-ketoglutarate were both examined as amino acceptors. For analysis, a mixture of 50 mM Tris-HCl pH 8.0, 0.05 mM PLP, 5 mM amino acceptor, 5 mM monatin and 25 μg of aminotransferase in 0.5 ml was used. The assay was incubated at 30 ° C. for 30 minutes before the reaction was stopped by addition of 0.5 ml isopropyl alcohol. Loss of monatin was monitored by LC / MS (Example 10). Maximal activity was shown by L. major BSAT using oxaloacetate as amino acceptor followed by the same enzyme using alpha-ketoglutarate as amino acceptor. The relative activity when using oxaloacetate was as follows: BSAT>AspC> Porcine IIa> Porcine I = TyrB. The relative activity with alpha-ketoglutarate was as follows: BSAT>AspC> Porcine I> Porcine IIa> TyrB.

Example  8

Pyruvate  With other C3 sources From tryptophan Monatin  Produce

As described above in Example 6, indole-3-pyruvate or tryptophan can be converted to monatin using pyruvate as the C3 molecule. In some cases, however, pyruvate may be undesirable as a raw material. For example, pyruvate may be more expensive than other C3 carbon sources, or may adversely affect fermentation when added to the medium. Alanine can be aminotransferred by a number of PLP enzymes to produce pyruvate.

Tryptopase-like enzymes perform beta-removal reactions faster than other PLP enzymes, such as aminotransferases. Enzymes of this class (4.1.99.-) are L-serine, L-cysteine, derivatives of serine and cysteine having good leaving groups such as O-methyl-L-serine, O-benzyl-L-serine, S- Ammonia and pyruvate can be produced from methylcysteine, S-benzylcysteine, S-alkyl-L-cysteine, O-acyl-L-serine, 3-chloro-L-alanine.

EC 4.1.99.- A method for producing monatin using a polypeptide is β-tyrosinase according to the method of Murato et al. (Mouratou et al., J. Biol. Chem. 274: 1320-5, 1999). (TPL) or tryptophanase can be mutated to improve. Murato et al. Describe the properties of converting β-tyrosinase to dicarboxylic amino acid β-degrading enzymes, which have not been reported to occur in nature. Changes in specificity were performed by replacing valine (V) 283 with arginine (R) and arginine (R) 100 with threonine (T). This amino acid change allows the enzyme to accept dicarboxylic amino acids (eg aspartate) in the hydrolytic deamination reaction. Thus, aspartate can be used as a source of pyruvate in the subsequent aldol condensation reaction.

The cell or enzymatic reactor may also be fed lactate and an enzyme that converts the lactate to pyruvate. Enzymes that can catalyze this reaction include lactate dehydrogenase and lactate oxidase.

Genome DNA Separation of

Tryptopase polypeptides have already been reported, for example, in Murateau et al. (JBC 274: 1320-5, 1999). Genomic DNA of Escherichia coli DH10B was used as a template for PCR as described in Example 1 to isolate genes encoding tryptopase polypeptides.

The tyrosine-phenol degrading enzyme gene was isolated from C. prundi (ATCC Catalog No. 8090, ATCC 13316; NCTC9750) and 37 ° C. until OD 2.0 in nutrient solid medium (Difco 0001) and nutrient liquid medium (Difco 0003). Proliferation at Genomic DNA was purified using the Quiagen Genomic-tip ™ 100 / G kit.

Code sequence PCR  Amplification

Primers were designed using the compatibility overhang of the pET 30 Xa / LIC vector (Novagen, Madison, Wisconsin) as described above in Example 1.

(서열번호 43). E. coli tna (SEQ ID NO: 41). N-terminal primers for pET30 Xa / LIC cloning:

(SEQ ID NO: 43).

(서열번호 44).C-terminal primers for pET30 Xa / LIC cloning:

(SEQ ID NO: 44).

(서열번호 45).C. Prundi tpl (SEQ ID NO: 42). N-terminal primers for pET30 Xa / LIC cloning:

(SEQ ID NO 45).

(서열번호 46).C-terminal primers for pET30 Xa / LIC cloning:

(SEQ ID NO 46).

For all PCR reactions, a thermocycler (Eppendorf Mastercycler ™ Gradient 5331 Thermal Cycler) was used. 50 μl contains 0.5 μg template (genomic DNA), 1.0 μM of each primer, 0.4 mM of each dNTP, 3.5 U Expand High Fidelity Polymerase (Roche), 1 × Expand buffer (containing Mg), and 5% DMSO (final concentration) One reaction was used. The thermocycler PCR program used was as follows: 96 ° C. high temperature start (5 min), 94 ° C.-30 sec, 40-60 ° C.-1 min 45 sec, 72 ° C.-2 min 15 sec; Repeat 30 times. The final polymerization step was carried out for 7 minutes and then the samples were stored at 4 ° C.

Cloning

The cloning and positive clone identification procedures described in detail in Example 1 were used to find suitable clones.

Gene Expression and Activity Analysis

Plasmid DNA (identified by sequencing) was subcloned into expression host BL21 (DE3) (Novagen). Cultures were grown in LB medium containing 30 mg / L kanamycin, plasmids were isolated using the Qiagen miniprep kit and then identified for restriction by restriction enzyme digestion.

Induction experiments were performed using a BL21 (DE3) expression host grown at 37 ° C. in LB medium containing 50 mg / L kanamycin. After OD ₆₀₀ of the culture reached about 0.6, protein expression was induced using 0.1 mM IPTG. Cells were grown for 4 hours at 30 ° C. and then harvested by centrifugation. Cells were then used with 5 ml (per g of wet cell weight) Bugbuster ™ reagent (Novagen) containing 5 μl / ml protease inhibitor mixture set #III (Calbiochem) and 1 μl / ml benzonase nuclease (Novagen). His-tag recombinant protein was purified using the His-Bind cartridge (Example 1) described above in Example 1. Purified protein was desalted using a PD-10 (G25 Sephadex, Amersham Biosciences) column and eluted with 100 mM Tris-Cl buffer (pH 8.0). Proteins were analyzed via SDS-PAGE of 4-15% gradient gels to detect the concentration of soluble protein at the estimated molecular weight of the recombinant fusion protein.

Mutagenesis

Some components of the polypeptide class 4.1.99 .- (tryptophanase and β-tyrosinase) use bepartates or similar amino acids to perform beta-degrading enzyme reactions without any modification. However, some components of this class may need to be mutated for substrate availability and / or creation of products. Moreover, in some cases polypeptides capable of performing the conversion may be further optimized through mutagenesis.

Therefore, site directed mutagenesis was performed based on the 3D structural analysis of the PLP-binding polypeptide. Two examples of changing the substrate specificity of a polypeptide are given below.

Tryptopase  Mutagenesis example 1

(서열번호 47) 및

(서열번호 48).The mutagenesis protocol presented below introduces two point mutations in the amino acid sequence. The first point mutation changed arginine (R) at position 103 to threonine (T) and the second point mutation changed valine (V) at position 299 to arginine (R). Use that). Then, mutagenesis experiments were performed by ATG Laboratories (Eden Prairie, MN). Mutations were then introduced into the PCR of the gene fragments and reassembly of the fragments was also performed by PCR. The primers used to convert arginine (R) 103 to threonine (T) are as follows:

(SEQ ID NO 47) and

(SEQ ID NO 48).

(서열번호 49) 및

(서열번호 50).Primers used to convert valine (V) 299 to arginine (R):

(SEQ ID NO 49) and

(SEQ ID NO 50).

Mutants were selected by restriction digestion with XbaI / HindIII and SphI and confirmed by sequencing.

Tyrosine  Phenolase ( β - Tyrosinase ) Mutagenesis example 2

Two point mutations were made to the tyrosine-phenol degrading enzyme amino acid sequence. This mutation converted arginine (R) at position 100 to threonine (T) and valine (V) at position 283 to arginine (R) (in the C. prundi mature protein sequence).

(서열번호 51) 및

(서열번호 52).Primers for R100T conversion are as follows:

(SEQ ID NO: 51) and

(SEQ ID NO 52).

(서열번호 53) 및

(서열번호 54).Primers for V283R conversion are as follows:

(SEQ ID NO: 53) and

(SEQ ID NO 54).

The method described above was used and clones were selected by KpnI / SacI digestion and BstXI digestion. The sequence was confirmed by dideoxy chain termination sequencing. Recombinant proteins were produced as described above relative to the wild type enzyme.

The reaction includes 50 mM Tri-Cl pH 8.3, 2 mM MgCl ₂ , 200 mM C3 carbon source, 5 mM alpha-ketoglutarate, sodium salt, 0.05 mM pyridoxal phosphate, degassing to bring the final volume to 0.5 ml after the addition of enzyme, 3 mM Potassium phosphate pH 7.5, crude recombinant C.testosterone ProA aldolase 25 μg as prepared in Example 4, crude L-aspartate aminotransferase (AspC) 500 as prepared in Example 1 A mixture consisting of μg, and solid tryptophan (saturated; partly undissolved during the reaction) was used to maintain a concentration of> 60 mM. The reaction mixture was incubated at 30 ° C. for 30 minutes with mixing. Serine, alanine and aspartate were fed to the C3 source. The presence of a second PLP enzyme (tablet) (tryptophanase (TNA), double mutant tryptophanase, β-tyrosinase (TPL)) capable of carrying out beta-removal reaction and beta-lyase enzyme reaction Analysis was performed in the absence and in the absence. The results are shown in Table 4:

Table 4

Monatin production using alternative C3 carbon sources

C3 carbon source Additional PLP enzymes Relative activity No addition No addition 0% Pyruvate No addition 100% Serine No addition 3% Serine 11 μg wild-type TNA (1U) 5.1% Serine 80 μg double mutant TNA 4.6% Alanine No addition 32% Alanine 11 μg wild-type TNA 41.7% Alanine 80 μg mutant TNA 43.9% Aspartate 110 μg wild-type TNA (10U) 7.7% Aspartate 5U wild type TPL (crude) 5.1% Aspartate 80 μg mutant TNA 3.3%

Monatine prepared using alanine and serine as the C3 source was confirmed by LC / MS / MS daughter scan analysis and was identical to the properties of monatin produced in Example 6. Alanine was aminotranslated by AspC enzyme as the best alternative source tested. The amount of monatin produced increased with the addition of tryptophanase, which can perform aminotransition with the second activity. The amount of monatin produced using serine as a carbon source was almost doubled by the addition of tryptophanase enzyme even when only 1/5 of tryptophanase enzyme was added to aminotransferase. AspC can only perform some beta clearance activity. The results with aspartate suggest that tryptophanase activity against aspartate is not increased by the same site directed mutations as previously proposed for β-tyrosinase. Mutant β-tyrosinase is expected to exhibit increased monatin production activity.                 

Example  9

Monatin  Chemical synthesis

The addition of alanine to indole-3-pyruvic acid produces monatin and this reaction can be carried out synthetically with Grignard or organolithium reagents.

For example, magnesium is added under anhydrous conditions to 3-chloro- or 3-bromo-alanine which is a suitable blocking of carboxyl and amino groups. Indole-3-pyruvate (suitably blocked) is then added to form the bound product, followed by removal of the protecting group to form monatin. Particularly useful protecting groups are THP (tetrahydropyranyl ether) which is easy to attach and remove.

Example  10

Monatin  And MP Detection of

This example describes the method used to detect the presence of monatin or its precursor 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid.

LC / MS analysis

Analysis of the mixture of alpha-ketosan form of monatin (monatin precursor, MP) and monatin induced by in vitro or in vivo biochemical reactions was performed using Waters 2690 liquid chromatograph and Waters 996 Photo-Diode Array (PDA) absorbance. This was performed using a Waters / Micromass Liquid Chromatography-Serial Mass Spectroscopy (LC / MS / MS) apparatus equipped with a monitor and a Micromass Quatto Ultima triple quadrupole mass spectrophotometer. LC separations were performed at room temperature using a Supelco Discovery C ₁₈ reverse phase chromatography column of 2.1 mm × 150 mm or Xterra MS C ₈ reverse phase chromatography column of 2.1 mm × 250 mm. The LC mobile phase was used by mixing A) water containing 0.05% (v / v) trifluoroacetic acid and B) methanol containing 0.05% (v / v) trifluoroacetic acid.

Gradient elution was linear from 5% B to 35% B (0 to 9 minutes), from 35% B to 90% B linear (9 to 16 minutes, 90% B isocratic (16 to 20 minutes), 90% B The solution was linearly configured at 5% B at, with a 10 min rebalance period for each elution, flow rate 0.25ml / min and PDA absorbance monitored from 200nm to 400nm.All parameters of ESI-MS were optimized, Selection was made based on the generation of protonated molecular ions ([M + H] ⁺ ) of the analyte and the generation of characteristic fragment ions.

LC / MS analysis of monatin was carried out with the following device parameters: capillary: 3.5 kV; Cone: 40 V; Hex1: 20 V; Aperture: 0V; Hex 2: 0 V; Source temperature: 100 ° C .; Desolvation temperature: 350 ° C .; Desolvation gas: 500 L / h; Cone gas: 50L / h; Low mass resolution (Q1): 15.0; High mass resolution (Q1): 15.0; Ion energy: 0.2; Inlet: 50V; Collision energy: 2; Outlet: 50V; Low mass resolution (Q2): 15; High mass resolution (Q2): 15; Ion energy (Q2): 3.5; Multiplier: 650. The reported mass / charge ratio (m / z) and uncertainty in molecular mass was ± 0.01%. Primary detection in the alpha-ketosan form (MP) of monatin and the mixture of monatin was achieved through LC / MS monitoring with collection of mass spectra in the range of m / z 150-400. Selected ion chromatograms for protonated molecular ions ([M + H] ⁺ = 292 for MP and [M + H] ⁺ = 293 for monatin) were performed in the mixture. Analytes can be identified directly.

MS / MS analysis

LC / MS / MS daughter ion experiments were performed on monatin as follows. Daughter ion analysis involves the transfer of the parent ions (eg m / z = 293 for monatin) from the first mass spectrometer (Q1) to the collision cell of the mass spectrophotometer, where argon is introduced to Chemical dissociation into fragment ions. These daughter ions can then be detected by a second mass spectrometer (Q2) and used to confirm the structural designation of the parent ion.

LC / MS / MS analysis of monatin was carried out with the following device parameters: capillary: 3.5 kV; Cone: 40 V; Hex1: 20 V; Aperture: 0V; Hex 2: 0 V; Source temperature: 100 ° C .; Desolvation temperature: 350 ° C .; Desolvation gas: 500 L / h; Cone gas: 50L / h; Low mass resolution (Q1): 13.0; High mass resolution (Q1): 13.0; Ion energy: 0.2; Inlet: -5V; Collision energy: 14; Outlet: 1V; Low mass resolution (Q2): 15; High mass resolution (Q2): 15; Ion energy (Q2): 3.5; Multiplier: 650.

Monatin High yield  analysis

In order to analyze monatin obtained in in vitro or in vivo reactions, high yield analysis of mixtures (<5 min / sample) was performed using the same parameters as described above in the instrument and LC / MS / MS. LC separation was carried out using Waters Xterra MS C ₈ (2.1 mm × 50 mm) chromatography at room temperature by isocratic elution of 15% MeOH aqueous solution, 0.25% acetic acid at a flow rate of 0.3 ml / min. Detection of monatin present in the mixture was performed using selective reaction monitoring (SRM) -serial mass spectroscopy. This includes the daughter ions of specific collision-induced ions ([M + H] ⁺ = 293.1) (eg, fragment ions at m / z = 168.1) to maximize the sensitivity, selectivity, and yield of detecting monatin. Monitoring the transition to -beta-1,3-dienyl-1H-indole carbonium ions). PDA absorbance data was collected in parallel to further demonstrate the homogeneity of monatin.

Example  11

Bacteria Monatin  production

This example describes a method for producing monatin in E. coli cells. Those skilled in the art will appreciate that similar methods can be used to produce monatin in other bacterial cells. It is also possible to use vectors containing other genes in the monatin synthesis pathway (FIG. 2).

Trp-1 + glucose medium (Zeman et al., Folia Microbiol. 35: 200-4, 1990), the smallest medium used to increase tryptophan production in E. coli cells, was prepared as follows. To 700 ml Nanopure water was added the following reagents: 2 g (NH ₄ ) ₂ SO ₄ , 13.6 g KH ₂ PO ₄ , 0.2 g MgSO ₄ · 7H ₂ O, 0.01 g CaCl ₂ · 2H ₂ O and 0.5 mg FeSO ₄ · 7H ₂ O. The pH was adjusted to 7.0 and the volume was increased to 850 ml and the medium autoclaved. 50% glucose solution was prepared separately and sterile filtered. 40 ml of this solution was added to the basal medium (850 ml) and the final volume was adjusted to 1 L.

10 g / L L-tryptophan solution was prepared at 0.1 M sodium phosphate pH 7 and sterile filtered. In general, 1/10 volume was added to the cultures indicated below. A 10% sodium pyruvate solution was also prepared and sterile filtered. Generally 10 ml aliquots were used for 1 L of culture. Ampicillin (100 mg / ml), kanamycin (25 mg / ml) and IPTG (840 mM) stocks were also prepared, sterilized and stored at −20 ° C. until use. Tween 20 (polyoxyethylene 20-sorbitan monolaurate) was used in an amount with a final concentration of 0.2% (v / v). Ampicillin was used at a non-fatal concentration, generally at a final concentration of 1-10 μg / ml.

A new plate of E. coli BL21 (DE3) :: C.testosteroney proA / pET30Xa / LIC (described in Example 4) was prepared in LB medium containing 50 μg / ml kanamycin. Inoculated with a single colony inoculated in kanamycin-added LB medium and incubated overnight at 30 ℃ to grow (5ml). In general, 1:50 inoculum was used for induction reaction in trp-1 + glucose medium. Fresh antibiotics were added at a final concentration of 50 mg / L. Shake flasks were grown at 37 ° C. prior to induction.

Cells were harvested every hour until OD ₆₀₀ reached 0.35 to 0.8. Cells were then induced with 0.1 mM IPTG and the temperature was reduced to 34 ° C. Samples (1 ml) were taken and centrifuged at 5000xg before induction. The supernatant was frozen at -20 ° C for LC / MS analysis. After 4 hours of induction, 1 ml of the sample was again taken and centrifuged to separate the cell pellet and the medium. Tryptophan, sodium pyruvate, ampicillin and Tween were added as described above.

Cells were proliferated for 48 hours after induction and again 1 ml of sample was prepared as described above. At 48 hours, additional aliquots of tryptophan and pyruvate were added. The entire volume of the culture was centrifuged for 20 minutes at 3500 rpm at 4 ° C. after about 70 hours of propagation (after induction). The supernatant was discarded and the medium and cells frozen at -80 ° C. Media fractions were filtered and analyzed by LC / MS. The height and area of the [M + H] ⁺ = 293 peaks were monitored as described in Example 10. The background value of the medium was subtracted. This data was normalized for cell proliferation by plotting the height of the [M + H] ⁺ = 293 peak divided by the optical density of the culture at 600 nm.

Four hours after induction, pyruvate, ampicillin and Tween were added to produce higher amounts of monatin. Other additives such as PLP, additional phosphate or additional MgCl ₂ did not increase the production of monatin. Higher titers of monatin were obtained when tryptophan was used in place of indole-3-pyruvate and when tryptophan was added after induction than at inoculation or at induction. Before and after 4 hours of induction (time of substrate addition), the concentration of monatin present in fermentation broth or cell extracts was generally undetectable. Negative controls were tested using cells with only the pET30a vector without the addition of tryptophan and pyruvate to the culture. The parent MS scan showed that the compound with (m + 1) / z = 293 was not induced in the larger molecule, and the daughter scan (running as in Example 10) was similar to the monatin prepared in vitro. .

The effect of tween was investigated with final concentrations of Tween-20 at 0, 0.2% (v / v) and 0.6%. The maximum amount of monatin produced by the shake flask was at 0.2% Tween. The concentration of ampicillin was used in various amounts from 0 to 10 µg / ml. The amount of monatin in the cell culture increased rapidly (2.5 ×) between 0 and 1 μg / ml and increased to 1.3 × when the ampicillin concentration increased from 1 μg / ml to 10 μg / ml.

Experimental results over time showing general results are shown in FIG. 10. The amount of monatin isolated in the cell culture was increased even when normalized to cell proliferation. Using the molar extinction coefficient of tryptophan, the amount of monatin in the culture was estimated to be less than 10 μg / ml. The same experiment was repeated with cells containing the vector without the proA insert. Most of the values were negative, so the peak height at m / z = 293 was lower than for the medium alone in this culture (FIG. 10). Even when tryptophan and pyruvate were not added, the levels were consistently lower, indicating that monatin production was the result of an enzymatic reaction catalyzed by the aldolase enzyme.

In vivo production of monatin in bacterial cells was repeated in an 800 ml shake flask experiment and fermentor. A 250 ml sample of monatin (cell-free culture) was purified by anion exchange chromatography and preparative reverse phase liquid chromatography. This sample was evaporated and treated by high resolution mass spectrometry (see Example 6). High resolution MS showed that the metabolite produced was monatin.

(서열번호 67) 및 3' 프라이머:

(서열번호 68). 5' 프라이머에는 클로닝을 위해 BamHI 부위를 포함시키고, 3' 프라이머에는 SalI 부위를 포함시켰다. PCR은 실시예 4에 기술한 바와 같이 수행하고 겔 정제하였다. aspC/pET30 Xa/LIC 작제물은 PCR 산물과 마찬가지로 BamHI 및 SalI으로 분해시켰다. 분해물은 퀴아겐 스핀 컬럼으로 정제하였다. proA PCR 산물은 Roche Rapid DNA Ligation 키트를 제조자의 지시에 따라 사용함으로써 벡터에 결찰시켰다. 그 다음, 실시예 1에 기술한 바와 같이 Novablues Sinles(Novagen)를 사용하여 화학적 변형을 수행하였다. 콜로니는 50mg/L 가나마이신을 함유하는 LB 배지에서 증식시키고 플라스미드 DNA는 퀴아겐 스핀 미니프렙 키트를 사용하여 정제하였다. 제한 효소 분석으로 클론을 선별하고 Seqwright(Houston, TX)로 서열을 확인하였다. 작제물을 BLR(DE3), BLR(DE3)pLysS, BL21(DE3) 및 BL21(DE3)pLysS(Novagen)에 서브클로닝하였다. proA/pET30Xa/LIC 작제물 또한 BL21(DE3)pLysS에 형질전환시켰다.In vitro analysis suggested that the aminotransferase needed to be present in greater amounts than aldolase, and overexpressed E. coli-derived aspartate aminotransferase with the aldolase gene which increases the production of monatin. The following primers were designed to introduce C. testosterone proA into operon with aspC / pET30 Xa / LIC: 5 'primer:

(SEQ ID NO: 67) and 3 'primer:

(SEQ ID NO: 68). The 5 'primer contained the BamHI site for cloning and the 3' primer contained the SalI site. PCR was performed as described in Example 4 and gel purified. aspC / pET30 Xa / LIC constructs were digested with BamHI and SalI as well as PCR products. The digest was purified by Qiagen spin column. The proA PCR product was ligated to the vector by using the Roche Rapid DNA Ligation Kit according to the manufacturer's instructions. Next, chemical modifications were performed using Novablues Sinles (Novagen) as described in Example 1. Colonies were grown in LB medium containing 50 mg / L kanamycin and plasmid DNA was purified using the Qiagen spin miniprep kit. Clones were selected by restriction enzyme analysis and sequenced by Seqwright (Houston, TX). Constructs were subcloned into BLR (DE3), BLR (DE3) pLysS, BL21 (DE3) and BL21 (DE3) pLysS (Novagen). The proA / pET30Xa / LIC construct was also transformed into BL21 (DE3) pLysS.

In the BLR (DE3) shake flask samples under the standard conditions described above, the addition of a second gene (aspC) was observed to increase the monatin production seven-fold when compared initially. BL21 (DE3) derived host strain was used to promote proliferation. The proA clone and two gene operon clones were derived from Trp-1 medium as described above, and chloramphenicol (34 mg / L) was also added to the pLysS host. Shake flask experiments were performed with addition and no addition of 0.2% Tween-20 and 1 mg / L ampicillin. The amount of monatin in the culture was calculated using in vitro produced purified monatin as a standard. SRM analysis was performed as described in Example 10. Cells were sampled at 0, 4, 24, 48, 72 and 96 hours of proliferation.

The results for the maximum amount produced in the culture are shown in Table 5. In most cases both gene constructs produced higher amounts of monatin than the proA construct alone. The pLysS strain, which could have a more leaky cell envelope, had more secretion of monatin even though the growth rate of this strain was generally slower. The addition of Tween and ampicillin had a beneficial effect.

Table 5

The amount of monatin produced by E. coli bacteria

Construct host Tween + Amp Monatin μg / ml Hours (hr) proA BL21 (DE3) - 0.41 72 proA BL21 (DE3) + 1.58 48 proA BL21 (DE3) pLysS - 1.04 48 proA BL21 (DE3) pLysS + 1.60 48 aspC: proA BL21 (DE3) - 0.09 48 aspC: proA BL21 (DE3) + 0.58 48 aspC: proA BL21 (DE3) pLysS - 1.39 48 aspC: proA BL21 (DE3) pLysS + 6.68 48

Example  12

In yeast Monatin  production

This example illustrates the method used to produce monatin in eukaryotic cells. Those skilled in the art will appreciate that similar methods can be used to produce monatin in all cells of interest. In addition to the gene described in this example, other genes may be used (as shown in FIG. 2), or other genes may be used instead of the gene described in this example.

The pESC Yeast Epitope Tagging Vector System (Stratagene, La Jolla, Calif.) Was used to clone and express the E. coli aspC and C. testosterone proA genes into Saccharomyces cerevisiae. The pESC vector can contain both GAL1 and GAL10 promoters with different multiple cloning sites on opposite strands to express both genes at the same time. The pESC-His vector also contains His3 gene to supplement histidine nutritional composition of the host (YPH500). GAL1 and GAL10 promoters are inhibited by glucose and induced by galactose; Kozak sequences are used for optimal expression in yeast. The pESC plasmid is a shuttle vector, and the first construct can be made in E. coli (including the bla gene for selection). However, it is good practice to remove bacterial ribosomal binding sites from multiple cloning sites.

(서열번호 69) 및

(서열번호 70). proA(EcoRI/NotI), GAL10:

(서열번호 71) 및

(서열번호 72).The following primers were designed to clone into pESC-His (restrictions are underlined and Kozak sequences are in bold): aspC (BamHI / SaclI), GAL1:

(SEQ ID NO: 69) and

(SEQ ID NO 70). proA (EcoRI / NotI), GAL10:

(SEQ ID NO: 71) and

(SEQ ID NO: 72).

The second codon of the two mature proteins changed from aromatic amino acid to valine due to the introduction of the Kozak sequence. The gene was amplified using pET30Xa / LIC miniprep DNA from the clones described in Examples 1 and 4 as templates. PCR was performed using the following protocol and thermocycler (Eppendorf Master cycler gradient thermocycler) in 50 μl reaction solution: 1.0 μl template, 1.0 μM each primer, 0.4 mM dNTP, 3.5 U Expand High Fidelity Polymerase (Roche) , Indianapolis, Indiana), and 1 × Expand ™ Buffer (with Mg). The thermocycler program used consisted of the following: 29 cycles consisting of a high temperature start of 94 ° C., 5 minutes, then 94 ° C. 30 seconds, 50 ° C. 1 minute, 72 ° C. 2 minutes. After 29 repetitions, the samples were kept at 72 ° C. for 10 minutes and then stored at 4 ° C. This PCR product was purified by 1% TAE-agarose gel separation and recovered using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA).

pESC-His vector DNA (2.7 μg) was digested with BamHI / SalI and gel purified as described above. The aspC PCR product was digested with BamHI / SalI and purified by QIAquick PCR Purification Column. Ligation was performed according to the manufacturer's protocol using the Roche Rapid DNA Ligation Kit. Desalted ligation was electropermeated into 40 μl Electromax DH10B competent cells (Invitrogen) in a 0.2 cm Biorad disposable cuvette using Biorad Gene Pulser II with Pulse Controller Plus according to the manufacturer's instructions. After 1 hour of recovery in 1 ml of SOC medium, transformants were plated in LB medium containing 100 µg / ml ampicillin. Plasmid DNA preparation of clones was performed using the QIAprep Spin Miniprep Kit. Plasmid DNA was screened by restriction digest and sequenced for confirmation using primers designed for this vector (Seqwright).                 

aspC / pESC-His clones were digested with EcoRI and NotI as well as proA PCR products. DNA was purified as described above and ligated as described above. DH10B cells were transformed with two gene constructs and selected by restriction digest and DNA sequencing.

This construct was used to transform S. cerevisiae strain YPH500 using S.c.EasyComp ™ Transformation Kit (Invitrogen). Transformants were plated in SC-His minimal medium (Invitrogen pYES2 manual) containing 2% glucose. Each yeast colony was selected for the presence of the proA and aspC genes by colony PCR using the PCR primers described above. Pelletized cells (2 μl) were suspended in 20 μl of Y-Lysis Buffer (Zymo Research) containing 1 μl Zymolase and heated at 37 ° C. for 10 minutes. 4 μl of this suspension was used for the 50 μl PCR reaction using the PCR reaction mixture and program described above.

5 ml of the culture was grown overnight at 30 ° C., 225 rpm on SC-His + glucose. Cells were slowly adjusted to proliferate on raffinose to minimize delays prior to galactose induction. After propagation for about 12 hours, the absorbance was measured at 600 nm, an appropriate amount of cells were precipitated and resuspended in fresh SC-His medium to OD 0.4. Then the following carbon sources were used: 1% raffinose + 1% glucose, 0.5% glucose + 1.5% raffinose, 2% raffinose and finally 1% raffinose + 2% galactose (for induction).

After about 16 hours of propagation in induction medium, 50 ml culture was divided into 25 ml cultures and the following components were added to only one culture: 1 g / L L-tryptophan, 5 mM sodium phosphate pH 7.1, 1 g / L pyruvic acid Sodium, 1 mM MgCl ₂ (final concentration). Culture medium and cell pellet samples derived from non-induction medium and culture medium and cell pellet samples derived from the culture cultured for 16 hours before being added to the substrate of the monatin pathway were stored as negative controls. In addition, constructs containing only the functional aspC gene (and truncated proA gene) were used as other negative controls. Cells were expanded for a total of 69 hours after induction. Sometimes, yeast cells were induced with lower OD and proliferated only for 4 hours before the addition of tryptophan and pyruvate. However, these monatin substrates appeared to inhibit proliferation and higher OD amounts were more effective.

Cell pellets obtained from the cultures were prepared according to the manufacturer's protocol with 5 ml of YeastBuster ™ + 50 μl THP (Novagen) / g of cells (wet weight) under the addition of a protease inhibitor and Benzonase nuclease as described in the previous examples. Lysate. Cultures and culture extracts were filtered and analyzed by SRM as described in Example 10. As a result of this method, no monatin was detected in the culture sample, indicating that the cells could not separate monatin under these conditions. Under these conditions, proton maneuverability may be insufficient or a general amino acid transfer factor may be saturated with tryptophan. Protein expression was not at levels detectable through SDS-PAGE.

When tryptophan and pyruvate were added to the medium, monatin was temporarily detected in the cell extracts of the cultures with the two functional genes (about 60 μg / ml). However, no monatin was detected in all negative control cell extracts. In vitro analysis of monatin was performed in two replicates with 4.4 mg / ml of total protein (typically about twice the amount used in E. coli cell extracts) using the optimal assay described in Example 6. Other assays were also performed by adding either 32 μg / ml C. Testosterone ProA aldolase or 400 μg / ml AspC aminotransferase to investigate the restriction enzymes in the cell extract. Negative controls were performed with no enzyme or only with AspC aminotransferase (aldol condensation can occur to some extent without enzyme). Positive controls were performed with 16 μg / ml aldolase and 400 μg / ml aminotransferase with partially purified enzyme (30-40%).

In vitro results were analyzed by SRM. As a result of the cell extract analysis, it was confirmed that tryptophan was effectively delivered to cells when added to the medium after induction, showing a second level of tryptophan level compared to cells to which tryptophan was not added. The results of in vitro monatin analysis are shown in Table 6 (values represent ng / ml).

Table 6

Monatine Production by Yeast Cell Extracts

aspC construct + Aldolase + AspC Two gene constructs + Aldolase + AspC Inhibition (glucose medium) 0 888.3 173.5 0 465.2 829 24-hour induction 0 2832.8 642.4 0 1375.6 9146.6 69 hours induction 0 4937.3 340.3 71.9 1652.8 23693.5 69hr + substitution 0 556.9 659.1 21.9 755.6 16688.2 + Control (tablet enzyme) 21853 21853 -Control (without enzyme) 0 254.3 0 254.3

Positive results were obtained with the full length two gene construct cell extracts with or without the addition of a substrate to the growth medium. These results indicate that the enzyme is expressed at about 1% of the total protein present in yeast compared to the positive control. When the cell extracts of the aspC construct (with truncated proA) were analyzed under the addition of aldolase, the amount of monatin produced was significantly higher than when only the cell extracts were analyzed, indicating that the recombinant AspC aminotransferase It represents about 1 to 2% of yeast total protein. Cell extracts of uninduced cultures exhibited a small amount of activity when analyzed under the addition of aldolase due to the presence of native aminotransferases in the cells. When analyzed under the addition of AspC aminotransferase, the activity of extracts derived from uninduced cells increased to the amount of monatin produced by the negative control with AspC (about 200 ng / ml). In contrast, the activity observed in the analysis of the two gene construct cell extracts was further increased with aminotransferase supplementation than with aldolase. Since both genes should be expressed at the same level, it is suggested that the production of monatin can be maximized when the concentration of aminotransferase is higher than that of aldolase, as shown in Example 6.

Example 13

Improvement of Enzyme Process Using Linked Reaction

In theory, if no side reaction or degradation of the substrate or intermediate occurs, the maximum amount of product formed through the enzymatic reaction illustrated in FIG. 1 is directly proportional to the equilibrium constant of each reaction and the concentrations of tryptophan and pyruvate. However, tryptophan is not a highly soluble substrate, and it is observed that the pyruvate concentration is more than 200 mM adversely affects the yield (Example 6).

Ideally, the concentration of monatin is maximized in proportion to the substrate to reduce the separation cost. Physical separation may be performed to remove monatin from the reaction mixture to block the possibility of adverse reactions. The raw material and the catalyst can then be produced again. Because monatin is similar in size, charge, and hydrophobicity to some reagents and intermediates, physical separation will be difficult unless there is a high affinity for monatin (eg, affinity chromatography techniques). However, the monatin reaction can be associated with other reactions that shift the equilibrium of the reaction system to the direction of monatin production. The following illustrates a method of improving the yield of monatin produced from tryptophan or indole-3-pyruvate.

Linked reaction with oxaloacetate decarboxylase (EC 4.1.1.3)

11 illustrates this reaction. In this reaction, tryptophan oxidase and catalase are used to drive the reaction towards indole-3-pyruvate production. Catalase is used in excess so that hydrogen peroxide cannot be used for adverse reactions or enzyme or intermediate damage. Oxygen is regenerated during the catalase reaction. Alternatively, indole-3-pyruvate may be used as the substrate.

Aspartate is used as an amino source in the amination of MP, and aspartate aminotransferase is used. Ideally, aspartate would not be used to reamine indole-3-pyruvate using aminotransferases with less specificity for tryptophan / indole-3-pyruvate reactions compared to MP-monatin reactions. . Oxaloacetate decarboxylase (from Pseudomonas species) may be added to convert oxaloacetate to pyruvate and carbon dioxide. Since CO ₂ is volatile, it is less likely to react with enzymes, reducing or even blocking back reactions. The pyruvate produced at this stage can be usefully used for the aldol condensation reaction. Other decarboxylase enzymes can also be used, and homologues are known to be present in the following microorganisms: Actinobacillus actinomycetem comitans, Aquifex aoricus, Akeoglobus fulgidus, azotobacter vine Randi, Bacterose prazilis, several Bordetella spp., Campylobacter jejuni, chlorobium tepidum, chloroplexus aurantius, enterococcus peculis, fusobacterium nucleatum, Krebsiella nu Monia, Legionella pneumophila, Magnetococcus MC-1, Manheimia haemolytica, Methylovacillus plagelatus KT, Pasteurella multocida Pm70, Petrotoga myothema, Popiromonas gingivalis, several Pseudomonas species, some Pyrococcus species, Rhodococcus species, some Salmonella species, some Streptococcus species, Thermochromatium tepidum, Thermomotoga marittima, Treponema parley And several Vibrio species.

Assays of tryptophan aminotransferase were described in E. coli aspartate aminotransferase (AspC), E. coli tyrosine aminotransferase (TyrB), L. major major substrate aminotransferase (BSAT) and as described in Example 1. Two commercialized porcine glutamate-oxaloacetate aminotransferases were performed. Oxaloacetate and alpha-ketoglutarate were tested as amino acceptors. Activity using monatin (Example 7): An enzyme having the highest specificity for the monatin aminotransferase reaction was determined by comparing the ratio of the activity using tryptophan. As a result, it was found that the enzyme having the highest specificity for the monatin reaction: tryptophan reaction was porcine II-A glutamate-oxaloacetate aminotransferase GOAT (Sigma G7005). This specificity was independent of the type of amino receptor used. Therefore, this enzyme was used for the linkage reaction with oxaloacetate decarboxylase.

Typical reactions starting with indole-3-pyruvate include 50 mM Tris-Cl pH 7.3, 6 mM indole-3-pyruvate, 6 mM sodium pyruvate, 6 mM aspartate, 0.05 mM PLP, 3 mM potassium phosphate, 3 mM MgCl ₂ , 25 μg / ml aminotransferase, 50 μg / ml C. testosterone ProA aldolase and 3 units / ml decarboxylase (Sigma O4878) were used. The reaction was carried out at 26 ° C. for 1 hour. In some cases the decarboxylase was excluded or alpha-ketoglutarate was used in place of aspartate (as negative control). The aminotransferases described above were used in place of GOAT to confirm conventional specificity experiments. Samples were filtered and analyzed by LC / MS as described in Example 10. As a result, it was confirmed that the GOAT enzyme produced the maximum amount of monatin per mg of protein and the minimum amount of tryptophan was produced as a by-product. It was also 2 to 3 times advantageous when decarboxylase was added. E. coli AspC enzyme also produced a higher amount of monatin than other aminotransferases.

Monatin production is performed by 1) adding indole-pyruvate, pyruvate and aspartate periodically (every 30 minutes to 1 hour), 2) carrying out the reaction in an anaerobic or degassed buffer, and 3) carrying out the reaction. Proceed overnight and 4) increased by using freshly prepared decarboxylase that was not freeze-thawed multiple times. Decarboxylase was inhibited when the pyruvate concentration was greater than 12 mM. When the concentration of indole-3-pyruvate was higher than 4 mM, the side reaction with indole-3-pyruvate was promoted. The amount of indole-3-pyruvate used in this reaction can also be increased if the amount of aldolase is increased. Large amounts of phosphate (50 mM) and aspartate (50 mM) were observed to inhibit this decarboxylase. The amount of decarboxylase could be reduced to 0.5 U / ml without a decrease in monatin production in the 1 hour reaction. The production of monatin also increased when the temperature was increased from 26 ° C to 30 ° C and from 30 ° C to 37 ° C. However, the side reaction of indole-3-pyruvate was also promoted at 37 ° C. Increasing the pH from 7.3 to 7.3 increased the monatin production and was relatively stable between pH 7.3 and 8.3.

Typical reactions initiated from tryptophan include 50 mM Tris-Cl pH 7.3, 20 mM tryptophan, 6 mM aspartate, 6 mM sodium pyruvate, 0.05 mM PLP, 3 mM potassium phosphate, 3 mM MgCl ₂ , 25 μg / ml aminotransferase, 50 μg / ml C Testosterone ProA aldolase, 4 units / ml decarboxylase, 5-200 mU / ml L-amino acid oxidase (Sigma A-2805), 168 U / ml catalase (Sigma C-3515) and 0.008 mg FAD It was. The reaction was run at 30 ° C. for 30 minutes. The reaction was improved upon addition of decarboxylase. The maximum amount of monatin was produced when 50mU / ml oxidase was used. Similar improvements were observed when indole-3-pyruvate was used as the substrate. In addition, 1) tryptophan concentration is low (i.e., less than K _{m of} aminotransferase and cannot compete with MP in the active site), and 2) oxidase: ratio of aldolase and aminotransferase indole-3- The production of monatin was increased when pyruvate was maintained at levels that could not accumulate.

Regardless of initiation from indole-3-pyruvate or tryptophan, the amount of monatin produced in the assay with incubation time of 1 to 2 hours increased when the amount of all enzymes was used 2 to 4 times while the enzyme ratio remained the same. It was. Whatever substrate was used, monatin at a concentration of about 1 mg / ml was obtained. The amount of tryptophan produced when starting from indole-pyruvate is generally less than 20% of the product amount, demonstrating the utility of the linkage reaction. Further optimization and control of intermediate and side reaction concentrations can significantly improve productivity and yield.

Linked reaction with lysine epsilon aminotransferase (EC 2.6.1.36)

Lysine epsilon aminotransferase (L-lysine 6-aminotransferase) is found in several organisms such as Rhodococcus, Mycobacterium, Streptomyces, Nocadia, Flavobacterium, Candida Utility, Streptomyces and the like. . As a first step, organisms used to produce some beta-lactam antibiotics are useful (Rius and Demain, J. Microbiol. Biotech., 7: 95-100, 1997). This enzyme converts lysine to L-2-aminoadipate 6-semialdehyde (allysine) by using PLP mediated aminotranslation of C6 of lysine with alpha-keto glutarate as an amino acceptor. Allicin is unstable and spontaneously undergoes intracellular dehydration to form the cyclic molecule 1-piperidine 6-carboxylate. This effectively suppresses any adverse reactions from occurring. This scheme is shown in FIG. 12. As an alternative enzyme lysine-pyruvate 6-aminotransferase (EC 2.6.1.71) can also be used.

Typical reactions include 50 mM Tris-Cl pH 7.3, 20 mM indole-3-pyruvate, 0.05 mM PLP, 6 mM potassium phosphate pH 8, 2 to 50 mM sodium pyruvate, 1.5 mM MgCl ₂ , 50 mM lysine, 100 μg aminotransferase ( Lysine epsilon aminotransferase LAT-101, BioCatalytics Pasadena, Calif.) And 200 μg C. Testosterone ProA aldolase were used. As the concentration of pyruvate increased, the production of monatin also increased. Under these reaction conditions (50 mM pyruvate), the maximum amount was less than 10 times less than that observed during the linkage reaction using oxaloacetate decarboxylase (about 0.1 mg / ml). [M + H] ⁺ = 293 The peak eluted at the expected time of monatin and contained several fragments as observed in other enzymatic reactions by mass spectra. The second peak, at an accurate mass / charge ratio of 293, eluted slightly faster than the time normally observed for S, S monatin observed in Example 6, which may suggest the presence of other isomers of monatin. The amount of tryptophan produced by this enzyme was very small. However, it is possible to have some activity on pyruvate (producing alanine as a byproduct). This enzyme is also known to be unstable. Thus, it may be improved by conducting directed evolution experiments to increase stability, reduce activity on pyruvate and increase activity on MP. This reaction can also be linked to L-amino acid oxidase / catalase as described above.

Other linkage reaction

Another linkage reaction that can increase the yield of monatin from tryptophan or indole-pyruvate is shown in FIG. 13. Formate dehydrogenase (EC 1.2.1.2 or 1.2.1.43) is a common enzyme. Some formate dehydrogenases require NADH, while other dehydrogenases require NADPH. Glutamic acid dehydrogenase catalyzed the interconversion between monatin precursor and monatin in the previous example using ammonium based buffer. The presence of ammonium formate and formate dehydrogenase is an efficient system for the regeneration of cofactors, and carbon dioxide production is an effective way to reduce the rate of reverse reactions (Bommarius et al., Biocatalysis 10:37, 1994 and Galkin et al. Appl. Environ. Microbiol. 63: 4651-6, 1997). In addition, large amounts of ammonium formate can be dissolved in the reaction buffer. The yield of monatin produced by the glutamic acid dehydrogenase reaction (or similar reductive amination) can be improved by the addition of formate dehydrogenase and ammonium formate.

Other methods of inducing equilibrium in the direction of monatin production may be used. For example, aminopropane is used as the amino acid source in the conversion of MP to monatin and with omega-amino acid aminotransferases (EC 2.6.1.18) as described in US Pat. Nos. 5,360,724 and 5,300,437. If so, one of the resulting products would be acetone, a product that is more volatile than the substrate aminopropane. To evaporate this acetone, the temperature can be periodically raised for a short time. This reduces equilibrium. The boiling point of acetone is 47 ° C, which is less likely to degrade the intermediate if used for a short time. Most aminotransferases that are active against alpha-ketoglutarate are also active with monatin precursors. Similarly, if glyoxylate / aromatic acid aminotransferase is used with glycine as the amino feeder, glyoxylate is produced, which is relatively unstable and has a markedly reduced boiling point compared to glycine.

Example 14

Recombinant Expression

Commercially available enzymes, cDNA and amino acid sequences and enzymes and sequences disclosed herein, such as SEQ ID NOs: 11 and 12, as well as variants, polymorphs, mutants, fragments and fusions thereof, can be used as standard laboratory techniques. Any protein such as can be expressed and purified. Those skilled in the art will appreciate that such enzymes and fragments thereof may be recombinantly produced in any cell or organism of interest and purified prior to use, such as before production of SEQ ID NO: 12 and derivatives thereof.

Methods of producing recombinant proteins are known in the art. In other words, the scope of the present invention includes recombinant expression of any protein such as enzyme or fragment thereof. See, eg, US Pat. No. 5,342,764 to Johnson et al .; US Patent No. 5,846,819 to Pausch et al .; See US Pat. No. 5,876,969 (Fleer et al.) And Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989, Ch. 17).

Briefly, a portion, full-length or modified cDNA sequence encoding a protein or peptide is ligated to an expression vector such as a bacterial or eukaryotic expression vector. Placing the promoter upstream of this cDNA sequence allows the production of proteins and / or peptides. Examples of promoters include lac, trp, tac, trc, main operator and promoter regions of phage lambda, regulatory regions of fd envelope proteins, early and late promoters of SV40, polyomas, adenoviruses, retroviruses, baculoviruses and simian Promoter derived from virus, promoter of 3-phosphoglycerate kinase, promoter of yeast acid kinase, promoter of yeast alpha matching factor and combinations thereof, but is not limited thereto.

Suitable vectors for the production of native natural proteins include pKC30 (Shimatake and Rosenberg, 1981, Nature 292: 128), pKK177-3 (Amann and Brosius, 1985, Gene 40: 183) and pET-3 (Studier and Moffatt, 1986). , J. Mol. Biol. 189: 113). DNA sequences can be transferred to other cloning media such as other plasmids, bacteriophages, cosmids, animal viruses and yeast synthetic chromosomes (YAC) and the like (Burke et al., 1987, Science 236: 806-12). These vectors are somatic cells. And simple or complex organisms such as bacteria, fungi (Timberlake and Marshall, 1989, Science 244: 1313-7), invertebrates, plants (Gasser and Fraley, 1989, Science 244: 1993) and mammals (Pursel et al., 1989, Science 244: 1281-8), and the like, which are mutated by the introduction of heterologous cDNA.

For expression in mammalian cells, the cDNA sequence is ligated to heterologous promoters such as the Simian virus SV40, a promoter in the pSV2 vector (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072-6), and the like. Transduction into cells, such as monkey COS-1 cells (Gluzman, 1981, Cell 23: 175-82), can achieve transient or long-term expression. Stable integration of chimeric genes can be achieved by biochemical selection markers such as neomycin (Southern and Berg, 1982, J. Mol. Appl. Genet. 1: 327-41) and mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072-6).

Transfer of DNA to human or other mammalian cells is a common technique. Vectors are introduced into receptor cells as pure DNA (transfection) by the following methods: precipitation with calcium phosphate (Graham and vander Eb, 1973, Virology 52: 466), precipitation with strontium phosphate (Brash et al. , 1987, Mol. Cell Biol. 7: 2013), electroporation (Neumann et al., 1982, EMBO J. 1: 841), lipofection (Felgner et al., 1987, Proc. Natl. Acad. Sci USA 84: 7413), DEAE dextran (McCuthan et al., 1968, J. Natl. Cancer Inst. 41: 351), microinjection (Mueller et al., 1978, Cell 15: 579), protoplast fusion (Schafenr, 1980 , Proc. Natl. Acad. Sci. USA 77: 2163-7), or pellet gun (Klein et al., 1987, Nature 327: 70). Alternatively, cDNA can be introduced through infection with viral vectors, examples of which include retroviruses (Bernstein et al., 1985, Gen. Engrg. 7: 235), adenoviruses (Ahmad et al., 1986, J. Virol. 57: 267) or herpes (Spaete et al., 1982, Cell 30: 295).

When reviewing as many embodiments as possible to which the principles of the invention may be applied, it will be apparent that the illustrated embodiments are merely some specific examples of the invention and should not be considered as limiting the scope of the invention. The scope of the invention should be defined according to the following claims. Accordingly, all inventions falling within the scope and spirit of this claim are claimed as applicant's inventions.

                         SEQUENCE LISTING <110> Cargill, Incorporated <120> Polypeptides and Biosynthetic Pathways <130> 6682-65741 <150> 60 / 374,831 <151> 2002-04-23 <160> 74 <170> PatentIn version 3.1 <210> 1 <211> 1170 <212> DNA <213> Sinorhizobium meliloti <400> 1 atgttcgacg ccctcgcccg ccaagccgac gatcccttgc ttttcctgat cggcctgttc 60 aggaaggatg agcgccccgg aaaggtcgat ctcggcgtag gagtctatcg cgacgagacc 120 ggacgcacgc cgatcttccg ggccgtcaag gcggcggaaa agcggcttct cgaaacacag 180 gacagcaagg cctatatcgg ccccgaaggg gacctcgtct ttctcgatcg gctctgggaa 240 ctcgtcggcg gcgacacgat cgagcggagc catgttgcgg gcgtccagac gcccggcggc 300 tccggcgcgc tccgtttggc ggcggacctc atcgcccgca tgggcggccg aggcatctgg 360 ctcgggctgc cgagctggcc gaaccacgcg ccgatcttca aggcggccgg gctcgatatc 420 gccacctacg acttcttcga cattccgtcg cagtcggtca tcttcgataa tctggtgagc 480 gcgctggaag gcgccgcatc cggcgatgcg gtgctgctgc atgcaagctg ccacaacccg 540 accggcggcg tcctgagcga agcacaatgg atggagatcg ccgcgctggt ggccgagcgc 600 ggcctgctgc cgctcgtcga tctcgcctat caggggttcg gccgcggcct cgaccaggat 660 gtcgcgggcc tccggcatct tctcggcgtg gtcccggaag cgctcgtcgc ggtttcctgc 720 tcgaagtcct tcgggcttta tcgcgagcgc gcgggcgcga tcttcgcgcg gaccagctcg 780 actgcctcgg cggacagggt gcgctcaaac ctcgcgggcc tcgcacgcac cagctattcc 840 atgccgccgg atcacggcgc agccgtcgtg cggacgatcc ttgacgaccc ggaactcagg 900 cgcgactgga cggaggagct cgagacgatg cggctcagga tgacgggcct ccggcggtcg 960 cttgccgagg gactccgcac ccgctggcag agcctcggcg cagtcgccga tcaggagggc 1020 atgttctcca tgctgccgct ttccgaagcg gaggttatgc ggctcaggac cgagcacggc 1080 atctatatgc cggcatccgg ccgcatcaac atcgccgggc tgaagacggc ggaagccgcc 1140 gagattgccg gcaagttcac cagtctctga 1170 <210> 2 <211> 389 <212> PRT <213> Sinorhizobium meliloti <400> 2 Met Phe Asp Ala Leu Ala Arg Gln Ala Asp Asp Pro Leu Leu Phe Leu 1 5 10 15 Ile Gly Leu Phe Arg Lys Asp Glu Arg Pro Gly Lys Val Asp Leu Gly             20 25 30 Val Gly Val Tyr Arg Asp Glu Thr Gly Arg Thr Pro Ile Phe Arg Ala         35 40 45 Val Lys Ala Ala Glu Lys Arg Leu Leu Glu Thr Gln Asp Ser Lys Ala     50 55 60 Tyr Ile Gly Pro Glu Gly Asp Leu Val Phe Leu Asp Arg Leu Trp Glu 65 70 75 80 Leu Val Gly Gly Asp Thr Ile Glu Arg Ser His Val Ala Gly Val Gln                 85 90 95 Thr Pro Gly Gly Ser Gly Ala Leu Arg Leu Ala Ala Asp Leu Ile Ala             100 105 110 Arg Met Gly Gly Arg Gly Ile Trp Leu Gly Leu Pro Ser Trp Pro Asn         115 120 125 His Ala Pro Ile Phe Lys Ala Ala Gly Leu Asp Ile Ala Thr Tyr Asp     130 135 140 Phe Phe Asp Ile Pro Ser Gln Ser Val Ile Phe Asp Asn Leu Val Ser 145 150 155 160 Ala Leu Glu Gly Ala Ala Ser Gly Asp Ala Val Leu Leu His Ala Ser                 165 170 175 Cys His Asn Pro Thr Gly Gly Val Leu Ser Glu Ala Gln Trp Met Glu             180 185 190 Ile Ala Ala Leu Val Ala Glu Arg Gly Leu Leu Pro Leu Val Asp Leu         195 200 205 Ala Tyr Gln Gly Phe Gly Arg Gly Leu Asp Gln Asp Val Ala Gly Leu     210 215 220 Arg His Leu Leu Gly Val Val Pro Glu Ala Leu Val Ala Val Ser Cys 225 230 235 240 Ser Lys Ser Phe Gly Leu Tyr Arg Glu Arg Ala Gly Ala Ile Phe Ala                 245 250 255 Arg Thr Ser Ser Thr Ala Ser Ala Asp Arg Val Arg Ser Asn Leu Ala             260 265 270 Gly Leu Ala Arg Thr Ser Tyr Ser Met Pro Pro Asp His Gly Ala Ala         275 280 285 Val Val Arg Thr Ile Leu Asp Asp Pro Glu Leu Arg Arg Asp Trp Thr     290 295 300 Glu Glu Leu Glu Thr Met Arg Leu Arg Met Thr Gly Leu Arg Arg Ser 305 310 315 320 Leu Ala Glu Gly Leu Arg Thr Arg Trp Gln Ser Leu Gly Ala Val Ala                 325 330 335 Asp Gln Glu Gly Met Phe Ser Met Leu Pro Leu Ser Glu Ala Glu Val             340 345 350 Met Arg Leu Arg Thr Glu His Gly Ile Tyr Met Pro Ala Ser Gly Arg         355 360 365 Ile Asn Ile Ala Gly Leu Lys Thr Ala Glu Ala Ala Glu Ile Ala Gly     370 375 380 Lys Phe Thr Ser Leu 385 <210> 3 <211> 1260 <212> DNA <213> Rhodobacter sphaeroides <400> 3 atgcgctcta cgacggctcc tggtccgagt ggggcatgta tgacgatctc aaggtcgcga 60 aaggatgacg aaggaatgct gaccgccctg aagccgcagc ccgcggacaa gatcctgcaa 120 ctgatccaga tgttccgcga ggatgcgcgc gcggacaaga tcgatctggg cgtgggcgtc 180 tacaaggacc cgaccgggct caccccggtc atgcgggccg tgaaggcggc cgagaagcgg 240 ctctgggagg tcgagaccac caagacctac accggccttg ccgacgagcc ggcctacaat 300 gccgcgatgg cgaagctgat cctcgcgggc gcggtcccgg ccgaccgggt ggcctcggtc 360 gccacccccg gcggcacggg cgcggtgcgt caggcgctcg agctgatccg catggcctcg 420 cccgaggcca ccgtctggat ctcgaacccg acctggccga accatctgtc gatcgtgaaa 480 tatctcggca tcccgatgcg ggaataccgc tatttcgacg ccgagaccgg cgccgtcgat 540 gccgagggca tgatggagga tctggcccag gtgaaggcgg gcgacgtggt gctgctgcac 600 ggctgctgcc acaacccgac cggcgccaac ccgaacccgg tgcagtggct ggccatctgc 660 gagagcctgg cccggacagg cgcggtgccg ctgatcgacc tcgcctatca gggcttcggc 720 gacgggctcg agatggatgc ggcggcgacg cggcttctgg ccaccagact gcccgaggtg 780 ctgatcgcgg cctcctgctc gaagaacttc ggcatctacc gcgagcgcac gggcatcctg 840 atcgccatcg gcgaggcggc gggccggggc acggtgcagg ccaacctcaa cttcctgaac 900 cggcagaact actccttccc gccggaccat ggcgcgcggc tcgtgaccat gatcctcgag 960 gacgagacgc tgagcgccga ctggaaggcg gaactcgagg aggtgcggct caacatgctg 1020 acactgcgcc gccagcttgc cgatgcgctg caggccgaga ccggctcgaa ccgcttcggc 1080 ttcgtggccg agcatcgcgg catgttctcg cgcctcggga tcacgcccgc cgaggtggag 1140 cggctgcgga ccgagcacgg ggtctacatg gtgggcgatt cgcggctgaa catcgcgggg 1200 ctgaaccgga cgaccgtgcc ggtgctggcg cgcgcggtgg ccaaggtgct gcgcggctga 1260 <210> 4 <211> 419 <212> PRT <213> Rhodobacter sphaeroides <400> 4 Met Arg Ser Thr Thr Ala Pro Gly Pro Ser Gly Ala Cys Met Thr Ile 1 5 10 15 Ser Arg Ser Arg Lys Asp Asp Glu Gly Met Leu Thr Ala Leu Lys Pro             20 25 30 Gln Pro Ala Asp Lys Ile Leu Gln Leu Ile Gln Met Phe Arg Glu Asp         35 40 45 Ala Arg Ala Asp Lys Ile Asp Leu Gly Val Gly Val Tyr Lys Asp Pro     50 55 60 Thr Gly Leu Thr Pro Val Met Arg Ala Val Lys Ala Ala Glu Lys Arg 65 70 75 80 Leu Trp Glu Val Glu Thr Thr Lys Thr Tyr Thr Gly Leu Ala Asp Glu                 85 90 95 Pro Ala Tyr Asn Ala Ala Met Ala Lys Leu Ile Leu Ala Gly Ala Val             100 105 110 Pro Ala Asp Arg Val Ala Ser Val Ala Thr Pro Gly Gly Thr Gly Ala         115 120 125 Val Arg Gln Ala Leu Glu Leu Ile Arg Met Ala Ser Pro Glu Ala Thr     130 135 140 Val Trp Ile Ser Asn Pro Thr Trp Pro Asn His Leu Ser Ile Val Lys 145 150 155 160 Tyr Leu Gly Ile Pro Met Arg Glu Tyr Arg Tyr Phe Asp Ala Glu Thr                 165 170 175 Gly Ala Val Asp Ala Glu Gly Met Met Glu Asp Leu Ala Gln Val Lys             180 185 190 Ala Gly Asp Val Val Leu Leu His Gly Cys Cys His Asn Pro Thr Gly         195 200 205 Ala Asn Pro Asn Pro Val Gln Trp Leu Ala Ile Cys Glu Ser Leu Ala     210 215 220 Arg Thr Gly Ala Val Pro Leu Ile Asp Leu Ala Tyr Gln Gly Phe Gly 225 230 235 240 Asp Gly Leu Glu Met Asp Ala Ala Ala Thr Arg Leu Leu Ala Thr Arg                 245 250 255 Leu Pro Glu Val Leu Ile Ala Ala Ser Cys Ser Lys Asn Phe Gly Ile             260 265 270 Tyr Arg Glu Arg Thr Gly Ile Leu Ile Ala Ile Gly Glu Ala Ala Gly         275 280 285 Arg Gly Thr Val Gln Ala Asn Leu Asn Phe Leu Asn Arg Gln Asn Tyr     290 295 300 Ser Phe Pro Pro Asp His Gly Ala Arg Leu Val Thr Met Ile Leu Glu 305 310 315 320 Asp Glu Thr Leu Ser Ala Asp Trp Lys Ala Glu Leu Glu Glu Val Arg                 325 330 335 Leu Asn Met Leu Thr Leu Arg Arg Gln Leu Ala Asp Ala Leu Gln Ala             340 345 350 Glu Thr Gly Ser Asn Arg Phe Gly Phe Val Ala Glu His Arg Gly Met         355 360 365 Phe Ser Arg Leu Gly Ile Thr Pro Ala Glu Val Glu Arg Leu Arg Thr     370 375 380 Glu His Gly Val Tyr Met Val Gly Asp Ser Arg Leu Asn Ile Ala Gly 385 390 395 400 Leu Asn Arg Thr Thr Val Pro Val Leu Ala Arg Ala Val Ala Lys Val                 405 410 415 Leu Arg Gly              <210> 5 <211> 1260 <212> DNA <213> Rhodobacter sphaeroides <400> 5 atgcgctcta cgacggctcc tggtccgagt ggggcatgta tgacgatctc aaggtcgcga 60 aaggatgacg aaggaatgct gaccgccctg aagccgcagc ccgcggacaa gatcctgcaa 120 ctgatccaga tgttccgcga ggatgcgcgc gcggacaaga tcgatctggg cgtgggcgtc 180 tacaaggacc cgaccgggct caccccggtc atgcgggccg tgaaggccgc cgagaagcgg 240 ctctgggagg tcgagaccac caagacctac accggccttg ccggcgagcc cgcctacaat 300 gccgcgatgg cgaagctgat cctcgcaggc gcggtcccgg ccgaccgggt ggcctcggtc 360 gccacccccg gcggcacggg cgcggtgcgt caggcgctcg agctgatccg catggcctcg 420 cccgaggcca ctgtctggat ctcgaacccg acctggccga accatctgtc gatcgtgaaa 480 tatctcggca tcccgatgcg ggaataccgc tatttcgacg ccgagaccgg cgccgtcgat 540 gccgagggct tgatggagga tctggcccag gtgaaggcgg gcgacgtggt gctgctgcac 600 ggctgctgcc acaacccgac cggcgccaac ccgaacccgg tgcagtggct ggccgtctgc 660 gagagcctgg cccggacagg cgcggtgccg ctgatcgacc tcgcctatca gggcttcggc 720 gacgggctcg agatggatgc ggcggcgacg cggcttctgg ccaccagact gcccgaggtg 780 ctgatcgcgg cctcctgctc gaagaacttc ggcatctacc gcgagcgaac gggcatcctg 840 atcgccatcg gcgaggcggc gggccggggc acggtgcagg ccaacctcaa cttcctgaac 900 cggcagaact actccttccc gccggaccat ggcgcgcggc tcgtgaccat gatcctcgag 960 gacgagacgc tgagcgccga ctggaaggcg gaactcgagg aggtgcggct caacatgctg 1020 acgctgcgcc gccagcttgc cgatgcgctg caggccgaga ccggctcgaa ccgcttcggc 1080 ttcgtggccg agcatcgcgg catgttctcg cgcctcggga tcacgcccgc cgaggtggag 1140 cggctgcgga ccgagcacgg ggtctacatg gtgggcgatt cgcggctgaa catcgcgggg 1200 ctgaaccgga cgaccgtgcc ggtgctggcg cgcgcggtgg ccaaggtgct gcgcggctga 1260 <210> 6 <211> 419 <212> PRT <213> Rhodobacter sphaeroides <400> 6 Met Arg Ser Thr Thr Ala Pro Gly Pro Ser Gly Ala Cys Met Thr Ile 1 5 10 15 Ser Arg Ser Arg Lys Asp Asp Glu Gly Met Leu Thr Ala Leu Lys Pro             20 25 30 Gln Pro Ala Asp Lys Ile Leu Gln Leu Ile Gln Met Phe Arg Glu Asp         35 40 45 Ala Arg Ala Asp Lys Ile Asp Leu Gly Val Gly Val Tyr Lys Asp Pro     50 55 60 Thr Gly Leu Thr Pro Val Met Arg Ala Val Lys Ala Ala Glu Lys Arg 65 70 75 80 Leu Trp Glu Val Glu Thr Thr Lys Thr Tyr Thr Gly Leu Ala Gly Glu                 85 90 95 Pro Ala Tyr Asn Ala Ala Met Ala Lys Leu Ile Leu Ala Gly Ala Val             100 105 110 Pro Ala Asp Arg Val Ala Ser Val Ala Thr Pro Gly Gly Thr Gly Ala         115 120 125 Val Arg Gln Ala Leu Glu Leu Ile Arg Met Ala Ser Pro Glu Ala Thr     130 135 140 Val Trp Ile Ser Asn Pro Thr Trp Pro Asn His Leu Ser Ile Val Lys 145 150 155 160 Tyr Leu Gly Ile Pro Met Arg Glu Tyr Arg Tyr Phe Asp Ala Glu Thr                 165 170 175 Gly Ala Val Asp Ala Glu Gly Leu Met Glu Asp Leu Ala Gln Val Lys             180 185 190 Ala Gly Asp Val Val Leu Leu His Gly Cys Cys His Asn Pro Thr Gly         195 200 205 Ala Asn Pro Asn Pro Val Gln Trp Leu Ala Val Cys Glu Ser Leu Ala     210 215 220 Arg Thr Gly Ala Val Pro Leu Ile Asp Leu Ala Tyr Gln Gly Phe Gly 225 230 235 240 Asp Gly Leu Glu Met Asp Ala Ala Ala Thr Arg Leu Leu Ala Thr Arg                 245 250 255 Leu Pro Glu Val Leu Ile Ala Ala Ser Cys Ser Lys Asn Phe Gly Ile             260 265 270 Tyr Arg Glu Arg Thr Gly Ile Leu Ile Ala Ile Gly Glu Ala Ala Gly         275 280 285 Arg Gly Thr Val Gln Ala Asn Leu Asn Phe Leu Asn Arg Gln Asn Tyr     290 295 300 Ser Phe Pro Pro Asp His Gly Ala Arg Leu Val Thr Met Ile Leu Glu 305 310 315 320 Asp Glu Thr Leu Ser Ala Asp Trp Lys Ala Glu Leu Glu Glu Val Arg                 325 330 335 Leu Asn Met Leu Thr Leu Arg Arg Gln Leu Ala Asp Ala Leu Gln Ala             340 345 350 Glu Thr Gly Ser Asn Arg Phe Gly Phe Val Ala Glu His Arg Gly Met         355 360 365 Phe Ser Arg Leu Gly Ile Thr Pro Ala Glu Val Glu Arg Leu Arg Thr     370 375 380 Glu His Gly Val Tyr Met Val Gly Asp Ser Arg Leu Asn Ile Ala Gly 385 390 395 400 Leu Asn Arg Thr Thr Val Pro Val Leu Ala Arg Ala Val Ala Lys Val                 405 410 415 Leu Arg Gly              <210> 7 <211> 1239 <212> DNA <213> Leishmania major <400> 7 atgtccatgc aggcggccat gaccacggcg gagcgctggc agaagattca ggcacaagct 60 cccgatgtca tcttcgatct cgcaaaacgc gccgccgctg ccaagggccc caaggccaac 120 ctcgtcattg gtgcctaccg cgacgagcag ggccgtccct atccgctacg cgtggtccgc 180 aaggctgagc agcttctctt ggacatgaat ctcgactacg agtacctacc catctcgggc 240 taccagccct tcatcgatga ggcggtaaag attatctacg gcaataccgt cgagctggag 300 aacctggttg cggtgcagac gctgagcggg accggtgctg tctctctcgg ggcgaagctg 360 ctgactcgcg tcttcgacgc tgagacgacg cccatctacc tttccgaccc cacgtggccc 420 aaccactacg gcgtcgtgaa ggctgctggc tggaagaaca tctgcacgta cgcctactac 480 gaccccaaga cggtcagcct gaatttcgag ggcatgaaga aagacattct ggcggcgccg 540 gacggctccg tgttcattct gcaccagtgc gcgcacaacc ccaccggcgt ggacccgtcg 600 caggagcagt ggaacgagat cgcgtcactg atgctggcca agcaccatca ggtgttcttc 660 gactccgcct accaaggcta tgcgagcggc agcctcgaca cggacgcgta tgctgcccgc 720 ctgtttgccc gccgcggcat cgaggtactg ctggcgcagt cgttctccaa gaacatgggc 780 ttgtacagcg agcgtgcagg cacgctgtcg ctgctcctca aggacaagac gaagcgcgcg 840 gatgtaaaga gcgtgatgga ttcgctgatc cgtgaggagt acacgtgccc cccagcccac 900 ggtgcccgct tagcccacct aatcctgagc aacaacgaac tgcgaaagga gtgggaggca 960 gagctatcag ccatggcaga gcgcatccgt acgatgcgcc gcaccgtgta cgacgagctg 1020 ctgcgcctgc agacgcccgg gagctgggaa catgtcatta accagattgg catgttttcc 1080 ttcctcgggc tgtcaaaggc gcagtgcgaa tactgccaaa accacaacat cttcatcaca 1140 gtgtcgggcc gcgctaacat ggcaggtctg acgcatgaga cggcgctgat gctagcacag 1200 acgatcaacg atgctgtgcg caatgtgaat cgtgagtga 1239 <210> 8 <211> 412 <212> PRT <213> Leishmania major <400> 8 Met Ser Met Gln Ala Ala Met Thr Thr Ala Glu Arg Trp Gln Lys Ile 1 5 10 15 Gln Ala Gln Ala Pro Asp Val Ile Phe Asp Leu Ala Lys Arg Ala Ala             20 25 30 Ala Ala Lys Gly Pro Lys Ala Asn Leu Val Ile Gly Ala Tyr Arg Asp         35 40 45 Glu Gln Gly Arg Pro Tyr Pro Leu Arg Val Val Arg Lys Ala Glu Gln     50 55 60 Leu Leu Leu Asp Met Asn Leu Asp Tyr Glu Tyr Leu Pro Ile Ser Gly 65 70 75 80 Tyr Gln Pro Phe Ile Asp Glu Ala Val Lys Ile Ile Tyr Gly Asn Thr                 85 90 95 Val Glu Leu Glu Asn Leu Val Ala Val Gln Thr Leu Ser Gly Thr Gly             100 105 110 Ala Val Ser Leu Gly Ala Lys Leu Leu Thr Arg Val Phe Asp Ala Glu         115 120 125 Thr Thr Pro Ile Tyr Leu Ser Asp Pro Thr Trp Pro Asn His Tyr Gly     130 135 140 Val Val Lys Ala Ala Gly Trp Lys Asn Ile Cys Thr Tyr Ala Tyr Tyr 145 150 155 160 Asp Pro Lys Thr Val Ser Leu Asn Phe Glu Gly Met Lys Lys Asp Ile                 165 170 175 Leu Ala Ala Pro Asp Gly Ser Val Phe Ile Leu His Gln Cys Ala His             180 185 190 Asn Pro Thr Gly Val Asp Pro Ser Gln Glu Gln Trp Asn Glu Ile Ala         195 200 205 Ser Leu Met Leu Ala Lys His His Gln Val Phe Phe Asp Ser Ala Tyr     210 215 220 Gln Gly Tyr Ala Ser Gly Ser Leu Asp Thr Asp Ala Tyr Ala Ala Arg 225 230 235 240 Leu Phe Ala Arg Arg Gly Ile Glu Val Leu Leu Ala Gln Ser Phe Ser                 245 250 255 Lys Asn Met Gly Leu Tyr Ser Glu Arg Ala Gly Thr Leu Ser Leu Leu             260 265 270 Leu Lys Asp Lys Thr Lys Arg Ala Asp Val Lys Ser Val Met Asp Ser         275 280 285 Leu Ile Arg Glu Glu Tyr Thr Cys Pro Pro Ala His Gly Ala Arg Leu     290 295 300 Ala His Leu Ile Leu Ser Asn Asn Glu Leu Arg Lys Glu Trp Glu Ala 305 310 315 320 Glu Leu Ser Ala Met Ala Glu Arg Ile Arg Thr Met Arg Arg Thr Val                 325 330 335 Tyr Asp Glu Leu Leu Arg Leu Gln Thr Pro Gly Ser Trp Glu His Val             340 345 350 Ile Asn Gln Ile Gly Met Phe Ser Phe Leu Gly Leu Ser Lys Ala Gln         355 360 365 Cys Glu Tyr Cys Gln Asn His Asn Ile Phe Ile Thr Val Ser Gly Arg     370 375 380 Ala Asn Met Ala Gly Leu Thr His Glu Thr Ala Leu Met Leu Ala Gln 385 390 395 400 Thr Ile Asn Asp Ala Val Arg Asn Val Asn Arg Glu                 405 410 <210> 9 <211> 1182 <212> DNA <213> Bacillus subtilis <400> 9 atggaacatt tgctgaatcc gaaagcaaga gagatcgaaa tttcaggaat acgcaaattc 60 tcgaatcttg tagcccaaca cgaagacgtc atttcactta caatcggcca gcctgatttt 120 ttcacaccgc atcatgtgaa agctgccgca aaaaaagcca ttgatgaaaa cgtgacgtca 180 tatactccga atgccggcta cctggagctg agacaagctg tgcagcttta tatgaagaaa 240 aaagcggatt tcaactatga tgctgaatct gaaattatca tcacaacagg cgcaagccaa 300 gccattgatg ctgcattccg gacgatttta tctcccggtg atgaagtcat tatgccaggg 360 cctatttatc cgggctatga acctattatc aatttgtgcg gggccaagcc tgtcattgtt 420 gatactacgt cacacggctt taagcttacc gcccggctga ttgaagatgc tctgacaccc 480 aacaccaagt gtgtcgtgct tccttatccg tcaaacccta ccggcgtgac tttatctgaa 540 gaagaactga aaagcatcgc agctctctta aaaggcagaa atgtcttcgt attgtctgat 600 gaaatataca gtgaattaac atatgacaga ccgcattact ccatcgcaac ctatttgcgg 660 gatcaaacga ttgtcattaa cgggttgtca aaatcacaca gcatgaccgg ttggagaatt 720 ggatttttat ttgcaccgaa agacattgca aagcacattt taaaggttca tcaatacaat 780 gtgtcgtgcg cctcatccat ttctcaaaaa gccgcgcttg aagctgtcac aaacggcttt 840 gacgatgcat tgattatgag agaacaatac aaaaaacgtc tggactatgt ttatgaccgt 900 cttgtttcca tgggacttga cgtagttaaa ccgtccggtg cgttttatat cttcccttct 960 attaaatcat ttggaatgac ttcatttgat tttagtatgg ctcttttgga agacgctggc 1020 gtggcactcg tgccgggcag ctcgttctca acatatggtg aaggatatgt aaggctgtct 1080 tttgcatgct caatggacac gctgagagaa ggcctagacc gtttagaatt atttgtatta 1140 aaaaaacgtg aagcaatgca gacgataaac aacggcgttt aa 1182 <210> 10 <211> 393 <212> PRT <213> Bacillus subtilis <400> 10 Met Glu His Leu Leu Asn Pro Lys Ala Arg Glu Ile Glu Ile Ser Gly 1 5 10 15 Ile Arg Lys Phe Ser Asn Leu Val Ala Gln His Glu Asp Val Ile Ser             20 25 30 Leu Thr Ile Gly Gln Pro Asp Phe Phe Thr Pro His His Val Lys Ala         35 40 45 Ala Ala Lys Lys Ala Ile Asp Glu Asn Val Thr Ser Tyr Thr Pro Asn     50 55 60 Ala Gly Tyr Leu Glu Leu Arg Gln Ala Val Gln Leu Tyr Met Lys Lys 65 70 75 80 Lys Ala Asp Phe Asn Tyr Asp Ala Glu Ser Glu Ile Ile Ile Thr Thr                 85 90 95 Gly Ala Ser Gln Ala Ile Asp Ala Ala Phe Arg Thr Ile Leu Ser Pro             100 105 110 Gly Asp Glu Val Ile Met Pro Gly Pro Ile Tyr Pro Gly Tyr Glu Pro         115 120 125 Ile Ile Asn Leu Cys Gly Ala Lys Pro Val Ile Val Asp Thr Thr Ser     130 135 140 His Gly Phe Lys Leu Thr Ala Arg Leu Ile Glu Asp Ala Leu Thr Pro 145 150 155 160 Asn Thr Lys Cys Val Val Leu Pro Tyr Pro Ser Asn Pro Thr Gly Val                 165 170 175 Thr Leu Ser Glu Glu Glu Leu Lys Ser Ile Ala Ala Leu Leu Lys Gly             180 185 190 Arg Asn Val Phe Val Leu Ser Asp Glu Ile Tyr Ser Glu Leu Thr Tyr         195 200 205 Asp Arg Pro His Tyr Ser Ile Ala Thr Tyr Leu Arg Asp Gln Thr Ile     210 215 220 Val Ile Asn Gly Leu Ser Lys Ser His Ser Met Thr Gly Trp Arg Ile 225 230 235 240 Gly Phe Leu Phe Ala Pro Lys Asp Ile Ala Lys His Ile Leu Lys Val                 245 250 255 His Gln Tyr Asn Val Ser Cys Ala Ser Ser Ile Ser Gln Lys Ala Ala             260 265 270 Leu Glu Ala Val Thr Asn Gly Phe Asp Asp Ala Leu Ile Met Arg Glu         275 280 285 Gln Tyr Lys Lys Arg Leu Asp Tyr Val Tyr Asp Arg Leu Val Ser Met     290 295 300 Gly Leu Asp Val Val Lys Pro Ser Gly Ala Phe Tyr Ile Phe Pro Ser 305 310 315 320 Ile Lys Ser Phe Gly Met Thr Ser Phe Asp Phe Ser Met Ala Leu Leu                 325 330 335 Glu Asp Ala Gly Val Ala Leu Val Pro Gly Ser Ser Phe Ser Thr Tyr             340 345 350 Gly Glu Gly Tyr Val Arg Leu Ser Phe Ala Cys Ser Met Asp Thr Leu         355 360 365 Arg Glu Gly Leu Asp Arg Leu Glu Leu Phe Val Leu Lys Lys Arg Glu     370 375 380 Ala Met Gln Thr Ile Asn Asn Gly Val 385 390 <210> 11 <211> 1176 <212> DNA <213> Lactobacillus amylovorus <400> 11 atgccagaat tagctaatga tttaggatta agcaaaaaga tcactgatgt aaaagcttca 60 ggaattagaa tctttgataa caaagtttca gctattcctg gcattatcaa attgactttg 120 ggtgaaccag atatgaatac tcctgagcat gttaagcaag cggctattaa gaatattgca 180 gataatgatt cacactatgc tccacaaaag ggaaagcttg aattaagaaa agctatcagt 240 aaatatttga aaaagattac tggaattgaa tatgatccag aaacagaaat cgtagtaaca 300 gttggtgcaa ctgaagcaat taacgctacc ttgtttgcta ttactaatcc gggtgacaag 360 gttgcaattc ctacgccagt cttttctcta tattggcccg tggctacact tgctgatgcc 420 gattatgttt tgatgaatac tgcagaagat ggttttaagt taacacctaa gaagttagaa 480 gaaactatca aagaaaatcc aacaattaaa gcagtaattt tgaattatcc aactaaccca 540 actggtgttg aatatagcga agatgaaatt aaagctttgg ctaaggtaat taaagataat 600 catctgtacg taattaccga tgaaatttac agtactttga cttacggtgt aaaacacttt 660 tcaattgcca gcttaattcc agaaagagca atttatatct ctggtttatc taaatcacat 720 gcgatgactg gttatcgttt aggctatgtt gccggacctg caaaaattat ggcagaaatt 780 ggtaaagttc atggccttat ggtgacgact acgacggatt catcacaagc tgccgcaatt 840 gaagcacttg aacacggact tgatgaccct gagaaatata gggaagttta tgaaaagcgt 900 cgtgactatg ttttaaagga attagccgag atagagatgc aagcagttaa gccagaaggt 960 gcattttata tctttgctaa aattccagct aagtatggca aagacgatat gaaatttgcc 1020 ttggatttag cttttaaaga aaaagtgggt atcactccag gtagtgcatt tggtcctggt 1080 ggtgaaggtc atattagatt atcttatgca tcaagtgatg aaaacttgca tgaggcaatg 1140 aagcgaatga agaaagtttt acaagaggac gaataa 1176 <210> 12 <211> 391 <212> PRT <213> Lactobacillus amylovorus <400> 12 Met Pro Glu Leu Ala Asn Asp Leu Gly Leu Ser Lys Lys Ile Thr Asp 1 5 10 15 Val Lys Ala Ser Gly Ile Arg Ile Phe Asp Asn Lys Val Ser Ala Ile             20 25 30 Pro Gly Ile Ile Lys Leu Thr Leu Gly Glu Pro Asp Met Asn Thr Pro         35 40 45 Glu His Val Lys Gln Ala Ala Ile Lys Asn Ile Ala Asp Asn Asp Ser     50 55 60 His Tyr Ala Pro Gln Lys Gly Lys Leu Glu Leu Arg Lys Ala Ile Ser 65 70 75 80 Lys Tyr Leu Lys Lys Ile Thr Gly Ile Glu Tyr Asp Pro Glu Thr Glu                 85 90 95 Ile Val Val Thr Val Gly Ala Thr Glu Ala Ile Asn Ala Thr Leu Phe             100 105 110 Ala Ile Thr Asn Pro Gly Asp Lys Val Ala Ile Pro Thr Pro Val Phe         115 120 125 Ser Leu Tyr Trp Pro Val Ala Thr Leu Ala Asp Ala Asp Tyr Val Leu     130 135 140 Met Asn Thr Ala Glu Asp Gly Phe Lys Leu Thr Pro Lys Lys Leu Glu 145 150 155 160 Glu Thr Ile Lys Glu Asn Pro Thr Ile Lys Ala Val Ile Leu Asn Tyr                 165 170 175 Pro Thr Asn Pro Thr Gly Val Glu Tyr Ser Glu Asp Glu Ile Lys Ala             180 185 190 Leu Ala Lys Val Ile Lys Asp Asn His Leu Tyr Val Ile Thr Asp Glu         195 200 205 Ile Tyr Ser Thr Leu Thr Tyr Gly Val Lys His Phe Ser Ile Ala Ser     210 215 220 Leu Ile Pro Glu Arg Ala Ile Tyr Ile Ser Gly Leu Ser Lys Ser His 225 230 235 240 Ala Met Thr Gly Tyr Arg Leu Gly Tyr Val Ala Gly Pro Ala Lys Ile                 245 250 255 Met Ala Glu Ile Gly Lys Val His Gly Leu Met Val Thr Thr Thr Thr             260 265 270 Asp Ser Ser Gln Ala Ala Ala Ile Glu Ala Leu Glu His Gly Leu Asp         275 280 285 Asp Pro Glu Lys Tyr Arg Glu Val Tyr Glu Lys Arg Arg Asp Tyr Val     290 295 300 Leu Lys Glu Leu Ala Glu Ile Glu Met Gln Ala Val Lys Pro Glu Gly 305 310 315 320 Ala Phe Tyr Ile Phe Ala Lys Ile Pro Ala Lys Tyr Gly Lys Asp Asp                 325 330 335 Met Lys Phe Ala Leu Asp Leu Ala Phe Lys Glu Lys Val Gly Ile Thr             340 345 350 Pro Gly Ser Ala Phe Gly Pro Gly Gly Glu Gly His Ile Arg Leu Ser         355 360 365 Tyr Ala Ser Ser Asp Glu Asn Leu His Glu Ala Met Lys Arg Met Lys     370 375 380 Lys Val Leu Gln Glu Asp Glu 385 390 <210> 13 <211> 1413 <212> DNA <213> R. sphaeroides <400> 13 atgcgcgagc ctcttgccct cgagatcgac ccgggccacg gcggcccgct gttcctcgcc 60 atcgccgagg cgatcaccct cgacatcacc cgcgggcggc tgaggcccgg agcgagactg 120 cccggcacac gcgcgctggc gcgggcgctc ggcgtgcatc gcaacacggt ggatgccgcc 180 tatcaggagt tgctgaccca gggctggctg caggccgagc ccgcgcgggg caccttcgtg 240 gcgcaggatc tgccgcaggg gatgctggtg cacaggcccg cgcccgcgcc ggtcgagccg 300 gtcgcgatgc gcgcggggct cgccttctcc gatggcgcgc cggaccccga gctggtgccc 360 gacaaggcgc tggcgcgggc ctttcgccgg gcgctcctgt cgcccgcctt ccgcgccgga 420 gcggattacg gcgatgcccg cggcacctcc tcgctgcggg aggcgctggc agcctatctc 480 gcctcggacc ggggcgtggt cgcggatcct gcgcggctgc tgatcgcgcg gggcagccag 540 atggcgctgt tcctggtagc ccgggcggcg ctggcgccgg gagaggcgat cgcggtcgag 600 gagccgggct atccgctggc ctgggaggcg ttccgcgcag cgggagcgga ggtgcgcggc 660 gtgccggtgg atggcggcgg cctcaggatc gacgcgctcg aggccgcgct ggcccgggat 720 ccgcgaatcc gggcggtcta tgtcacgccc catcaccagt atccgacgac cgtcaccatg 780 ggcgcggcgc ggcggttgca gcttctggaa ctggcagagc gccaccggct cgcgctgatc 840 gaggacgact acgaccacga ataccgcttc gagggccgtc cggtgctgcc gctggctgcc 900 cgcgcgccgg aaggtctgcc gctgatctat gtgggctcgc tgtcgaaact gctctcgccc 960 ggtatccggc tgggatacgc gctggcgccc gagcggctgc tgacccgcat ggccgcggcg 1020 cgcgccgcca tcgaccggca gggcgacgcg ccgctcgagg cggcgctggc cgagctgatc 1080 cgcgacggcg atctgggccg tcatgcccgc aaggcgcgca gggtctaccg ggcgcggcgg 1140 gatctgctgg cggagcgtct cacggcgcag ctggccgggc gcgccgcctt cgatctgccg 1200 gccgggggcc tcgcgctgtg gctgcgctgc gcgggcgtct cggccgagac ctgggccgaa 1260 gccgcagggc aggcggggct cgccctgctg ccgggcacgc gcttcgcgct ggagagcccg 1320 gcgccgcagg ccttccggct gggctatgcg gcgctggacg aggggcagat cgcccgggcg 1380 gtggagatcc tcgcccggag cttccccggc tga 1413 <210> 14 <211> 470 <212> PRT <213> R. sphaeroides <400> 14 Met Arg Glu Pro Leu Ala Leu Glu Ile Asp Pro Gly His Gly Gly Pro 1 5 10 15 Leu Phe Leu Ala Ile Ala Glu Ala Ile Thr Leu Asp Ile Thr Arg Gly             20 25 30 Arg Leu Arg Pro Gly Ala Arg Leu Pro Gly Thr Arg Ala Leu Ala Arg         35 40 45 Ala Leu Gly Val His Arg Asn Thr Val Asp Ala Ala Tyr Gln Glu Leu     50 55 60 Leu Thr Gln Gly Trp Leu Gln Ala Glu Pro Ala Arg Gly Thr Phe Val 65 70 75 80 Ala Gln Asp Leu Pro Gln Gly Met Leu Val His Arg Pro Ala Pro Ala                 85 90 95 Pro Val Glu Pro Val Ala Met Arg Ala Gly Leu Ala Phe Ser Asp Gly             100 105 110 Ala Pro Asp Pro Glu Leu Val Pro Asp Lys Ala Leu Ala Arg Ala Phe         115 120 125 Arg Arg Ala Leu Leu Ser Pro Ala Phe Arg Ala Gly Ala Asp Tyr Gly     130 135 140 Asp Ala Arg Gly Thr Ser Ser Leu Arg Glu Ala Leu Ala Ala Tyr Leu 145 150 155 160 Ala Ser Asp Arg Gly Val Val Ala Asp Pro Ala Arg Leu Leu Ile Ala                 165 170 175 Arg Gly Ser Gln Met Ala Leu Phe Leu Val Ala Arg Ala Ala Leu Ala             180 185 190 Pro Gly Glu Ala Ile Ala Val Glu Glu Pro Gly Tyr Pro Leu Ala Trp         195 200 205 Glu Ala Phe Arg Ala Ala Gly Ala Glu Val Arg Gly Val Pro Val Asp     210 215 220 Gly Gly Gly Leu Arg Ile Asp Ala Leu Glu Ala Ala Leu Ala Arg Asp 225 230 235 240 Pro Arg Ile Arg Ala Val Tyr Val Thr Pro His His Gln Tyr Pro Thr                 245 250 255 Thr Val Thr Met Gly Ala Ala Arg Arg Leu Gln Leu Leu Glu Leu Ala             260 265 270 Glu Arg His Arg Leu Ala Leu Ile Glu Asp Asp Tyr Asp His Glu Tyr         275 280 285 Arg Phe Glu Gly Arg Pro Val Leu Pro Leu Ala Ala Arg Ala Pro Glu     290 295 300 Gly Leu Pro Leu Ile Tyr Val Gly Ser Leu Ser Lys Leu Leu Ser Pro 305 310 315 320 Gly Ile Arg Leu Gly Tyr Ala Leu Ala Pro Glu Arg Leu Leu Thr Arg                 325 330 335 Met Ala Ala Ala Arg Ala Ala Ile Asp Arg Gln Gly Asp Ala Pro Leu             340 345 350 Glu Ala Ala Leu Ala Glu Leu Ile Arg Asp Gly Asp Leu Gly Arg His         355 360 365 Ala Arg Lys Ala Arg Arg Val Tyr Arg Ala Arg Arg Asp Leu Leu Ala     370 375 380 Glu Arg Leu Thr Ala Gln Leu Ala Gly Arg Ala Ala Phe Asp Leu Pro 385 390 395 400 Ala Gly Gly Leu Ala Leu Trp Leu Arg Cys Ala Gly Val Ser Ala Glu                 405 410 415 Thr Trp Ala Glu Ala Ala Gly Gln Ala Gly Leu Ala Leu Leu Pro Gly             420 425 430 Thr Arg Phe Ala Leu Glu Ser Pro Ala Pro Gln Ala Phe Arg Leu Gly         435 440 445 Tyr Ala Ala Leu Asp Glu Gly Gln Ile Ala Arg Ala Val Glu Ile Leu     450 455 460 Ala Arg Ser Phe Pro Gly 465 470 <210> 15 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 15 ggtattgagg gtcgcatgaa ggttttagtc aatgg 35 <210> 16 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 16 agaggagagt tagagcctta tgaaatgcta gcagcct 37 <210> 17 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 17 ggtattgagg gtcgcatgtt cgacgccctc gcccg 35 <210> 18 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 18 agaggagagt tagagcctca gagactggtg aacttgc 37 <210> 19 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 19 ggtattgagg gtcgcatgga acatttgctg aatcc 35 <210> 20 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 20 agaggagagt tagagcctta aacgccgttg tttatcg 37 <210> 21 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 21 ggtattgagg gtcgcatgcg cgagcctctt gccct 35 <210> 22 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 22 agaggagagt tagagcctca gccggggaag ctccggg 37 <210> 23 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 23 ggtattgagg gtcgcatgtc cacgcaggcg gccat 35 <210> 24 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 24 agaggagagt tagagcctca ctcacgattc acattgc 37 <210> 25 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 25 ggtattgagg gtcgcatgcc agaattagct aatga 35 <210> 26 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 26 agaggagagt tagagcctta ttcgtcctct tgtaaaa 37 <210> 27 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 27 ggtattgagg gtcgcatgcg ctctacgacg gctcc 35 <210> 28 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 28 agaggagagt tagagcctca gccgcgcagc accttgg 37 <210> 29 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 29 ggtattgagg gtcgcatgtt tgagaacatt accgc 35 <210> 30 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 30 agaggagagt tagagcctta cagcactgcc acaatcg 37 <210> 31 <211> 1194 <212> DNA <213> E. coli <400> 31 gtgtttcaaa aagttgacgc ctacgctggc gacccgattc ttacgcttat ggagcgtttt 60 aaagaagacc ctcgcagcga caaagtgaat ttaagtatcg gtctgtacta caacgaagac 120 ggaattattc cacaactgca agccgtggcg gaggcggaag cgcgcctgaa tgcgcagcct 180 catggcgctt cgctttattt accgatggaa gggcttaact gctatcgcca tgccattgcg 240 ccgctgctgt ttggtgcgga ccatccggta ctgaaacaac agcgcgtagc aaccattcaa 300 acccttggcg gctccggggc attgaaagtg ggcgcggatt tcctgaaacg ctacttcccg 360 gaatcaggcg tctgggtcag cgatcctacc tgggaaaacc gcgtagcaat attcgccggg 420 gctggattcg aagtgagtac ttacccctgg tatgacgaag cgactaacgg cgtgcgcttt 480 aatgacctgt tggcgacgct gaaaacatta cctgcccgca gtattgtgtt gctgcatcca 540 tgttgccaca acccaacggg tgccgatctc actaatgatc agtgggatgc ggtgattgaa 600 attctcaaag cccgcgagct tattccattc ctcgatattg cctatcaagg atttggtgcc 660 ggtatggaag aggatgccta cgctattcgc gccattgcca gcgctggatt acccgctctg 720 gtgagcaatt cgttctcgaa aattttctcc ctttacggcg agcgcgtcgg cggactttct 780 gttatgtgtg aagatgccga agccgctggc cgcgtactgg ggcaattgaa agcaacagtt 840 cgccgcaact actccagccc gccgaatttt ggtgcgcagg tggtggctgc agtgctgaat 900 gacgaggcat tgaaagccag ctggctggcg gaagtagaag agatgcgtac tcgcattctg 960 gcaatgcgtc aggaattggt gaaggtatta agcacagaga tgccagaacg caatttcgat 1020 tatctgctta atcagcgcgg catgttcagt tataccggtt taagtgccgc tcaggttgac 1080 cgactacgtg aagaatttgg tgtctatctc atcgccagcg gtcgcatgtg tgtcgccggg 1140 ttaaatacgg caaatgtaca acgtgtggca aaggcgtttg ctgcggtgat gtaa 1194 <210> 32 <211> 397 <212> PRT <213> E. coli <400> 32 Val Phe Gln Lys Val Asp Ala Tyr Ala Gly Asp Pro Ile Leu Thr Leu 1 5 10 15 Met Glu Arg Phe Lys Glu Asp Pro Arg Ser Asp Lys Val Asn Leu Ser             20 25 30 Ile Gly Leu Tyr Tyr Asn Glu Asp Gly Ile Ile Pro Gln Leu Gln Ala         35 40 45 Val Ala Glu Ala Glu Ala Arg Leu Asn Ala Gln Pro His Gly Ala Ser     50 55 60 Leu Tyr Leu Pro Met Glu Gly Leu Asn Cys Tyr Arg His Ala Ile Ala 65 70 75 80 Pro Leu Leu Phe Gly Ala Asp His Pro Val Leu Lys Gln Gln Arg Val                 85 90 95 Ala Thr Ile Gln Thr Leu Gly Gly Ser Gly Ala Leu Lys Val Gly Ala             100 105 110 Asp Phe Leu Lys Arg Tyr Phe Pro Glu Ser Gly Val Trp Val Ser Asp         115 120 125 Pro Thr Trp Glu Asn Arg Val Ala Ile Phe Ala Gly Ala Gly Phe Glu     130 135 140 Val Ser Thr Tyr Pro Trp Tyr Asp Glu Ala Thr Asn Gly Val Arg Phe 145 150 155 160 Asn Asp Leu Leu Ala Thr Leu Lys Thr Leu Pro Ala Arg Ser Ile Val                 165 170 175 Leu Leu His Pro Cys Cys His Asn Pro Thr Gly Ala Asp Leu Thr Asn             180 185 190 Asp Gln Trp Asp Ala Val Ile Glu Ile Leu Lys Ala Arg Glu Leu Ile         195 200 205 Pro Phe Leu Asp Ile Ala Tyr Gln Gly Phe Gly Ala Gly Met Glu Glu     210 215 220 Asp Ala Tyr Ala Ile Arg Ala Ile Ala Ser Ala Gly Leu Pro Ala Leu 225 230 235 240 Val Ser Asn Ser Phe Ser Lys Ile Phe Ser Leu Tyr Gly Glu Arg Val                 245 250 255 Gly Gly Leu Ser Val Met Cys Glu Asp Ala Glu Ala Ala Gly Arg Val             260 265 270 Leu Gly Gln Leu Lys Ala Thr Val Arg Arg Asn Tyr Ser Ser Pro Pro         275 280 285 Asn Phe Gly Ala Gln Val Val Ala Ala Val Leu Asn Asp Glu Ala Leu     290 295 300 Lys Ala Ser Trp Leu Ala Glu Val Glu Glu Met Arg Thr Arg Ile Leu 305 310 315 320 Ala Met Arg Gln Glu Leu Val Lys Val Leu Ser Thr Glu Met Pro Glu                 325 330 335 Arg Asn Phe Asp Tyr Leu Leu Asn Gln Arg Gly Met Phe Ser Tyr Thr             340 345 350 Gly Leu Ser Ala Ala Gln Val Asp Arg Leu Arg Glu Glu Phe Gly Val         355 360 365 Tyr Leu Ile Ala Ser Gly Arg Met Cys Val Ala Gly Leu Asn Thr Ala     370 375 380 Asn Val Gln Arg Val Ala Lys Ala Phe Ala Ala Val Met 385 390 395 <210> 33 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 33 ggtattgagg gtcgcgtgtt tcaaaaagtt gacgc 35 <210> 34 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 34 agaggagagt tagagcctta catcaccgca gcaaacg 37 <210> 35 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 35 ggtattgagg gtcgcatgga gtccaaagtc gttga 35 <210> 36 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 36 agaggagagt tagagcctta cacttggaaa acagcct 37 <210> 37 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 37 ggtattgagg gtcgcatgaa aaactggaaa acaag 35 <210> 38 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 38 agaggagagt tagagcctta cagcttagcg ccttcta 37 <210> 39 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 39 ggtattgagg gtcgcatgcg aggggcatta ttcaa 35 <210> 40 <211> 36 <212> DNA <213> Artificial sequence <220> <223> primer <400> 40 agaggagagt tagagcctca gcccttgagc gcgaag 36 <210> 41 <211> 1416 <212> DNA <213> E. coli <400> 41 atggaaaact ttaaacatct ccctgaaccg ttccgcattc gtgttattga gccagtaaaa 60 cgtaccaccc gcgcttatcg tgaagaggca attattaaat ccggtatgaa cccgttcctg 120 ctggatagcg aagatgtttt tatcgattta ctgaccgaca gcggcaccgg ggcggtgacg 180 cagagcatgc aggctgcgat gatgcgcggc gacgaagcct acagcggcag tcgtagctac 240 tatgcgttag ccgagtcagt gaaaaatatc tttggttatc aatacaccat tccgactcac 300 cagggccgtg gcgcagagca aatctatatt ccggtactga ttaaaaaacg cgagcaggaa 360 aaaggcctgg atcgcagcaa aatggtggcg ttctctaact atttctttga taccacgcag 420 ggccatagcc agatcaacgg ctgtaccgtg cgtaacgtct atatcaaaga agccttcgat 480 acgggcgtgc gttacgactt taaaggcaac tttgaccttg agggattaga acgcggtatt 540 gaagaagttg gtccgaataa cgtgccgtat atcgttgcaa ccatcaccag taactctgca 600 ggtggtcagc cggtttcact ggcaaactta aaagcgatgt acagcatcgc gaagaaatac 660 gatattccgg tggtaatgga ctccgcgcgc tttgctgaaa acgcctattt catcaagcag 720 cgtgaagcag aatacaaaga ctggaccatc gagcagatca cccgcgaaac ctacaaatat 780 gccgatatgc tggcgatgtc cgccaagaaa gatgcgatgg tgccgatggg cggcctgctg 840 tgcatgaaag acgacagctt ctttgatgtg tacaccgagt gcagaaccct ttgcgtggtg 900 caggaaggct tcccgacata tggcggcctg gaaggcggcg cgatggagcg tctggcggta 960 ggtctgtatg acggcatgaa tctcgactgg ctggcttatc gtatcgcgca ggtacagtat 1020 ctggtcgatg gtctggaaga gattggcgtt gtctgccagc aggcgggcgg tcacgcggca 1080 ttcgttgatg ccggtaaact gttgccgcat atcccggcag accagttccc ggcacaggcg 1140 ctggcctgcg agctgtataa agtcgccggt atccgtgcgg tagaaattgg ctctttcctg 1200 ttaggccgcg atccgaaaac cggtaaacaa ctgccatgcc cggctgaact gctgcgttta 1260 accattccgc gcgcaacata tactcaaaca catatggact tcattattga agcctttaaa 1320 catgtgaaag agaacgcggc gaatattaaa ggattaacct ttacgtacga accgaaagta 1380 ttgcgtcact tcaccgcaaa acttaaagaa gtttaa 1416 <210> 42 <211> 1371 <212> DNA Citrobacter freundii <400> 42 atgaattatc cggcagaacc cttccgtatt aaaagcgttg aaactgtatc tatgatcccg 60 cgtgatgaac gcctcaagaa aatgcaggaa gcgggttaca atactttcct gttaaattcg 120 aaagatattt atattgacct gctgacagac agtggcacta acgcaatgag cgacaagcag 180 tgggccggaa tgatgatggg tgatgaagcg tacgcgggca gcgaaaactt ctatcatctg 240 gaaagaaccg tgcaggaact gtttggcttt aaacatattg ttccgactca ccaggggcgt 300 ggcgcagaaa acctgttatc gcagctggct attaaacctg ggcaatatgt tgccgggaat 360 atgtatttca ctaccacccg ttatcaccag gaaaaaaatg gtgcggtgtt tgtcgatatc 420 gttcgtgacg aagcgcacga tgccggtctg aatattgcgt ttaaaggtga tatcgatctt 480 aaaaaattac aaaagctgat tgatgaaaaa ggcgcagaga atattgcgta tatctgcctg 540 gcggtgacgg ttaacctcgc gggcggccaa ccggtctcga tggctaacat gcgtgcggtg 600 cgtgaactga cagaagcgca tggcattaaa gtgttctacg acgctacccg ctgcgtagaa 660 aacgcctact ttatcaaaga gcaagagcag ggctttgaga acaagagcat cgccgagatc 720 gtgcatgaga tgttcagcta cgccgacggt tgtaccatga gtggtaaaaa agactgtctg 780 gtgaacatcg gcggcttcct gtgcatgaac gatgacgaaa tgttctcttc tgccaaagag 840 ttagtcgtgg tctacgaagg gatgccatct tacggcggcc tggccggacg tgatatggaa 900 gcgatggcga ttggcctgcg tgaagccatg cagtacgaat atattgagca ccgcgtgaag 960 caggttcgct acctgggcga taagctgaaa gccgctggcg taccgattgt tgaaccggta 1020 ggcggtcacg cggtattcct cgatgcgcgt cgcttctgcg agcatctgac gcaagatgag 1080 ttcccggcac aaagtctggc tgccagcatc tatgtggaaa ccggcgtgcg cagtatggag 1140 cgcggaatta tctctgcggg ccgtaataac gtgaccggtg aacaccacag accgaaactg 1200 gaaaccgtgc gtctgactat tccacgtcgt gtttatacct acgcacatat ggatgttgtg 1260 gctgacggta ttattaaact ttaccagcac aaagaagata ttcgcgggct gaagtttatt 1320 tacgagccga agcagttgcg tttctttact gcacgctttg attacatcta a 1371 <210> 43 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 43 ggtattgagg gtcgcatgga aaactttaaa catct 35 <210> 44 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 44 agaggagagt tagagcctta aacttcttta agttttg 37 <210> 45 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 45 ggtattgagg gtcgcatgaa ttatccggca gaacc 35 <210> 46 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 46 agaggagagt tagagcctta gatgtaatca aagcgtg 37 <210> 47 <211> 31 <212> DNA <213> Artificial sequence <220> <223> primer <400> 47 ccagggcacc ggcgcagagc aaatctatat t 31 <210> 48 <211> 30 <212> DNA <213> Artificial sequence <220> <223> primer <400> 48 tgcgccggtg ccctggtgag tcggaatggt 30 <210> 49 <211> 32 <212> DNA <213> Artificial sequence <220> <223> primer <400> 49 tcctgcacgc ggcaaagggt tctgcactcg gt 32 <210> 50 <211> 30 <212> DNA <213> Artificial sequence <220> <223> primer <400> 50 ctttgccgcg tgcaggaagg cttcccgaca 30 <210> 51 <211> 29 <212> DNA <213> Artificial sequence <220> <223> primer <400> 51 aggggaccgg cgcagaaaac ctgttatcg 29 <210> 52 <211> 32 <212> DNA <213> Artificial sequence <220> <223> primer <400> 52 tctgcgccgg tcccctggtg agtcggaaca at 32 <210> 53 <211> 29 <212> DNA <213> Artificial sequence <220> <223> primer <400> 53 gttagtccgc gtctacgaag ggatgccat 29 <210> 54 <211> 29 <212> DNA <213> Artificial sequence <220> <223> primer <400> 54 gtagacgcgg actaactctt tggcagaag 29 <210> 55 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 55 ggtattgagg gtcgcatgta cgaactggga gttgt 35 <210> 56 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 56 agaggagagt tagagcctta gtcaatatat ttcaggc 37 <210> 57 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 57 ggtattgagg gtcgcatgtc cggcatcgtt gtcca 35 <210> 58 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 58 agaggagagt tagagcctca gacatatttc agtccca 37 <210> 59 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 59 ggtattgagg gtcgcatgcg actgaacaac ctcgg 35 <210> 60 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 60 agaggagagt tagagcctca gttctccacg tattcca 37 <210> 61 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 61 ggtattgagg gtcgcatgag cgtggttcac cggaa 35 <210> 62 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 62 agaggagagt tagagcctca atcgatatat ttcagtc 37 <210> 63 <211> 35 <212> DNA <213> Artificial sequence <220> <223> primer <400> 63 ggtattgagg gtcgcatgag cctggttaat atgaa 35 <210> 64 <211> 37 <212> DNA <213> Artificial sequence <220> <223> primer <400> 64 agaggagagt tagagcctta tgactttaac gcgttga 37 <210> 65 <211> 684 <212> DNA C. testosteroni <400> 65 atgtacgaac tgggagttgt ctaccgcaat atccagcgcg ccgaccgcgc tgctgctgac 60 ggcctggccg ccctgggctc cgccaccgtg cacgaggcca tgggccgcgt cggtctgctc 120 aagccctata tgcgccccat ctatgccggc aagcaggtct cgggcaccgc cgtcacggtg 180 ctgctgcagc ccggcgacaa ctggatgatg catgtggctg ccgagcagat tcagcccggc 240 gacatcgtgg tcgcagccgt caccgcagag tgcaccgacg gctacttcgg cgatctgctg 300 gccaccagct tccaggcgcg cggcgcacgt gcgctgatca tcgatgccgg cgtgcgcgac 360 gtgaagacgc tgcaggagat ggactttccg gtctggagca aggccatctc ttccaagggc 420 acgatcaagg ccaccctggg ctcggtcaac atccccatcg tctgcgccgg catgctggtc 480 acgcccggtg acgtgatcgt ggccgacgac gacggcgtgg tctgcgtgcc cgccgcgcgt 540 gccgtggaag tgctggccgc cgcccagaag cgtgaaagct tcgaaggcga aaagcgcgcc 600 aagctggcct cgggcatcct cggcctggat atgtacaaga tgcgcgagcc cctggaaaag 660 gccggcctga aatatattga ctaa 684 <210> 66 <211> 227 <212> PRT C. testosteroni <400> 66 Met Tyr Glu Leu Gly Val Val Tyr Arg Asn Ile Gln Arg Ala Asp Arg 1 5 10 15 Ala Ala Ala Asp Gly Leu Ala Ala Leu Gly Ser Ala Thr Val His Glu             20 25 30 Ala Met Gly Arg Val Gly Leu Leu Lys Pro Tyr Met Arg Pro Ile Tyr         35 40 45 Ala Gly Lys Gln Val Ser Gly Thr Ala Val Thr Val Leu Leu Gln Pro     50 55 60 Gly Asp Asn Trp Met Met His Val Ala Ala Glu Gln Ile Gln Pro Gly 65 70 75 80 Asp Ile Val Val Ala Ala Val Thr Ala Glu Cys Thr Asp Gly Tyr Phe                 85 90 95 Gly Asp Leu Leu Ala Thr Ser Phe Gln Ala Arg Gly Ala Arg Ala Leu             100 105 110 Ile Ile Asp Ala Gly Val Arg Asp Val Lys Thr Leu Gln Glu Met Asp         115 120 125 Phe Pro Val Trp Ser Lys Ala Ile Ser Ser Lys Gly Thr Ile Lys Ala     130 135 140 Thr Leu Gly Ser Val Asn Ile Pro Ile Val Cys Ala Gly Met Leu Val 145 150 155 160 Thr Pro Gly Asp Val Ile Val Ala Asp Asp Asp Gly Val Val Cys Val                 165 170 175 Pro Ala Ala Arg Ala Val Glu Val Leu Ala Ala Ala Gln Lys Arg Glu             180 185 190 Ser Phe Glu Gly Glu Lys Arg Ala Lys Leu Ala Ser Gly Ile Leu Gly         195 200 205 Leu Asp Met Tyr Lys Met Arg Glu Pro Leu Glu Lys Ala Gly Leu Lys     210 215 220 Tyr Ile Asp 225 <210> 67 <211> 42 <212> DNA <213> Artificial sequence <220> <223> primer <400> 67 actcggatcc gaaggagata tacatatgta cgaactggga ct 42 <210> 68 <211> 33 <212> DNA <213> Artificial sequence <220> <223> primer <400> 68 cggctgtcga ccgttagtca atatatttca ggc 33 <210> 69 <211> 31 <212> DNA <213> Artificial sequence <220> <223> primer <400> 69 cgcggatcca taatggttga gaacattacc g 31 <210> 70 <211> 30 <212> DNA <213> Artificial sequence <220> <223> primer <400> 70 acgcgtcgac ttacagcact gccacaatcg 30 <210> 71 <211> 32 <212> DNA <213> Artificial sequence <220> <223> primer <400> 71 ccggaattca taatggtcga actgggagtt gt 32 <210> 72 <211> 33 <212> DNA <213> Artificial sequence <220> <223> primer <400> 72 gaatgcggcc gcttagtcaa tatatttcag gcc 33 <210> 73 <211> 15 <212> DNA <213> Artificial sequence <220> <223> primer <400> 73 ggtattgagg gtcgc 15 <210> 74 <211> 17 <212> DNA <213> Artificial sequence <220> <223> primer <400> 74 agaggagagt tagagcc 17  

Claims (57)

A method for preparing monatin, comprising converting glucose into monatin, the method comprising: Converting glucose into tryptophan; Contacting the tryptophan with a first polypeptide, wherein the first polypeptide is an aminotransferase and converts tryptophan to indole-3-pyruvate; Contacting the indole-3-pyruvate with a second polypeptide and a C3 carbon source selected from the group consisting of pyruvate, phosphoenolpyruvate, alanine, serine, cysteine and combinations thereof, wherein the second polypeptide Is aldolase and a C3 carbon source selected from the group consisting of indole-3-pyruvate and pyruvate, phosphoenolpyruvate, alanine, serine, cysteine and combinations thereof is 2-hydroxy 2- (indole- Converting to 3-ylmethyl) -4-keto glutaric acid; And Contacting the 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid with a third polypeptide, wherein the third polypeptide is an aminotransferase and 2-hydroxy 2- (indole-3 -Ylmethyl) -4-keto glutaric acid is converted to monatin Monatine manufacturing method comprising a. A process for preparing monatin comprising converting tryptophan or indole-3-lactic acid to monatin, the method comprising Contacting tryptophan or indole-3-lactic acid with a first polypeptide, wherein when the tryptophan is contacted with the first polypeptide, the first polypeptide is selected from aminotransferase, dehydrogenase, or oxidase, and the indole- When 3-lactic acid is contacted with a first polypeptide, the first polypeptide is selected from oxidase, dehydrogenase or reductase, and the first polypeptide converts tryptophan or indole-3-lactic acid to indole-3-pyruvate To make; Contacting the indole-3-pyruvate with a second polypeptide and a C3 carbon source selected from the group consisting of pyruvate, phosphoenolpyruvate, alanine, serine, cysteine and combinations thereof, wherein the second polypeptide Is aldolase and a C3 carbon source selected from the group consisting of indole-3-pyruvate and pyruvate, phosphoenolpyruvate, alanine, serine, cysteine and combinations thereof is 2-hydroxy 2- (indole- Converting to 3-ylmethyl) -4-keto glutaric acid; And Contacting the 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid with a third polypeptide, wherein the third polypeptide is an aminotransferase or a dehydrogenase and 2-hydroxy 2- ( Converting indol-3-ylmethyl) -4-keto glutaric acid to monatin Monatine manufacturing method comprising a. The enzyme of claim 2, wherein when the tryptophan is contacted with the first polypeptide, the first polypeptide is tryptophan aminotransferase from enzyme class EC 2.6.1.27, tryptophan dehydrogenase from enzyme class EC 1.4.1.19, enzyme class EC 2.6. Tyrosine (aromatic) aminotransferase derived from 1.5, tryptophan-phenylpyruvate aminotransferase derived from enzyme class EC 2.6.1.28, aspartate aminotransferase from enzyme class EC 2.6.1.1, tryptophan oxidase, enzyme class EC 1.4 .3.2 L-amino acid oxidase derived from enzyme class, D-amino acid dehydrogenase derived from enzyme class EC 1.4.99.1, D-amino acid oxidase derived from enzyme class EC 1.4.3.3, D-tryptophan aminotransferase, enzyme class EC 2.6. D-alanine aminotransferase from 1.21, glutamate dehydrogenase from enzyme class EC 1.4.1.2, enzyme class EC 1.4.1.3 or enzyme class EC 1.4.1.4, As being selected from the group consisting of bovine class phenylalanine dehydrogenase, and combinations thereof of the EC 1.4.1.20 pedigree of monatin manufacturing method of converting tryptophan to indole-3-pyruvate. 3. The method of claim 2, wherein when contacting indole-3-lactic acid with the first polypeptide, the first polypeptide is indole lactate dehydrogenase from enzyme class EC 1.1.1.110, R-4-hydroxy from enzyme class EC 1.1.1.222 Hydroxyphenyl lactate dehydrogenase, 3- (4) -hydroxyphenylpyruvate reductase from enzyme class EC 1.1.1.237, enzyme class EC 1.1.1.27, enzyme class EC 1.1.1.28, or enzyme class EC 1.1.2.3 Lactate dehydrogenase, (3-imidazol-5-yl) lactate dehydrogenase from enzyme class EC 1.1.1.111 and combinations thereof, wherein indole-3-pyruvate is selected from indole-3-pyruvate. Method of producing monatin is converted to. The Genbank access according to claim 2 or 3, wherein the first polypeptide, the third polypeptide or the first polypeptide and the third polypeptide are SEQ ID NOs: 2,4,6,8,10,12,14,32 Comprising the amino acid sequence set forth in No. NP_388848.1, GenBank Accession No. ZP00005082.1 or GenBank Accession No. AAC74014.1, said amino acid sequence converting tryptophan to indole-3-pyruvate Phosphorus monatin production method. The method of claim 2, wherein said second polypeptide is an aldolase from enzyme class EC 4.1.3.16 or enzyme class EC 4.1.3.17. The method of claim 6, wherein the aldolase is 4-hydroxy-2-oxoglutarate glyoxylate degrading enzyme from enzyme class EC 4.1.3.16, 4-hydroxy-4- from enzyme class EC 4.1.3.17. Method for producing monatin, which is methyl-2-oxoglutarate pyruvate degrading enzyme or a combination thereof. The method of claim 2, wherein the third polypeptide is a tyrosine (aromatic) aminotransferase from enzyme class EC 2.6.1.5, tryptophan dehydrogenase from enzyme class EC 1.4.1.19, tryptophan-phenyl from enzyme class EC 2.6.1.28 Pyruvate aminotransferase, tryptophan aminotransferase from enzyme class EC 2.6.1.27, aspartate aminotransferase from enzyme class EC 2.6.1.1, D-alanine aminotransferase from enzyme class EC 2.6.1.21, D- Tryptophan aminotransferase, enzyme class EC 1.4.1.2, enzyme class EC 1.4.1.3, or glutamate dehydrogenase from enzyme class EC 1.4.1.4, phenylalanine dehydrogenase from enzyme class EC 1.4.1.20, enzyme class EC 1.4. A method for producing monatin, selected from the group consisting of D-amino acid dehydrogenase derived from 99.1 and combinations thereof. The method of claim 2, wherein the first polypeptide comprises an amino acid oxidase from enzyme class EC 1.4.3.2 or enzyme class EC 1.4.3.3 and a catalase from enzyme class EC 1.11.1.6, wherein tryptophan is selected from indole-3-pyruvic acid. Method of producing monatin that is converted to a salt. 10. The method of claim 9, wherein said amino acid oxidase comprises tryptophan oxidase. delete The method of claim 2, wherein the pyruvate is a polypeptide capable of performing the amino transfer of alanine and alanine; Serine and polypeptides capable of beta-removing serine; Polypeptides capable of beta-removing cysteine and cysteine; Polypeptides capable of beta-degrading enzyme reactions with aspartate and dicarboxylic amino acids; Lactate dehydrogenase from lactate and enzyme class EC 1.1.1.27, enzyme class EC 1.1.1.28, or enzyme class EC 1.1.2.3; Or a combination thereof. The method of claim 2, further comprising contacting the resulting hydrogen peroxide with catalase to reduce the amount of hydrogen peroxide when the tryptophan is converted to indole-3-pyruvate. The amino acid sequence of claim 2, wherein the second polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 66, Genbank Accession No. CAC46344, Genbank Accession No. CAB14127.1, Genbank Accession No. AAC74920.1, or Genbank Accession No. CAC47463.1. Wherein said amino acid sequence converts indole-3-pyruvate and pyruvate to 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid. The oxaloacetic acid salt of claim 8, wherein the third polypeptide comprises aspartate aminotransferase, such that when 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid is contacted with aspartate. Monatine production method to produce and monatin. The method of claim 15, further comprising contacting oxaloacetate with a decarboxylase. The monatin preparation of claim 2 wherein the third polypeptide is lysine epsilon aminotransferase and further contacts 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid with lysine. Way. The method of claim 2, wherein the third polypeptide is an enzyme having reductive amination activity and is capable of recycling NAD (P) H, wherein the third polypeptide is glutamate dehydrogenase, phenylalanine dehydrogenase, tryptophan dehydrogenase, form A method for producing monatin, selected from the group consisting of an acid dehydrogenase or a combination thereof, wherein the third polypeptide produces carbon dioxide if the third polypeptide is formate dehydrogenase. delete delete delete The method of claim 2, wherein the method comprises converting indole-3-lactic acid to monatin. The method of claim 2, wherein monatin is produced at least 10 g / L. A method for preparing monatin, comprising converting indole-3-pyruvate to monatin, the method comprising Contacting the indole-3-pyruvate with a first polypeptide and a C3 carbon source selected from the group consisting of pyruvate, phosphoenolpyruvate, alanine, serine, cysteine and combinations thereof, wherein the first polypeptide Is aldolase and a C3 carbon source selected from the group consisting of indole-3-pyruvate and pyruvate, phosphoenolpyruvate, alanine, serine, cysteine and combinations thereof is 2-hydroxy 2- (indole- Converting to 3-ylmethyl) -4-keto glutaric acid; And Contacting the 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid with a second polypeptide, wherein the second polypeptide is an aminotransferase and 2-hydroxy 2- (indole-3 -Ylmethyl) -4-keto glutaric acid monatin conversion Monatine manufacturing method comprising a. A method for producing monatin, comprising converting tryptophan to monatin, wherein the method Contacting the tryptophan with a first polypeptide, wherein the first polypeptide is an aminotransferase and converts tryptophan to indole-3-pyruvate; Contacting the indole-3-pyruvate with a second polypeptide and a C3 carbon source selected from the group consisting of pyruvate, phosphoenolpyruvate, alanine, serine, cysteine and combinations thereof, wherein the second polypeptide Is aldolase and a C3 carbon source selected from the group consisting of indole-3-pyruvate and pyruvate, phosphoenolpyruvate, alanine, serine, cysteine and combinations thereof is 2-hydroxy 2- (indole- Converting to 3-ylmethyl) -4-keto glutaric acid; And Contacting the 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid with a third polypeptide, wherein the third polypeptide is an aminotransferase and 2-hydroxy 2- (indole-3 -Ylmethyl) -4-keto glutaric acid is converted to monatin Monatine manufacturing method comprising a. As a host cell capable of producing monatin, The cells Tryptophan is converted to indole-3-pyruvate, tryptophan aminotransferase from enzyme class EC 2.6.1.27, tryptophan dehydrogenase from enzyme class EC 1.4.1.19, tyrosine (aromatic) amino from enzyme class EC 2.6.1.5 Transferase, tryptophan-phenylpyruvate aminotransferase derived from enzyme class EC 2.6.1.28, aspartate aminotransferase, tryptophan oxidase derived from enzyme class EC 2.6.1.1, L-amino acid oxidation from enzyme class EC 1.4.3.2 Enzyme, D-amino acid dehydrogenase from enzyme class EC 1.4.99.1, D-amino acid oxidase from enzyme class EC 1.4.3.3, D-tryptophan aminotransferase, D-alanine aminotransferase from enzyme class EC 2.6.1.21 Glutamate dehydrogenase from enzyme, enzyme class EC 1.4.1.2, enzyme class EC 1.4.1.3 or enzyme class EC 1.4.1.4 and phenyl derived from enzyme class EC 1.4.1.20 A first nucleic acid encoding a first polypeptide selected from the group consisting of alanine dehydrogenases, Indole-3-pyruvate is converted to 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid and composed of aldolase from enzyme class EC 4.1.3.16 or enzyme class EC 4.1.3.17 A second nucleic acid encoding a second polypeptide selected from the group consisting of: and 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid is converted to monatin and tyrosine (aromatic) aminotransferase from enzyme class EC 2.6.1.5, enzyme class EC 1.4.1.19 Tryptophan dehydrogenase derived, tryptophan-phenylpyruvate aminotransferase derived from enzyme class EC 2.6.1.28, tryptophan aminotransferase derived from enzyme class EC 2.6.1.27, aspartate aminotransferase derived from enzyme class EC 2.6.1.1, D-alanine aminotransferase from enzyme class EC 2.6.1.21, D-tryptophan aminotransferase, glutamate dehydrogenase from enzyme class EC 1.4.1.2, enzyme class EC 1.4.1.3, or enzyme class EC 1.4.1.4, A third nucleic acid encoding a third polypeptide selected from the group consisting of phenylalanine dehydrogenase from enzyme class EC 1.4.1.20 and D-amino acid dehydrogenase from enzyme class EC 1.4.99.1 Including, At least one of said first to third nucleic acids is exogenous to said host cell. The method of claim 26, wherein at least one of the first to third polypeptides is from tryptophan aminotransferase from enzyme class 2.6.1.27, tryptophan dehydrogenase from enzyme class EC 1.4.1.19, from enzyme class EC 2.6.1.5 Tyrosine (aromatic) aminotransferase, tryptophan-phenylpyruvate aminotransferase from enzyme class EC 2.6.1.28, tryptophan oxidase, L-amino acid oxidase from enzyme class EC 1.4.3.2, enzyme class EC 2.6.1.1 Aspartate aminotransferase, D-amino acid dehydrogenase derived from enzyme class EC 1.4.99.1, D-amino acid oxidase derived from enzyme class EC 1.4.3.3, D-alanine aminotransferase derived from enzyme class EC 2.6.1.21, D-tryptophan aminotransferase, indole lactate dehydrogenase from enzyme class EC11.1.1.110, R-4-hydroxyphenyl lactate from enzyme class EC 1.1.1.222 Hydrogenase, 3- (4) -hydroxyphenylpyruvate reductase from enzyme class EC 1.1.1.237, lactate dehydrogen from enzyme class EC 1.1.1.27, enzyme class EC 1.1.1.28, or enzyme class EC 1.1.2.3 Enzyme, (3-imidazol-5-yl) lactate dehydrogenase from enzyme class EC 1.1.1.111, aldolase from enzyme class EC 4.1.3.16 or enzyme class EC 4.1.3.17, derived from enzyme class EC 1.4.1.20 A host cell selected from the group consisting of phenylalanine dehydrogenase, enzyme class EC 1.4.1.2, enzyme class EC 1.4.1.3, or glutamate dehydrogenase from enzyme class EC 1.4.1.4 and combinations thereof. The method of claim 27, wherein at least one of the first to third polypeptides is from tryptophan aminotransferase from enzyme class EC 2.6.1.27, tryptophan dehydrogenase from enzyme class EC 1.4.1.19, enzyme class EC 2.6.1.5 Tyrosine (aromatic) aminotransferase, tryptophan-phenylpyruvate aminotransferase from enzyme class EC 2.6.1.28, aspartate aminotransferase, enzyme tryptophan oxidase from enzyme class EC 2.6.1.1, enzyme class EC 1.4.3.2 L-amino acid oxidase derived from, D-amino acid dehydrogenase derived from enzyme class EC 1.4.99.1, D-amino acid oxidase derived from enzyme class EC 1.4.3.3, D-alanine aminotransferase derived from enzyme class EC 2.6.1.21 , D-tryptophan aminotransferase, aldolase from enzyme class EC 4.1.3.16 or enzyme class EC 4.1.3.17, phenylalanine deactivation from enzyme class EC 1.4.1.20 A host cell selected from the group consisting of hydrogenase, enzyme class EC 1.4.1.2, enzyme class EC 1.4.1.3, or glutamate dehydrogenase from enzyme class EC 1.4.1.4 and combinations thereof. The method of claim 27, wherein at least one of the first to third polypeptides is an indole lactate dehydrogenase derived from enzyme class EC 1.1.1.110, an R-4-hydroxyphenyl lactate dehydrogenase derived from enzyme class EC 1.1.1.222, 3- (4) -hydroxyphenylpyruvate reductase from enzyme class EC 1.1.1.237, lactate dehydrogenase, enzyme from enzyme class EC 1.1.1.27, enzyme class EC 1.1.1.28, or enzyme class EC 1.1.2.3 (3-imidazol-5-yl) lactate dehydrogenase from class EC 1.1.1.111, aldolase from enzyme class EC 4.1.3.16 or enzyme class EC 4.1.3.17, tryptophan dehydrogen from enzyme class EC 1.4.1.19 Enzyme, tryptophan-phenylpyruvate aminotransferase derived from enzyme class EC 2.6.1.28, tryptophan aminotransferase derived from enzyme class EC 2.6.1.27, tyrosine (aromatic) aminotransferase derived from enzyme class EC 2.6.1.5, Aspartate aminotransferase from bovine class EC 2.6.1.1, glutamate dehydrogenase from enzyme class EC 1.4.1.2, or enzyme class EC 1.4.1.3, or enzyme class EC 1.4.1.4 Selected from the group consisting of phenylalanine dehydrogenase, D-tryptophan aminotransferase, D-amino acid dehydrogenase from enzyme class EC 1.4.99.1, D-alanine aminotransferase from enzyme class EC 2.6.1.21 and combinations thereof Phosphorus host cell. The method of claim 27, wherein the aldolase is 4-hydroxy-2-oxoglutarate aldolase from enzyme class EC 4.1.3.16 or 4-hydroxy-4-methyl from enzyme class EC 4.1.3.17 The host cell being 2-oxoglutarate aldolase. The host cell of claim 26, wherein the host cell is a bacterial cell. The host cell of claim 26, wherein the host cell is a yeast cell. The cell of claim 27, wherein the cell comprises one or more additional polypeptides encoded by one or more additional exogenous nucleic acid sequences, wherein the additional polypeptide is catalase, oxaloacetate decarboxylase, lysine epsilon aminotransferase. And formate dehydrogenase; and a host cell comprising an activity selected from the group consisting of. delete delete delete delete delete delete delete -Converting tryptophan or indole-3-lactic acid to indole-3-pyruvate using a first polypeptide, wherein when the tryptophan contacts the first polypeptide, the first polypeptide is derived from enzyme class EC 2.6.1.27 Tryptophan aminotransferase, tryptophan dehydrogenase derived from enzyme class EC 1.4.1.19, tyrosine (aromatic) aminotransferase derived from enzyme class EC 2.6.1.5, tryptophan-phenylpyruvate aminotransferase derived from enzyme class EC 2.6.1.28 Aspartate aminotransferase, tryptophan oxidase from enzyme class EC 2.6.1.1, L-amino acid oxidase from enzyme class EC 1.4.3.2, D-amino acid dehydrogenase from enzyme class EC 1.4.99.1, enzyme class EC D-amino acid oxidase derived from 1.4.3.3, D-tryptophan aminotransferase, D-alanine aminotransferase derived from enzyme class EC 2.6.1.21, enzyme Selected from the class EC 1.4.1.2, EC 1.4.1.3 class enzyme or enzyme class phenylalanine dehydrogenase, and combinations thereof of the group consisting of the EC 1.4.1.4 derived glutamate dehydrogenase, the enzyme class EC 1.4.1.20 is derived; When indole-3-lactic acid contacts the first polypeptide, the first polypeptide is indole lactate dehydrogenase from enzyme class EC 1.1.1.110, R-4-hydroxyphenyl lactate dehydrogen from enzyme class EC 1.1.1.222. Enzyme, 3- (4) -hydroxyphenylpyruvate reductase from enzyme class EC 1.1.1.237, lactate dehydrogenase from enzyme class EC 1.1.1.27, enzyme class EC 1.1.1.28, or enzyme class EC 1.1.2.3 , (3-imidazol-5-yl) lactate dehydrogenase from enzyme class EC 1.1.1.111 and combinations thereof; Converting indole-3-pyruvate to 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid by aldol condensation reaction; And Converting 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid to monatin using a second polypeptide, wherein the second polypeptide is of enzyme class EC 2.6.1.5 Tyrosine (aromatic) aminotransferase, tryptophan dehydrogenase derived from enzyme class EC 1.4.1.19, tryptophan-phenylpyruvate aminotransferase derived from enzyme class EC 2.6.1.28, tryptophan aminotransferase derived from enzyme class EC 2.6.1.27, Aspartate aminotransferase from enzyme class EC 2.6.1.1, D-alanine aminotransferase from enzyme class EC 2.6.1.21, D-tryptophan aminotransferase, enzyme class EC 1.4.1.2, enzyme class EC 1.4.1.3, Or glutamate dehydrogenase from enzyme class EC 1.4.1.4, phenylalanine dehydrogenase from enzyme class EC 1.4.1.20, D-amino acid dehydrogenase from enzyme class EC 1.4.99.1 and its derivatives Which is selected from the group consisting of the steps Monatine manufacturing method comprising a. delete delete delete delete delete delete delete delete delete delete delete The method of claim 2, wherein the method further comprises the step of detecting 2-hydroxy 2- (indol-3-ylmethyl) -4-keto glutaric acid by mass spectrometry. delete The method of claim 2, wherein said tryptophan is produced from indole using a polypeptide from enzyme class EC 4.1.99.1. delete delete

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