CN1894415A

CN1894415A - Methods for the preparation of a fine chemical by fermentation

Info

Publication number: CN1894415A
Application number: CNA2004800378081A
Authority: CN
Inventors: O·策尔德尔; C·克洛普罗格; H·施罗德; S·哈夫纳; B·克勒格尔; P·基弗; E·海因茨勒; C·维特曼
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 2003-12-18
Filing date: 2004-12-17
Publication date: 2007-01-10

Abstract

The present invention features methods of increasing the production of a fine chemical, e.g., lysine from a microorganism, e.g., Corynebacterium by way of deregulating an enzyme encoding gene, i.e., lactate dehydrogenase. In a preferred embodiment, the invention provides methods of increasing the production of lysine in Corynebacterium glutamicum by way of the expression of lactate dehydrogenase activity. The invention also provides a novel process for the production of lysine by way of regulating carbon flux towards oxaloacetate (OAA). In a preferred embodiment, the invention provides methods for the production of lysine by way of utilizing fructose or sucrose as a carbon source.

Description

Method by the fermentative preparation fine chemicals

Background of invention

The industrial production of Methionin has become important economically commercial run.Because Methionin can improve feeding quality by increasing other amino acid whose absorption, so Methionin is at the commercial animal feedstuff additive that can be used as, Methionin also can be used in the human medicine, particularly as the composition of infusion solution, and can be used for pharmaceutical industries.

The commercial production of Methionin mainly utilizes gram positive bacterium Corynebacterium glutamicum (Corynebacterium glutamicum), brevibacterium flavum (Brevibacterium flavum) and brevibacterium lactofermentum (Brevibacterium lactofermentum) to carry out (Kleemann, A. wait the people, " Amino Acids; " ULLMANN ' S ENCYCLOPEDIA OF INDUSTRIALCHEMISTRY, the A2 volume, the 57-97 page or leaf, Weinham:VCH-Verlagsgesellschaft (1985)).At present, these organisms are being undertaken the production of annual about 250,000 Methionins.A large amount of research is devoted to separate on the mutant strain that can produce greater amount Methionin.The microorganism of adopting at the microbial process that is used for amino acid production is divided into 4 classes: wild type strain, auxotrophic mutation body, regulate mutant and auxotroph and regulate mutant (people such as K.Nakayama,: Nutritional Improvement of Food and Feed Proteins, M.Friedman compiles, (1978), 649-661 page or leaf).The mutant of Corynebacterium (Corynebacterium) and related organisms can be by direct fermentation from cheap carbon source, and for example molasses, acetate and ethanol cheapness are produced amino acid.In addition, it is more favourable to make this method compare with synthetic method by fermentative production stereospecificity amino acid (L isomer).

The another kind of method of improving Methionin commercial production efficient is by studying realization to Methionin production with through the dependency between the metabolic flux of pentose-phosphate pathway.Consider by fermentation process and produce the importance of Methionin at economic aspect, so Methionin synthetic bio-chemical pathway is furtherd investigate, obviously this is to produce the total amount of Methionin in order improving and to reduce production costs (by people such as Sahm, (1996) Ann.N.Y.Acad.Sci.782:25-39 summary).Using metabolic engineering to instruct glucose source carbon to flow to more existing successes (Flores, people such as N., (1996) Nature Biotechnol.14:620-623) aspect the aromatic amino acid formation.In case glucose is absorbed by cell, then make glucose phosphorylation (phosphotransferase system) (Malin and Bourd by consuming phosphoenolpyruvic acid, (1991) Journal of Applied Bacteriology 71,517-523), utilized by cell as G-6-P then.By phosphotransferase system (people such as Shio, (1990) Agriculturaland Biological Chemistry 54,1513-1519) and saccharase reaction (people such as Yamamoto, (1986) Journal of Fermentation Technology 64, and 285-291) sucrose is transformed into fructose and G-6-P.

In the breakdown of glucose metabolic process, glucose-6-phosphate dehydrogenase (G6PD) (EC 1.1.14.9) and G-6-P isomerase (EC 5.3.1.9) competition substrate G-6-P.First reactions steps in G-6-P isomerase catalysis Embden-Meyerhof-Parnas approach or the glycolysis-promptly is transformed into fructose-6-phosphate.First reactions steps of oxidized portion promptly is transformed into the 6-phosphogluconolactone in the circulation of glucose-6-phosphate dehydrogenase (G6PD) catalysis pentose phosphate.

In pentose phosphate round-robin oxidized portion, G-6-P is transformed into ribulose-5-phosphoric acid, therefore produces the reducing equivalent of NADPH form.Along with the pentose phosphate round-robin further carries out, pentose phosphate, phosphohexose and triose phosphate change mutually.In the Nucleotide biosynthesizing, need pentose phosphate such as ribose 5-phosphate-1-tetra-sodium.In addition, ribose 5-phosphate-1-tetra-sodium is the precursor of aromatic amino acid and L-Histidine.NADPH is used as reducing equivalent in numerous anabolic biosynthesizing.At the NADPH that from oxaloacetic acid biosynthesizing a part Methionin, will consume 4 molecules.Therefore, the carbon flux that flows to oxaloacetate (OAA) still keeps constant, and not guard system whether be interfered (people such as J.Vallino, (1993) Biotechnol.Bioeng., 41,633-646).

The invention summary

The present invention has promptly found the gene of coding pentose-phosphate pathway key enzyme (for example serum lactic dehydrogenase) and has found the adjusting (deregulation) (reducing and active reduction as expression) of going of serum lactic dehydrogenase has been caused the increase of Methionin production at least in part based on following discovery in Corynebacterium glutamicum.In addition, have been found that by going to regulate (as reducing) lactic dehydrogenase expression of enzymes and the active increase that makes that the increase of carbon output can cause Methionin to be produced in the Methionin production process.In one embodiment, carbon source is fructose and sucrose.Therefore, the invention provides that to be used to improve with fructose or sucrose be the method that the microorganism (for example Corynebacterium glutamicum) of substrate produces Methionin.

Therefore, in one aspect, the invention provides and be used for increasing the flow through method of metabolic flux of pentose-phosphate pathway of microorganism, this method comprises that culture bag is contained in the microorganism of the gene that is gone to regulate (so that having increased the metabolic flux of the pentose-phosphate pathway of flowing through) under the certain condition.In one embodiment, microorganism is used for the fermentative production fine chemicals, as Methionin.In another embodiment, fructose or sucrose are as carbon source.In another embodiment, gene is a lactate dehydrogenase gene.In relevant embodiment, lactate dehydrogenase gene is from Corynebacterium, for example Corynebacterium glutamicum.In another embodiment, lactate dehydrogenase gene is expressed not enough.In another embodiment, lactate dehydrogenase gene encoded protein matter has the activity of reduction.

In another embodiment, microorganism also comprises one or more extra regulatory gene of going.One or more other regulatory gene of going include, but is not limited to ask gene, dapA gene, asd gene, dapB gene, ddh gene, lysA gene, lysE gene, pycA gene, zwf gene, pepCL gene, gap gene, zwa1 gene, tkt gene, tad gene, mqo gene, tpi gene, pgk gene and sigC gene.In specific embodiment, gene can be express or express not enough.In addition, go regulatory gene to encode and be selected from following protein: feedback opposing E.C. 2.7.2.4., the dihydrodipicolinate synthase, aspartate-semialdehyde dehydrogenase, the dihydrodipicolinate reductase, diaminopimelate dehydrogenase, the diaminopimelic acid epimerase, Methionin output (exporter), pyruvate carboxylase, glucose-6-phosphate dehydrogenase (G6PD), Phosphoenolpyruvate carboxylase, glyceraldehyde-3-phosphate dehydrogenase, the RPF protein precursor, transketolase, transaldolase, menaquinone oxydo-reductase (menaquinine oxidoreductase), triose-phosphate isomerase, glycerol 3-phosphate acid kinase and RNA polymerase Sigma Factors sigC.In specific embodiments, protein has activity increase or that reduce.

According to the inventive method, the regulatory gene of going that one or more are other also includes, but is not limited to pepCK gene, mal E gene, glgA gene, pgi gene, dead gene, menE gene, citE gene, mikE17 gene, poxB gene, zwa2 gene and sucC gene.In specific embodiments, at least a expression of gene is raised, is weakened, is reduced, is reduced or suppressed.In addition, go regulatory gene to encode and be selected from following protein: rna helicase enzyme, o-succinyl-phenylformic acid-CoA ligase enzyme, citrate lyase β chain, transcriptional, pyruvic oxidase, RPF protein precursor and succinyl CoA synthetase that phosphoenolpyruvate carboxykinase, malic enzyme, Glycogensynthase, G-6-P isomerase, ATP rely on.In specific embodiments, protein has activity reduction or that increase.

In one embodiment, the microorganism that is used for the inventive method belongs to Corynebacterium, for example Corynebacterium glutamicum.

In yet another aspect, the invention provides the method that is used to produce fine chemicals, it comprise serum lactic dehydrogenase removed the microbial fermentation regulated and in substratum or microorganism cells accumulation fine chemicals such as Methionin, produce fine chemicals thus.In one embodiment, method comprises the recovery fine chemicals.In another embodiment, lactic dehydrogenase expression of enzymes deficiency.In another embodiment, fructose or sucrose are as carbon source.

In one aspect, serum lactic dehydrogenase is from Corynebacterium glutamicum and comprise nucleotide sequence SEQID NO:1 and aminoacid sequence SEQ ID NO:2.

From following be described in detail with claim as seen, further feature of the present invention and advantage are conspicuous.

The accompanying drawing summary

Fig. 1: pentose biosynthetic pathway synoptic diagram.

Fig. 2: at Corynebacterium glutamicum ATCC 21526 serves as that the basis produces in the chase experiment of Methionin with glucose and fructose, the comparison of relative mass isotope-isomerism thing (isotopomer) fraction in secreted Methionin of measuring by GC/MS and the trehalose.

Fig. 3: be that the basis produces in the process of Methionin with glucose, distribute in the body of carbon flux in the Corynebacterium glutamicum ATCC21526 center metabolism, this integrated approach that is to use the balanced and isotope-isomerism object model of combination metabolite carries out to experimental result that best-fit infers out, and wherein the isotope-isomerism object model is used for respectively secreted Methionin and trehalose being carried out mark mensuration by GC/MS ¹³The C chase experiment.Net flux provides in square symbol, and for reversible reaction, the direction of net flux marks with the other arrow of corresponding black surround.Numeral under transaldolase, transketolase and glucose 6-phosphoric acid isomerase flux in the square brackets has shown the reversibility flux.All flux all are expressed as average special glucose uptake rate (1.77mmol g ^-1h ^-1) molecular fraction.

Fig. 4: be that the basis produces in the process of Methionin with fructose, distribute in the body of carbon flux in the Corynebacterium glutamicum ATCC21526 center metabolism, this integrated approach that is to use the balanced and isotope-isomerism object model of combination metabolite carries out to experimental result that best-fit infers out, and wherein the isotope-isomerism object model is used for respectively secreted Methionin and trehalose being carried out mark mensuration by GC/MS ¹³The C chase experiment.Net flux provides in square symbol, and for reversible reaction, the direction of net flux marks with the other arrow of corresponding black surround.Numeral under transaldolase, transketolase and glucose 6-phosphoric acid isomerase flux in the square brackets has shown the reversibility flux.All flux all are expressed as average special fructose uptake ratio (1.93mmol g ^-1h ^-1) molecular fraction.

Fig. 5: grow in glucose (A) and grow in the metabolic metabolism network in center of the Corynebacterium glutamicum of the production Methionin in the fructose (B), comprise the flux between transhipment flux, assimilation flux and intermediary metabolism pond.

Detailed Description Of The Invention

The present invention is at least in part based on the gene of coding pentose-phosphate pathway indispensable enzyme, as the evaluation of Corynebacterium glutamicum gene.The present invention is feature with the method, and wherein said method comprises the pentose phosphate biosynthetic pathway in controlling microbial such as the Corynebacterium glutamicum so that carbon output increases and generation (for example producing with the output that increases) some purpose fine chemicals such as Methionin.Particularly, the present invention includes the method for producing fine chemicals such as Methionin by fermentation with (as reduce) lactic dehydrogenase expression of enzymes of regulating and active microorganism (as Corynebacterium glutamicum).In one embodiment, use fructose or sucrose carbon source as microbial fermentation.Verified, the production in microorganism is the relatively poor substrate of efficient to fructose for fine chemicals (as Methionin).Yet, the invention provides that to be used for optimizing with fructose or sucrose be the microorganism of substrate such as the method that Corynebacterium glutamicum Methionin produces.Lactic dehydrogenase expression of enzymes or actively go to regulate (as reducing) and cause the higher flux through pentose-phosphate pathway increases and the lysine production increase thereby cause NADPH to produce.

Term " pentose-phosphate pathway " comprise that fine chemicals such as Methionin form or synthetic in the approach that participated in of the pentose phosphate enzyme polypeptide of the coded by said gene of encoding human synthetic enzyme (for example by), compound (for example precursor, substrate, intermediate or product), cofactor of being utilized or the like.Pentose-phosphate pathway changes the glucose glycan molecule into biological chemistry available less molecule.

In order more easily to understand the present invention, at first define some term in the literary composition.

Term " pentose phosphate biosynthetic pathway " comprise that fine chemicals such as Methionin form or synthetic in the biosynthetic pathway that participated in of pentose phosphate biosynthesis gene, the enzyme polypeptide of the coded by said gene of encoding human synthetic enzyme (for example by), compound (for example precursor, substrate, intermediate or product), cofactor of being utilized or the like.Term " pentose phosphate biosynthetic pathway " comprise cause fine chemicals such as Methionin in microorganism synthetic (for example in the body) biosynthetic pathway and cause fine chemicals such as the external synthetic biosynthetic pathway of Methionin.

Term " pentose phosphate biosynthetic pathway protein " or " pentose phosphate biosynthetic pathway enzyme " comprise those peptides, polypeptide, protein, enzyme and its fragment, for example serum lactic dehydrogenase of direct or indirect participation pentose phosphate biosynthetic pathway.

Term " pentose phosphate biosynthetic pathway gene " comprises that coding directly or indirectly participates in those genes and the gene fragment of peptide, polypeptide, protein and the enzyme of pentose phosphate biosynthetic pathway, for example lactate dehydrogenase gene.

Term " amino acid biosynthetic pathway gene " meaning is to comprise encoding participating in those genes and the gene fragment of amino acid synthetic peptide, polypeptide, protein and enzyme (for example serum lactic dehydrogenase) directly.These genes can with host cell in naturally occurring and participate in the host cell arbitrary amino acid and particularly those genes of Methionin synthetic are identical.

Term " Methionin biosynthetic pathway gene " comprises that coding directly or indirectly participates in those genes and the gene fragment of Methionin synthetic peptide, polypeptide, protein and enzyme (for example serum lactic dehydrogenase).These genes can with host cell in naturally occurring and to participate in the host cell those genes of Methionin synthetic identical.Alternatively, can modify or suddenly change this kind gene, for example gene can comprise and has no significant effect bioactive modification of coded protein or sudden change.For example, can be by mutagenesis or by introducing or substitute one or more Nucleotide or natural gene being modified by the nonessential zone of removing in the gene.This type of modification can easily realize according to standard technique.

Term " the Methionin biosynthetic pathway protein " meaning is to comprise participating in those peptides of Methionin synthetic, polypeptide, protein, enzyme and its fragment directly.These protein can with host cell in naturally occurring and to participate in the host cell those protein of Methionin synthetic identical.Alternatively, can modify or suddenly change this proteinoid, for example protein can comprise and has no significant effect bioactive modification of protein or sudden change.For example, can be by mutagenesis or by introducing or substitute one or more amino acid (preferably substituting) or natural protein being modified by the nonessential zone of removing in the protein by conserved amino acid.This type of modification can easily realize according to standard technique.Alternatively, Methionin biosynthesizing protein can be the protein of relative particular host cell for external source.This proteinoid can carry any biology that coding has the gene of same or similar biosynthesizing action protein matter.

Term " carbon flux " refers to compare along the quantity of specific pathways metabolism progressive glucose molecule with competitive approach.Particularly, the increase of NADPH can realize by the carbon Flux Distribution that changes between interior glycolysis-of this organism and pentose-phosphate pathway in the microorganism.

" lactate dehydrogenase activity " comprises as according in the standard technique body or any activity of bringing into play of lactic dehydrogenase zymoprotein, polypeptide or the nucleic acid molecule of external test.Serum lactic dehydrogenase is present in protokaryon and the eukaryote.Preferably, lactate dehydrogenase activity comprises that reversible NAD is dependent pyruvic acid and lactic acid is transformed mutually.In vertebrates muscle and lactic-acid-bacterium, it is representing the glucolytic final step of anaerobism.

Term " fine chemicals " is art-recognized and comprises by the biogenic for example molecule of (but being not limited to) pharmacy industry, agricultural, cosmetic industry of multiple industry that is used for.This compounds includes for example tartrate of machine acid, methylene-succinic acid, diaminopimelic acid, raw albumen amino acid (proteinogenicamimo acid) and non-raw albumen amino acid (non-proteinogenic amino acid), purine and pyrimidine bases, nucleosides and Nucleotide (as are described in Kuninaka, A. (1996) Nucleotides andrelated compounds, the 561-612 page or leaf, Biotechnology the 6th volume, people such as Rehm compile, VCH:Weinheim and the reference that is comprised therein), lipid, saturated and unsaturated fatty acids (for example arachidonic acid), glycols (for example propylene glycol and butyleneglycol), carbohydrate (for example hyaluronic acid and trehalose), aromatic compound (aromatic amine for example, Vanillin and indigo), VITAMIN and cofactor (as are described in Ullmann ' s Encyclopedia of Industrial Chemistry, the A27 volume, " Vitamins ", 443-613 volume (1996) VCH:Weinheim and reference wherein; And Ong, A.S., Niki, E. and Packer, L. (1995) " Nutrition; Lipids; Health; and Disease " UNESCO procceedings/Confederation of Scientific andTechnological Associations in Malaysia and Society for Free RadicalResearch-Asia, hold in Malaysian Penang in 1-3 day in September, 1994, AOCS press, (1995)), enzyme, polyketide (people such as Cane, Science 282:63-68) and be described in Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, all other chemical substances in ISBN:0818805086 and the reference wherein (1998).The metabolism and the purposes of some fine chemicals in the middle of describing in detail below.

Amino acid metabolism and purposes

Therefore amino acid has constituted the basic structural unit of all proteins, and amino acid is that the normal cell functions of all organisms is necessary.Term " amino acid " is art-recognized.Raw albumen amino acid has 20 kinds, they are proteinic structural units, in protein, amino acid is connected by peptide bond, but not raw albumen amino acid (known wherein hundreds of) is non-existently in the protein under the normal circumstances (to see Ulmann ' s Encyclopedia of Industrial Chemistry, the A2 volume, 57-97 page or leaf VCH:Weinheim (1985)).Though L-amino acid is common in the natural unique type that exists in the protein that exists, amino acid can be D-optical configuration and L-optical configuration.Each amino acid whose biosynthesizing and degradation pathway have characterized (seeing for example Stryer, L.Biochemistry, the 3rd edition, 578-590 page or leaf, (1988)) well in 20 kinds of raw albumen amino acid in prokaryotic cell prokaryocyte and eukaryotic cell." essential " amino acid (Histidine, Isoleucine, leucine, Methionin, methionine(Met), phenylalanine, Threonine, tryptophane and Xie Ansuan) can easily be transformed into 11 kinds of " nonessential " amino acid (L-Ala, arginine, l-asparagine, aspartic acid, halfcystine, L-glutamic acid, glutamine, glycine, proline(Pro), Serine and tyrosine) by the single creature route of synthesis, and why " essential " amino acid is so named is because these amino acid bio synthetic complicacy make them be generally nutritional needs.Higher animal has kept some the amino acid whose abilities in synthetic these amino acid really, still then must supply with from diet for the synth essential amino acid of realizing normal protein matter.

Except their functions in the protein biosynthesizing, these amino acid itself are interesting chemical substance still, and has been found that many amino acid can have multiple application in grocery trade, feed industry, chemical industry, cosmetic industry, agricultural and pharmacy industry.Methionin is the important amino acid in the human nutrition still not, and it still is the important amino acid in monogastric animal such as poultry and the pig nutrition.(msg powder type, MSG) and be widely used in whole grocery trade, aspartic acid, phenylalanine, glycine and halfcystine also are like this to L-glutamic acid as odor control additive the most commonly.Glycine, L-methionine(Met) and tryptophane all are used for pharmacy industry.Glutamine, Xie Ansuan, leucine, Isoleucine, Histidine, arginine, proline(Pro), Serine and L-Ala can be used for pharmacy industry and cosmetic industry.Threonine, tryptophane and D/L-methionine(Met) are common fodder additives (Leuchtenberger, W. (1996) Amino aids-technical production and use, the 466-502 page or leaf,: people such as Rehm (editor) Biotechnology the 6th volume, the 14a chapter, VCH:Weinheim).In addition, have been found that these amino acid can be used as the precursor of synthesizing amino acid and protein synthesis, for example N-acetylcystein, S-carboxymethyl-L-halfcystine, (S)-5-hydroxyryptophan and be described in Ulmann ' sEncyclopedia of Industrial Chemistry, the A2 volume, the 57-97 page or leaf, VCH:Weinheim, other material in 1985.

The biosynthesizing of these natural amino acids in organism that can produce them such as bacterium carried out characterizing (summary synthetic for the bacterium amino acid bio and that regulate is seen Umbarger, H.E. (1978) Ann.Rev.Biochem.47:533-606) well.L-glutamic acid is synthetic by the reduction amination effect of tricarboxylic acid cycle intermediate α-Tong Wuersuan.Produce glutamine, proline(Pro) and the arginine each from L-glutamic acid subsequently.The biosynthesizing of Serine is with three step process of 3-phoshoglyceric acid (glycolysis-intermediate) beginning, has produced Serine after oxygenizement, transamination and hydrolysing step.Halfcystine and glycine produce from Serine; Halfcystine forms by the condensation of homocysteine and Serine, and glycine is to form by be transferred to tetrahydrofolic acid (THFA) through the side chain beta carbon in the catalytic reaction of serine transhydroxymethylase.Phenylalanine and tyrosine are synthetic from glycolysis-and pentose-phosphate pathway precursor erythrose 4-phosphoric acid and phosphoenolpyruvic acid in 9 step biosynthetic pathways, and they are the latter two steps difference after prephenic acid is synthetic only.Tryptophane can also produce from these two starting molecules, but its synthetic be the approach in one 11 step.Tyrosine can also be synthetic from phenylalanine in the catalytic reaction of Phenylalanine hydroxylase.L-Ala, Xie Ansuan and leucine all are the biosynthetic products of glycolysis-end product pyruvic acid.Aspartic acid forms from tricarboxylic acid cycle intermediate oxaloacetic acid.In l-asparagine, methionine(Met), Threonine and the Methionin each produces by the aspartic acid conversion.Isoleucine forms from Threonine.Histidine produces through 9 step approach of complexity from activating sugared ribose 5-phosphate-1-tetra-sodium.

Surpassing the amino acid that protein synthesis needs in the cell can not be stored, but degraded (is seen Stryer for summary with the main metabolic pathway that provides intermediate to be used for cell, L.Biochemistry, the 3rd edition, the 21st chapter, " Amino Acid Degradation and the Urea Cycle ", 495-516 page or leaf, (1988)).Though cell can be transformed into unnecessary amino acid useful metabolism intermediate, from synthesizing amino acid energy needed, precursor molecule and enzyme aspect, amino acid whose production is expensive.Therefore, not surprised, amino acid bio is synthetic to be regulated by feedback inhibition, wherein the existence of specific amino acids is used for slowing down and stops fully himself producing that (summary for the Feedback mechanism in the amino acid biosynthetic pathway is seen Stryer, L.Biochemistry, the 3rd edition, the 24th chapter,: " Biosynthesis of Amino Acids and Heme ", 575-600 page or leaf, (1988)).Therefore, the turnout of any specific amino acids is by this amino acid whose quantity limitation of existing in the cell.The metabolism and the purposes of VITAMIN, cofactor and nutriment (Nutraceutical)

VITAMIN, cofactor and nutriment comprise the molecule of another group, and higher animal has been lost the ability of synthesizing them, thus must absorb, but it can be biological synthetic as bacterium by other easily.These molecules are biologically active molecule own or are the biologically active substance precursor as electron carrier in many pathways metabolisms or intermediate.These compounds are except having nutritive value, and they also have tangible industrial value as tinting material, antioxidant and catalyzer or other processing auxiliary material.(for the summary of structure, activity and the industrial application of these compounds, seeing for example Ullmann ' sEncyclopedia of Industrial Chemistry, " Vitamins ", A27 volume, 443-613, VCH:Weinheim, 1996).Term " VITAMIN " is known in the art and comprise that biological normal function is required but can't be by the synthetic nutrition of this biology own.The VITAMIN group can comprise cofactor and nutriment compound.Term " cofactor " comprises the active required non-protein compound of normal enzyme.These compounds can be organic or inorganic; Cofactor molecule of the present invention is preferably organic.Term " nutriment " is included in the food additive that plant and animal, particularly philtrum have the health-promoting effect.The example of this quasi-molecule is VITAMIN, antioxidant and some lipid (as polyunsaturated fatty acid).

The biosynthesizing of these molecules in the biology that can produce them (as bacterium) at length characterized (Ullmann ' s Encyclopedia of Industrial Chemistry, " Vitamins ", the A27 volume, the 443-613 page or leaf, VCH:Weinheim, 1996, Michal, G (1999) BiochemicalPathways:An Atlas of Biochemistry and Molecular Biology, John Wilely﹠amp; Sons; Ong, A.S., Niki, E. and Packer, L. (1995) " Nutrition; Lipids, Healthand Disease " UNESCO procceedings/Confederation of Scientific andTech nological Associations in Malaysia and the Society for Free RadicalResearch-Asia, 1-3 day in September, 1994 holds in Malaysian Penang, AOCS Press, Champaign, IL X, 374S).

Thiamines (vitamins B ₁) form by pyrimidine and the chemical coupling of thiazole part.Riboflavin (vitamins B ₂) synthetic from guanosine 5 '-triphosphoric acid (GTP) and ribose-5 '-phosphoric acid.Riboflavin is used for synthetic flavine mononucleotide (FMN) and flavin adenine dinucleotide (FAD).This compound family is referred to as " vitamin B6 " (for example pyridoxol, Pyridoxylamine, pyridoxal 5 '-phosphoric acid and commercial pyridoxine hydrochloride that uses), and it is all the derivative of common structure unit 5-hydroxyl-6-picoline.Pantothenate (pantothenic acid, R-(+)-N-(2,4-dihydroxyl-3,3-dimethyl-1-oxygen-butyl)-Beta-alanine) can be by chemosynthesis or fermentative preparation.The biosynthetic final step of pantothenic acid is the Beta-alanine of ATP driving and the condensation of pantoic acid.The enzyme of being responsible for being transformed into pantoic acid and being transformed into Beta-alanine and being condensed into the biosynthesizing step of pantothenic acid is known.The metabolic activity form of pantothenic acid is a coenzyme A, and its biosynthesizing is produced by 5 enzymatic steps.Pantothenic acid, pyridoxal phosphate 5 '-phosphoric acid, halfcystine and ATP are the precursor of coenzyme A.These enzymes are the formation of catalysis pantothenic acid not only, also catalysis (R)-pantoic acid, (R)-pantoyl lactone, (R)-panthenol (pro-vitamin B ₅), the formation of pantetheine (and derivative) and coenzyme A.

Study in the microorganism vitamin H in great detail from the biosynthesizing of precursor molecule pimeloyl-CoA, and identified several related genes.It is synthetic and be the protein-based member of nifS to have shown that many respective egg white matters participate in the Fe duster compounds.Thioctic Acid is derived from sad and as the coenzyme in the energy metabolism, it becomes the part of pyruvate dehydrogenase complex and ketoglurate dehydrogenase mixture.The folic acid class is for all being derived from one group of material of folic acid, and folic acid is derived from L-L-glutamic acid, para-amino benzoic acid and 6-methyl petrin successively.The folic acid that begins from metabolic intermediate guanosine 5 '-triphosphoric acid (GTP), L-L-glutamic acid and para-amino benzoic acid and the biosynthesizing of derivative thereof in certain micro-organisms, have been studied in great detail.

Corrinoid (as cobalami, vitamins B particularly ₁₂) and porphyrin belong to the one group of chemical that characterizes by the tetrapyrrole loop systems.Vitamins B ₁₂The biosynthesizing too complex so that understand fully as yet, but existing known many relevant enzymes and substrate.

Nicotinic acid (nicotinate) and niacinamide are pyridine derivate, are also referred to as " nicotinic acid ".Nicotinic acid is the precursor of important coenzyme NAD (Reduced nicotinamide-adenine dinucleotide) and NADP (Triphosphopyridine nucleotide, reduced) and reduction form thereof.

Though have some to produce in these compounds, as riboflavin, vitamins B by extensive microorganism culturing ₆, pantothenic acid and vitamin H, but the production of these compounds on technical scale is mainly based on acellular chemosynthesis.Has only vitamins B ₁₂Can only pass through fermentative production owing to its synthetic complicacy.In vitro method needs sizable input material and time, and cost is usually very high.

The metabolism of purine, pyrimidine, nucleosides and Nucleotide and purposes

Being used for the gene of purine and pyrimidine metabolic and corresponding protein thereof, to verify in the treatment of tumor disease and virus infection be important target.Term " purine " or " pyrimidine " comprise nitrogenous base, and it is the moiety of nucleic acid, coenzyme and Nucleotide.Term " Nucleotide " comprises the infrastructure element of nucleic acid molecule, and it is made up of nitrogenous base, pentose (this sugar is ribose in RNA, and this sugar is the D-ribodesose in DNA) and phosphoric acid.Term " nucleosides " comprises the molecule as nucleotide precursor, but it lacks the phosphoric acid part that Nucleotide had.Biosynthesizing by suppressing these molecules or mobilize and form nucleic acid molecule by suppressing it, it is synthetic to suppress RNA and DNA; In cancer cells, this active target is suppressed then can suppress tumour cell division and the ability of duplicating.In addition, also have some Nucleotide not form nucleic acid molecule but as energy storage (being AMP) or coenzyme (being FAD and NAD).

Several publications have been described these chemical by influencing purposes in the medical science indication of purine and/or pyrimidine metabolic (Christophrson for example, R.I. and Lyons, S.D. (1990), " Potentinhibitors of de novo pyrimidine and purine biosynthesis aschemotherapeutic agents ", Med.Res.Reviews 10:505-548).The research of the enzyme that participates in purine and pyrimidine metabolic is concentrated on can be as (Smith, J.L. (1995) " Enzymes in Nucleotide Synthesis " Curr.Opin.Struct.Biol.5 752-757 on the new drug development of for example immunosuppressor or antiproliferative; (1995) Biochem.Soc.Transact.23877-902).Yet, purine and pyrimidine bases, nucleosides and Nucleotide also have other purposes: as the intermediate in the multiple fine chemicals biosynthesizing (as thiamines, S-adenosylmethionine, folic acid or riboflavin), as the energy carrier (for example ATP or GTP) of cell and for chemical self usually as odorant (for example IMP or GMP) or be used for many medical uses and (see for example Kuninaka, A., (1996) " Nucleotidesand Related Compounds in Biotechnology ", the 6th volume, editor Rehm etc., VCH:Weinheim, the 561-612 page or leaf).The enzyme that participates in purine, pyrimidine, nucleosides or nucleotide metabolism also day by day become the chemical (comprising mycocide, weedicide and sterilant) developed for Crop protection at target.

The metabolism of these compounds in bacterium characterized (for summary, see for example Zalkin, H. and Dixon, J.E. (1992) " De novo purine nucleotide biosynthesis ",: Progressin Nucleic Acids Research and Molecular biology, the 42nd volume, AcademicPress, 259-287 page or leaf; And Michal, G. (1999), " Nucleotides andNucleosides "; The 8th chapter: Biochemical Pathways:An Atlas of Biochemistryand Molecular Biology, Wiley, New York).Purine metabolism is the problem of further investigation, and it is that the cell normal function is necessary.Purine metabolism is impaired in the higher animal may cause serious disease, for example gout.By series of steps from ribose 5-phosphoric acid via midbody compound inosine 5 '-phosphoric acid (IMP) purine biosynthesis Nucleotide, cause producing guanosine 5 '-single phosphoric acid (GMP) or adenosine 5 '-single phosphoric acid (AMP), can form the triphosphoric acid form of using as Nucleotide thus easily.These compounds can also be used as energy storage, so that their degraded can provide energy for the many different Biochemical processes in the cell.The pyrimidine biosynthesizing forms uridine 5 '-single phosphoric acid (UMP) from ribose 5-phosphoric acid and carries out.Afterwards, UMP is transformed into cytidine 5 '-triphosphoric acid (CTP).The deoxidation form of all these Nucleotide all is to become the bisphosphate ribodesose form of Nucleotide by the bisphosphate ribose form of Nucleotide and produce in a step reduction reaction.After the phosphorylation, it is synthetic that these molecules can participate in DNA.

Trehalose Metabolism and purposes

Trehalose is by with α, and two glucose molecules that α-1,1 key links together are formed.It is used as the additive in sweeting agent, drying or frozen product and the beverage usually in grocery trade.Yet it also is used for pharmacy industry or cosmetic industry and biotechnology industry and (for example sees people such as Nishimoto, (1998) U.S. Patent number 5 759 610; Singer, M.A. and Lindquist, S. (1998) Trends Biotech.16460-467; Paiva, C.L.A and Panek, A.D. (1996) Biotech Ann.Rev.2293-314; And Shiosaka, M. (1997) J.Japan 172 97-102).Trehalose is released into substratum on every side by the enzyme production of many microorganisms and in natural mode, can separate trehalose from described substratum by methods known in the art.

Thereby recombinant microorganism and be used for the method that culturing micro-organisms produces fine chemicals I.

The characteristic of the inventive method is a microorganism, the recombinant microorganism that for example preferably includes carrier described in the literary composition or gene (for example gene of wild type gene and/or sudden change) and/or cultivate in the mode that causes purpose fine chemicals such as Methionin to produce.Term " reorganization " microorganism comprise with its from natural have microorganism to compare to carry out hereditary change, modification or engineered (for example genetically engineered transformation) thus make it present genotype change, that modify or different and/or the microorganism (for example bacterium, yeast cell, fungal cell or the like) of phenotype (for example, when genetic modification influences the nucleic acid sequence encoding of microorganism).Preferably, the present invention's " reorganization " thus microorganism has carried out genetically engineered transformation makes bacterial gene or gene product expression deficiency described in its at least a literary composition, biosynthesizing enzyme coding gene for example lactate dehydrogenase gene and/or the biosynthetic enzyme such as the serum lactic dehydrogenase of expressing from recombinant vectors that is contained in the recombinant vectors preferably as described herein.Those of ordinary skill will recognize, gene product expression or express that insufficient microorganism can produce gene product or owing to the nucleotide sequence and/or the not enough gene product expression deficiency that makes of expression of gene of encoding gene product.In one embodiment, recombinant microorganism has biosynthetic enzyme (for example serum lactic dehydrogenase) activity of reduction.

In certain embodiments of the invention, except lactate dehydrogenase gene or enzyme, at least a gene or protein can go to regulate, thereby increase the amino acid whose production of L-.For example, gene or the enzyme such as the biosynthetic pathway of glycolysis-, anaplerosis, tricarboxylic acid cycle, pentose phosphate circulation or amino acid output can be gone to regulate.In addition, regulatory gene or protein can be gone to regulate.

In multiple embodiments, expression of gene can increase so that increase proteinic intracellular reactive of this coded by said gene or concentration, finally improves the amino acid whose production of purpose thus.Each technician of this area can use multiple technologies to obtain expected results.For example the skilled work personnel can increase copy number, use effectively start of a certain gene or a plurality of genes and/or use coding to have the gene or the allelotrope of highly active corresponding enzyme.Use method of the present invention, for example cross the expression specific gene, the activity of respective egg white matter and concentration can be based on initial activity or concentration increases about at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500%, 1000% or 2000%.

In multiple embodiments, go regulatory gene can include, but is not limited to one of following at least gene or protein:

● the ask gene of encoder feedback resistance E.C. 2.7.2.4. (as disclosed among the international publication number WO2004069996);

● coding dihydrodipicolinate synthase's dapA gene (as being disclosed in the SEQ ID NO:55 and 56 among the international publication number WO200100843 respectively);

● the asd gene of coding aspartate-semialdehyde dehydrogenase (as being disclosed in the SEQ ID NO:3435 and 6935 in the European publication numbers 1108790 respectively);

● coding dihydrodipicolinate reductase's dapB gene (as being disclosed in the SEQ ID NO:35 and 36 among the international publication number WO200100843 respectively);

● the ddh gene of coding diaminopimelate dehydrogenase (as being disclosed in the SEQ ID NO:3444 and 6944 in the European publication numbers 1108790 respectively);

● the lysA gene of coding diaminopimelic acid epimerase (as being disclosed in the SEQ ID NO:3451 and 6951 in the European publication numbers 1108790 respectively);

● the lysE gene of coding Methionin output (as being disclosed in the SEQ ID NO:3455 and 6955 in the European publication numbers 1108790 respectively);

● the pycA gene of coding pyruvate carboxylase (as being disclosed in the SEQ ID NO:765 and 4265 in the European publication numbers 1108790 respectively);

● the zwf gene (as being disclosed in the SEQ ID NO:243 and 244 among the international publication number WO200100844 respectively) of coding glucose-6-phosphate dehydrogenase (G6PD);

● the pepCL gene of coding Phosphoenolpyruvate carboxylase (as being disclosed in the SEQ ID NO:3470 and 6970 in the European publication numbers 1108790 respectively);

● the gap gene of encoding glycerol aldehyde-3-phosphate dehydrogenase (as being disclosed in the SEQ ID NO:67 and 68 among the international publication number WO200100844 respectively);

● the zwa1 gene of coding RPF protein precursor (as being disclosed in the SEQ ID NO:917 and 4417 in the European publication numbers 1108790 respectively);

● the tkt gene (as being disclosed in the SEQ ID NO:247 and 248 among the international publication number WO200100844 respectively) of coding transketolase;

● the tad gene (as being disclosed in the SEQ ID NO:245 and 246 among the international publication number WO200100844 respectively) of coding transaldolase;

● the mqo gene (as being disclosed in the SEQ ID NO:569 and 570 among the international publication number WO200100844 respectively) of coding menaquinone oxydo-reductase;

● the tpi gene (as being disclosed in the SEQ ID NO:61 and 62 among the international publication number WO200100844 respectively) of coding triose-phosphate isomerase;

● the coding kinase whose pgk gene of 3-phoshoglyceric acid (as being disclosed in the SEQ ID NO:69 and 70 among the international publication number WO200100844 respectively); With

● the sigC gene of coding RNA polymerase sigma factor sigC (as being disclosed in the SEQ ID NO:284 and 3784 in the European publication numbers 1108790 respectively).

In specific embodiments, gene can be express and/or protein active can increase.

Alternatively, in other embodiments, expression of gene can be that weaken, that reduce or downtrod, by the proteinic intracellular reactive or the concentration of this coded by said gene, finally improves the amino acid whose production of purpose so that reduce (for example eliminating) thus.For example, those skilled in the art can use weak promoter.Alternatively or in combination, the skilled work personnel can use coding to have the gene of SA corresponding enzyme or the gene or the allelotrope of allelotrope or inactivation corresponding gene or enzyme.Utilize method of the present invention, the activity of respective egg white matter or concentration can be reduced to about 0-50%, 0-25%, 0-10%, 0-9%, 0-8%, 0-7%, 0-6%, 0-5%, 0-4%, 0-3%, 0-2% or the 0-1% of wild-type protein activity or concentration.

In certain embodiments, go regulatory gene can include, but is not limited to one of following at least gene or protein:

● the pepCK gene (as being disclosed in the SEQ ID NO:179 and 180 among the international publication number WO200100844 respectively) of coding phosphoenolpyruvate carboxykinase;

● the mal E gene of coding malic enzyme (as being disclosed in the SEQ ID NO:3328 and 6828 in the European publication numbers 1108790 respectively);

● the glgA gene of encoding glycogen synthase (as being disclosed in the SEQ ID NO:1239 and 4739 in the European publication numbers 1108790 respectively);

● the pgi gene (as being disclosed in the SEQ ID NO:41 and 42 among the international publication number WO200100844 respectively) of coding G-6-P isomerase;

● the dead gene of the rna helicase enzyme that coding ATP relies on (as being disclosed in the SEQ ID NO:1278 and 4778 in the European publication numbers 1108790 respectively);

● the menE gene of coding o-succinyl-phenylformic acid-CoA ligase enzyme (as being disclosed in the SEQ ID NO:505 and 4005 in the European publication numbers 1108790 respectively);

● the citE gene (as being disclosed in the SEQ ID NO:547 and 548 among the international publication number WO200100844 respectively) of coding citrate lyase β chain;

● the mikE17 gene of encoding transcription instrumentality (as being disclosed in the SEQ ID NO:411 and 3911 in the European publication numbers 1108790 respectively);

● the poxB gene (as being disclosed in the SEQ ID NO:85 and 86 among the international publication number WO200100844 respectively) of coding pyruvic oxidase;

● the zwa2 gene of coding RPF protein precursor (as disclosed in the European publication numbers 1106693); With

● the sucC gene of coding succinyl CoA synthetase (as disclosed in the European publication numbers 1103611).

In specific embodiments, expression of gene can be that weaken, that reduce or downtrod and/or activity of proteins can reduce.

Term " through the microorganism of operation " comprises engineered (for example genetically engineered transformation) thereby or modifies so that cause the microorganism that pathways metabolism is destroyed or change causes the carbon metabotic change.When enzyme with than its expression in comparable wild-type cell more during low expression level, then enzyme " is expressed not enough " in the metabolic engineering engineered cells, the situation that it includes, but is not limited to not express completely.The genetic expression deficiency can cause being reduced by the activity of the protein of this coded by said gene (for example serum lactic dehydrogenase).

The modification of this quasi-microorganism or engineered can carrying out, method according to any means described in the literary composition include, but is not limited to biosynthetic pathway go regulate and/or the expression deficiency of at least a biosynthetic enzyme." through what operate " enzyme (for example " through what operate " biosynthetic enzyme) comprises a kind of like this enzyme, be that its expression or produce has changed or modifies so that compare upstream or downstream precursor, substrate or the product of at least a enzyme with for example wild-type or naturally occurring enzyme and be changed or modify, for example have the activity of reduction.

Term " is expressed insufficient " or " expressing not enough " comprises that gene product (for example pentose phosphate biosynthetic enzyme) is to express than the microorganism before operation or without the lower level of expression in the comparable microorganism of operation.In one embodiment, (for example genetically engineered transformation) of microorganism hereditary operation is so that with than the microorganism before operation or without the lower horizontal expression gene product of expression level in the comparable microorganism of operation.Genetic manipulation can include, but is not limited to change or modify to be regulated sequence or expresses relevant site (for example by removing strong promoter with specific gene, inducible promoters or multiple promoter), modify the chromosomal foci of specific gene, change the nucleotide sequence adjacent for example ribosome bind site or transcription terminator with specific gene, reduce the copy number of specific gene, modify the protein that the participation specific gene is transcribed and/or the specific gene product is translated and (for example regulate protein, inhibition, enhanser, transcriptional activator or the like), perhaps go to regulate any other ordinary method (include but not limited to use antisense nucleic acid molecule, perhaps knock out or seal other method that target protein is expressed) that specific gene is expressed in this area.

In another embodiment, microorganism can be on self or the environment through operation so that make the gene product expression deficiency than the microorganism before operation or without the higher level of expression in the comparable microorganism of operation.For example, microorganism can reduce the reagent that specific gene is transcribed and/or the specific gene product is translated with known or suspection to be handled or cultivates in the presence of it, transcribes and/or translate reduction thereby make.Alternatively, microorganism can be cultivated under selected temperature to increase transcribing and/or the translation of specific gene product of specific gene, transcribes and/or translates thereby reduce.

Term " goes to regulate " or " going to regulate " comprises the change or the modification of the gene of enzyme at least one encoding human route of synthesis in the microorganism, so that the level of biosynthetic enzyme or activity are changed or modify in the microorganism.Preferably, at least one encoding human route of synthesis the gene of enzyme be changed or modification so that gene product reduces, reduce the activity of this gene product thus.Term " go regulate approach " also can comprise a kind of like this biosynthetic pathway, and wherein the gene of enzyme is changed or modifies so that the level of more than one biosynthetic enzymes or activity are changed or modify in more than one encoding human route of synthesis." go to regulate " phenomenon of the sub-microorganism in ability source of approach (for example removing to regulate more than one genes in the given biosynthetic pathway simultaneously) in microorganism, wherein more than one enzyme (for example two or three biosynthetic enzyme) is coded by the gene (being called " operon ") of existence adjacent one another are on the sequential portion of genetic material.

Term " operon " comprises the collaborative unit of genetic expression, its comprise promotor and possible with one or more, the preferred relevant regulatory element of at least two kinds of structure genes (for example gene of codase such as biosynthetic enzyme).The expression of structure gene can be worked in coordination with adjusting, for example by regulating protein bound to regulatory element or the antitermination by transcribing.Structure gene can be transcribed and be formed the proteinic single mRNA of coding entire infrastructure.Because the collaborative adjusting of the included gene of operon, the change of single promotor and/or regulatory element or modification can cause change and the modification by each coded gene product of this operon.The change of regulatory element and modification can include, but is not limited to remove endogenous promotor and/or regulatory element; Add strong promoter, inducible promoters or multiple promoter or remove the adjusting sequence so that the expression of gene product obtains modifying; Modify the chromosome position of operon; Change the nucleotide sequence ribosome bind site for example in or the operon adjacent with operon; Reduce the operon copy number; Modify and participate in the protein (for example regulating protein, inhibition, reinforce (enhancer), transcriptional activator or the like) that operon is transcribed and/or the operon gene product is translated, any other ordinary method (including but not limited to use antisense nucleic acid molecule) of perhaps going regulatory gene to express in this area as the expression of sealing arrestin matter.Go to regulate and also relate to the enzyme that the coding region that changes one or more genes for example has the feedback resistance with generation or has higher or lower activity specific.

The present invention's particularly preferred " reorganization " microorganism has carried out genetically engineered transformation, so that make the gene or the gene product expression deficiency of bacterial origin.Term " bacterial origin " or " from " for example naturally be present in the gene of bacterium or comprising of bacterium by the coded gene product of bacterial gene (for example lactate dehydrogenase gene).

Methodological characteristic of the present invention is that wherein one or more genes such as lactate dehydrogenase gene are expressed not enough or had the recombinant microorganism of the lactate dehydrogenase activity of reduction.The particularly preferred recombinant microorganism of the present invention (for example Corynebacterium glutamicum, acetylglutamate rod bacillus (Corynebacteriumacetoglutamicum), Corynebacterium acctoacidophlum (Corynebacterium acetoacidophilum) and heat are produced ammonia rod bacillus (Corynebacterium thermoaminogenes) or the like) has carried out genetically engineered transformation makes biosynthetic enzyme (for example serum lactic dehydrogenase, aminoacid sequence is SEQ ID NO:2 or coded by nucleic acid sequence SEQ ID NO:1) express deficiency.

Other preferred " reorganization " microorganism of the present invention goes enzyme to regulate in pentose-phosphate pathway.Term " microorganism with pentose-phosphate pathway of regulating " is included in the gene of enzyme of at least a coding pentose-phosphate pathway to have the microorganism that changes or modify or have the microorganism that changes or modify in the operon of the gene that comprises more than one coding pentose-phosphate pathway enzymes.Preferably " microorganism with pentose-phosphate pathway of regulating " carried out genetically engineered transformation so that excellent bacillus (for example Corynebacterium glutamicum) biosynthesizing expression of enzymes deficiency (for example engineered so that lactic dehydrogenase expression of enzymes deficiency).

In another preferred embodiment, recombinant microorganism is designed or engineered so that one or more pentose phosphate biosynthesizing expression of enzymes not enough or gone to regulate.

In another preferred embodiment, microorganism of the present invention is expressed not enough to the gene of bacterial origin or biosynthetic enzyme (for example pentose phosphate biosynthetic enzyme) or it is suddenlyd change.Term " bacterial origin " or " from " for example bacterium comprises by the coded gene product of bacterial gene (for example serum lactic dehydrogenase).

In one embodiment, recombinant microorganism of the present invention is gram-positive microorganism (for example microorganism that can keep basic dyestuff such as Viola crystallina owing to the existence of Gram-positive wall around the microorganism).An embodiment preferred, recombinant microorganism is to belong to be selected from bacillus (Bacillus), brevibacterium sp (Brevibacterium), Corynebacterium (Cornyebacterium), lactobacillus (Lactobacillus), lactococcus (Lactococci) and streptomyces (Streptomyces) microorganism.In the embodiment that is more preferably, recombinant microorganism is the Corynebacterium microorganism.In a further preferred embodiment, recombinant microorganism is selected from Corynebacterium glutamicum, acetylglutamate rod bacillus, Corynebacterium acctoacidophlum or heat and produces ammonia rod bacillus.In particularly preferred embodiments, recombinant microorganism is a Corynebacterium glutamicum.

The important aspect of the present invention relates to cultivates recombinant microorganism described in the literary composition, thereby produces purpose compound (for example purpose fine chemicals).Term " cultivation " comprises is maintained live microorganism of the present invention and/or grow (culture or bacterial strain are maintained and/or grow).In one embodiment, microorganism culturing of the present invention is in liquid nutrient medium.In another embodiment, microorganism culturing of the present invention is in solid medium or semisolid medium.In preferred embodiments, microorganism culturing of the present invention in comprise that microorganism is kept and/or the necessary or favourable nutraceutical substratum (for example aseptic liquid nutrient medium) of growing in.Operable carbon source comprises carbohydrate and carbohydrate, for example glucose, sucrose, lactose, fructose, maltose, molasses, starch and Mierocrystalline cellulose; Oil ﹠ fat, for example soybean oil, Oleum Helianthi, peanut oil and Oleum Cocois; Lipid acid, for example Palmiticacid, stearic acid and linolic acid; Alcohols, for example glycerine and ethanol; And organic acid acetic acid for example.In preferred embodiments, fructose or sucrose are as carbon source.These materials can use separately or use as mixture.

Operable nitrogenous source comprises and comprises nitrogen organic compound for example peptone, yeast extract, meat extract, malt extract, corn steep liquor, soyflour and urea, perhaps for example ammonium sulfate, ammonium chloride, ammonium phosphate, volatile salt and ammonium nitrate of mineral compound.Nitrogenous source can use separately or use as mixture.Operable phosphorus source is phosphoric acid, potassium primary phosphate or dipotassium hydrogen phosphate or corresponding sodium salts.In addition, substratum also must comprise growth necessary metal-salt, for example sal epsom or ferrous sulfate.At last, except that above-mentioned substance, can also use essential growth helping matter, for example amino acid and VITAMIN.And, suitable precursor can be added in the substratum.Described supply material can single batch adds in the substratum or in culturing process suitably to be supplied with.

Preferably, microorganism of the present invention is cultivated under control pH.Term " pH that controls " comprises any pH that causes purpose fine chemicals such as Methionin to be produced.In one embodiment, microorganism culturing is in being approximately under 7 the pH condition.In another embodiment, under the pH condition between the microorganism culturing in 6.0 to 8.5.Expect that pH can keep by any means well known by persons skilled in the art.For example, use basic cpd, perhaps suitably control the pH of culture such as phosphoric acid or vitriolic acidic cpd such as sodium hydroxide, potassium hydroxide, ammonium or ammoniacal liquor.

Equally preferably, microorganism of the present invention can be cultivated under control venting condition.Term " inflation of controlling " comprises that abundant inflation (for example oxygen) is to cause expecting the generation of fine chemicals such as Methionin.In one embodiment, inflation is to control by the oxygen level of regulating in the culture, for example is dissolved in the amount of the oxygen in the substratum by adjusting.Preferably, the inflation of culture is controlled by stirring culture.Stirring can be by water screw or similar mechanical agitation device, carry out by rotation or oscillating growth container (for example fermentor tank) or by multiple pumping unit.Can further control inflation by making sterile air or oxygen pass substratum (for example passing fermenting mixture).Equally preferably, microorganism of the present invention (for example, by adding defoamer such as fatty acid polyglycol ester) under the situation that does not have too much foam to form is cultivated.

In addition, microorganism of the present invention can be cultivated under institute's controlled temperature condition.Term " institute's controlled temperature " comprises the arbitrary temp that causes expecting that fine chemicals such as Methionin produce.In one embodiment, institute's controlled temperature comprises the temperature between 15 ℃ and 95 ℃.In another embodiment, institute's controlled temperature comprises the temperature between 15 ℃ and 70 ℃.Preferred temperature is more preferably between 30 ℃ and 45 ℃ or between 30 ℃ and 50 ℃ between 20 ℃ and 55 ℃.

Microorganism can be cultivated (for example keep and/or grow) in liquid nutrient medium, and preferably by conventional cultural method such as static cultivation, test tube cultivate, shaking culture (for example rotational oscillation is cultivated, shake-flask culture or the like), the inflation turn is cultivated or fermentation is cultivated constantly or off and on.In preferred embodiments, microorganism is cultivated in shaking bottle.In the embodiment that is more preferably, microorganism is cultivated (for example fermentation process) in fermentor tank.Fermentation process of the present invention includes, but is not limited in batches, the fermentation process of batch feeding and continuous feeding.Term " batch process " or " batch fermentation " refer to a kind of system of sealing, wherein the composition of substratum, nutrition, additive or the like is put into when the fermentation beginning and is remained unchanged during the fermentation, yet, still can attempt to control factor such as pH and oxygen concn so that prevent excessive acidifying of substratum and/or microbial death.Term " fed-batch process " or " batch feeding " fermentation are meant the batch fermentation except add one or more substrates or additive (for example increment or adding continuously) along with fermenting process.Term " continuation method " or " continuously fermenting " are meant a kind of like this system, fermention medium defined in it joins in the fermentor tank continuously and removes the used of equivalent simultaneously or " condition " substratum, is preferably used for the recovery of purpose fine chemicals such as Methionin.Developed multiple these class methods and be well-known in the art.

Phrase " under the condition of Methionin production, cultivate causing the purpose fine chemicals " be included in be suitable for or be enough to obtain the purpose fine chemicals produce or obtain the expection turnout make under the condition (for example temperature, pressure, pH, time length or the like) of the specific fine chemicals of production (as Methionin) microorganism keep and/or grow.For example, cultured continuously reaches the time that is enough to produce desired amount fine chemicals (for example Methionin).Preferably, cultured continuously reaches the time that is enough to realize substantially fine chemicals (for example Methionin) largest production.In one embodiment, cultivate lasting about 12-24 hour.In another embodiment, cultivate to continue about 24-36 hour, 36-48 hour, 48-72 hour, 72-96 hour, 96-120 hour, 120-144 hour, perhaps greater than 144 hours.In another embodiment, cultured continuously reaches the time that is enough to realize the fine chemicals maximum output, for example culturing cell is so that produce at least approximately 15-20g/L fine chemicals, produce at least approximately 20-25g/L fine chemicals, produce at least approximately 25-30g/L fine chemicals, produce at least approximately 30-35g/L fine chemicals, produce at least approximately 35-40g/L fine chemicals, produce at least approximately 40-50g/L fine chemicals, produce at least approximately 50-60g/L fine chemicals, produce at least approximately 60-70g/L fine chemicals, produce at least approximately 70-80g/L fine chemicals, produce at least approximately 80-90g/L fine chemicals, produce at least approximately 90-100g/L fine chemicals, produce at least approximately 100-110g/L fine chemicals, produce at least approximately 110-120g/L fine chemicals, produce at least approximately 120-130g/L fine chemicals, produce at least approximately 130-140g/L fine chemicals, perhaps produce at least approximately 140-160g/L fine chemicals.In another embodiment, microorganism make in about 24 hours, in about 36 hours, in about 40 hours, in about 48 hours, in about 72 hours, in about 96 hours, in about 108 hours, in about 122 hours or produce in about 144 hours under the condition of preferred turnout fine chemicals (turnout in for example above-mentioned listed scope) and cultivate.

Methodology of the present invention may further include the step that reclaims purpose fine chemicals such as Methionin.Term " recovery " purpose fine chemicals such as Methionin comprise from substratum extraction, results, isolated or purified compound.Can carry out the recovery of compound according to any conventional isolated or purified method known in the art, these methods include, but is not limited to conventional resin (for example negatively charged ion or Zeo-karb, non-ionic adsorption resin or the like) handle, with conventional sorbent material (for example gac, silicic acid, silica gel, Mierocrystalline cellulose, aluminium or the like) handle, pH change, solvent extraction (for example using conventional solvent such as ethanol, ethyl acetate, hexane or the like), dialysis, filtration, concentrate, crystallization, recrystallization, pH regulator, freeze-drying or the like.For example, can from substratum, reclaim fine chemicals such as Methionin by at first from culture, reclaiming microorganism.Then with substratum through Zeo-karb removing unwanted positively charged ion, and make then its through anionite-exchange resin to remove unwanted inorganic anion and to have more highly acid organic acid than purpose fine chemicals (as Methionin).

Preferably, the object of the invention fine chemicals is " extraction ", " isolating " or " purifying ", so that essentially no other composition of resulting preparation (for example not having medium component and/or fermentation by-product).Phrase " essentially no other composition " comprises the purpose compound formulation, and wherein compound is to separate (for example purifying or partial purification) from medium component or the culture fermentation by-product that produces it to obtain.In one embodiment, preparation contains greater than the purpose compound of about 80% (with dry weight basis) (for example being less than about 20% other medium component or fermentation by-product), purpose compound more preferably greater than about 90% (for example being less than about 10% other medium component or fermentation by-product), still more preferably greater than about 95% purpose compound (for example being less than about 5% other medium component or fermentation by-product), and most preferably greater than the purpose compound (other medium component or the fermentation by-product that for example are less than about 1-2%) of about 98-99%.

In alternate embodiment, the purpose fine chemicals is not to obtain from the microorganism purifying as Methionin, for example when microorganism when biologically harmless (for example safety).For example, all culture (perhaps culture supernatant) can be used as product source (for example thick product).In one embodiment, culture (culture supernatants) can not deal with and use.In another embodiment, culture (culture supernatants) is spissated.In another embodiment, culture (culture supernatants) is an exsiccant or cryodesiccated.

II. do not rely on the method for the production fine chemicals of precursor feed supplement demand

Depend on the combination of operated biosynthetic enzyme or biosynthetic enzyme, provide (for example feed supplement) at least a pentose-phosphate pathway biosynthesizing precursor so that the generation fine chemicals is desirable or essential as Methionin to microorganism of the present invention.Term " pentose-phosphate pathway biosynthesizing precursor " or " precursor " comprise such reagent or compound, when they are provided to microbiological culture media, they contact with microbiological culture media or microbiological culture media when comprising them, they can strengthen or improve the pentose phosphate biosynthesizing.In one embodiment, pentose phosphate biosynthesizing precursor or precursor are glucose.In another embodiment, pentose phosphate biosynthesizing precursor is a fructose.The glucose that is added or the amount of fructose are preferably and cause concentration in the substratum to be enough to strengthen the microorganisms producing power amount of (for example being enough to strengthen the concentration that fine chemicals such as Methionin are produced).Pentose phosphate biosynthesizing precursor of the present invention can concentrated solution or the form (for example being dissolved in appropriate solvent such as water or damping fluid) of suspension or add with solid form (for example with powder type).And pentose phosphate biosynthesizing precursor of the present invention can be used as single aliquots containig and adds continuously or at interval in stage preset time.

In learning, pentose phosphate biosynthetic means of the present invention provide the pentose phosphate biosynthesizing precursor may be with expensive relevant, for example when methodology is used to produce the high yield fine chemicals.Therefore, preferable methods of the present invention is characterised in that the microorganism with at least a operated biosynthetic enzyme or biosynthetic enzyme combination (for example at least a pentose phosphate biosynthetic enzyme), and described operation makes microorganism produce Methionin or other purpose fine chemicals in the mode that does not rely on the precursor feed supplement.When referring to be used for the method for production purpose compound, term " does not rely on the mode of precursor feed supplement " and comprises the method or the pattern of production purpose compound, and it does not rely on or be not fixed against the precursor that (for example feed supplement) is provided to the Institute of Micro-biology that is used to production purpose compound.For example, the microorganism that plays an important role in the inventive method is used for producing fine chemicals in the mode that does not need precursor glucose or fructose feed supplement.

The alternative preferred method of the present invention is learned and is characterised in that the microorganism with at least a operated biosynthetic enzyme or biosynthetic enzyme combination, and described operation makes Methionin or other fine chemicals produce in the mode that does not rely on the precursor feed supplement basically.Phrase " does not rely on the mode of precursor feed supplement basically " and comprises less degree and relies on or be fixed against to the utilization microorganism and provide the precursor of (for example feed supplement) and the approach or the method for production purpose compound.For example, the microorganism that plays an important role in the inventive method is used for providing the precursor glucose of basic reduction quantity or the mode of fructose to produce fine chemicals with needs.

In the mode that do not rely on the precursor feed supplement or alternatively relate to culturing micro-organisms with the preferred method of the mode production purpose fine chemicals that do not rely on the precursor feed supplement basically, these microorganisms have carried out operating (for example design or engineered is as genetically engineered transformation) so that make the expression of at least a pentose phosphate biosynthetic enzyme obtain modifying.For example, in one embodiment, microorganism is operated (for example design or engineered) so that make at least a pentose phosphate biosynthetic enzyme be gone to regulate.In preferred embodiments, microorganism is operated (for example design or engineered) so that make it have the biosynthetic pathway of regulating, for example definedly in the literary composition remove to regulate the pentose phosphate biosynthetic pathway.In another preferred embodiment, microorganism is operated (for example design or engineered) so that make at least a pentose phosphate biosynthetic enzyme such as lactic dehydrogenase expression of enzymes deficiency.

III. the high yield producer science of law

Particularly preferred embodiment of the present invention is the high yield production method that is used to produce fine chemicals such as Methionin, and it is included under the condition that causes with remarkable high yield production Methionin and cultivates operated microorganism.Term " high yield production method " for example is used for the high yield production method of production purpose fine chemicals such as Methionin, comprises causing with level that improves or the method that surpasses the level production purpose fine chemicals of common comparable production method.Preferably, the high yield production method causes with remarkable high yield production purpose compound.Term " significantly high yield " comprise be enough to improve or surpass the production or the yield level that can compare production method usually, for example it is increased to the level (for example producing product with the cost of commericially feasible) that is enough to commercial production purpose product.In one embodiment, the present invention is characterised in that the high yield production method of producing Methionin, and it is included in to cultivate under the condition that makes Methionin produce with the level greater than 2g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L, 50g/L, 55g/L, 60g/L, 65g/L, 70g/L, 75g/L, 80g/L, 85g/L, 90g/L, 95g/L, 100g/L, 110g/L, 120g/L, 130g/L, 140g/L, 150g/L, 160g/L, 170g/L, 180g/L, 190g/L or 200g/L has operated microorganism.

The present invention has further characterized the high yield production method that is used for production purpose fine chemicals such as Methionin, this method relates to cultivating has under certain condition operated microorganism, and described condition makes produces the compound of improving the standard in commercial stage expected time.In representative embodiment, the present invention has characterized the high yield production method of producing Methionin, and this method comprises cultivating has under certain condition operated microorganism, and described condition makes produced the Methionin that is higher than the 15-20g/L level in 5 hours.In another embodiment, the present invention has characterized the high yield production method of producing Methionin, and this method comprises cultivating has under certain condition operated microorganism, and described condition makes produced the Methionin that is higher than the 25-40g/L level in 10 hours.In another embodiment, the present invention has characterized the high yield production method of producing Methionin, and this method comprises cultivating has under certain condition operated microorganism, and described condition makes produced the Methionin that is higher than the 50-100g/L level in 20 hours.In another embodiment, the present invention has characterized the high yield production method of producing Methionin, this method comprises cultivating has under certain condition operated microorganism, and described condition makes produced the Methionin (for example producing the Methionin that is higher than the 150g/L level in 40 hours) that is higher than the 140-160g/L level in 40 hours.In another embodiment, the present invention has characterized the high yield production method of producing Methionin, this method comprises under certain condition cultivating has operated microorganism, and described condition makes produced the Methionin that is higher than the 130-160g/L level (for example produced be higher than 135,145 or the Methionin of 150g/L level) in 40 hours in 40 hours.Be included in and/or in literary composition the numerical value between the listed scope and scope also be intended to be included in the scope of the present invention.For example, with in 40 hours at least 140,141,142,143,144,145,146,147,148,149 and the level of 150g/L produce Methionin and be intended to be included in the scope of 140-150g/L in 40 hours.In another example, the scope of 140-145g/L or 145-150g/L is intended to be included in the scope of 140-150g/L in 40 hours.In addition, those of skill in the art will recognize, microorganism has been operated in cultivation, and the production level of " 140-150g/L in 40 hours " comprises for some time that microorganism culturing is extra (for example than 40 hours longer time periods) for example to obtain, and randomly causes the lysine production of producing even higher.

IV. isolated nucleic acid molecule and gene

The present invention has characterized the coded protein (for example Corynebacterium glutamicum protein) that the is used for the inventive method isolated nucleic acid molecule as Corynebacterium pentose phosphate biosynthetic enzyme (for example Corynebacterium glutamicum pentose phosphate enzyme) on the other hand.In one embodiment, the isolated nucleic acid molecule that is used for the inventive method is the serum lactic dehydrogenase nucleic acid molecule.

Term " nucleic acid molecule " comprises dna molecular (for example linear, cyclic cDNA or chromosomal DNA) and RNA molecule (for example tRNA, rRNA, mRNA) and the DNA of use nucleotide analog deposits yields or the analogue of RNA.Nucleic acid molecule can be strand or two strands, but is preferably double-stranded DNA.Term " isolating " nucleic acid molecule comprises the naturally occurring sequence in organic chromosomal DNA amplifying nucleic acid molecule both sides that do not contain nucleic acid molecule and originate (for example be positioned at nucleic acid molecule 5 ' and 3 ' terminal sequence).In multiple embodiments, isolated nucleic acid molecule may comprise naturally occurring about 10kb, 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb, 0.1kb, 50bp, 25bp or the 10bp nucleotide sequence of being less than in chromosomal DNA amplifying nucleic acid molecule both sides of the microorganism that nucleic acid molecule originates.And " isolating " nucleic acid molecule for example cDNA molecule is possible be substantially free of other cellular material when producing by recombinant technology, is to be substantially free of precursor or other compound when producing by chemosynthesis perhaps.

Such as in the literary composition use, term " gene " be included in the biology by intergenic DNA (be among the biological stain body DNA natural be positioned at the gene both sides and/or with separated DNA of interleaving of gene or spacer DNA) nucleic acid molecule (for example dna molecular or its fragment) that separates with another gene or other gene, for example nucleic acid molecule of coded protein or RNA.Gene can instruct the synthetic of enzyme or other protein molecule (for example can comprise encoding sequence, as the uninterrupted open reading-frame (ORF) (ORF) of coded protein) or himself have effect in vivo.Gene in the organism can be in operon cluster exist, such as in the literary composition definition, described operon separates by intergenic DNA and other gene and/or operon.Each gene that is comprised in the operon can be overlapping, and do not contain intergenic DNA between described each gene.Such as in the literary composition use, " isolating gene " comprises that being substantially free of gene originates among the biological stain body DNA the natural gene that is positioned at the sequence of gene both sides (promptly, do not contain adjacent encoder sequence, adjacent structure sequence of coding second or different proteins or RNA molecule or the like), and comprise that randomly 5 ' and 3 ' regulates sequence, for example promoter sequence and/or terminator sequence.In one embodiment, isolating gene mainly comprises proteinic encoding sequence (sequence of the excellent thuringiensis protein of for example encoding).In another embodiment, isolating gene comprises that the encoding sequence of protein (for example excellent thuringiensis protein) and gene are originated among the biological stain body DNA and contiguous 5 ' and/or 3 ' regulates sequence (for example contiguous 5 ' and/or 3 ' the excellent bacillus is regulated sequence).Preferably, isolating gene comprises natural the originate nucleotide sequence that is less than about 10kb, 5kb, 2kb, 1kb, 0.5kb, 0.2kb, 0.1kb, 50bp, 25bp or 10bp of biological stain body DNA gene both sides of gene that is arranged in.

In one aspect, method feature of the present invention is the purposes of isolating serum lactic dehydrogenase nucleotide sequence or gene.

In preferred embodiments, nucleic acid or gene are from excellent bacillus (for example, for excellent bacillus source).Term " from excellent bacillus " or " excellent bacillus source " comprise natural nucleic acid or the gene that is present in the Corynebacterium microorganism.Preferably, nucleic acid or gene are from being selected from following microorganism: Corynebacterium glutamicum, acetylglutamate rod bacillus, Corynebacterium acctoacidophlum or heat are produced ammonia rod bacillus.In particularly preferred embodiments, nucleic acid or gene are from Corynebacterium glutamicum (for example, for Corynebacterium glutamicum source).In another preferred embodiment, nucleic acid or gene are excellent bacillus gene homologue (for example from the species different with excellent bacillus but have remarkable homology with the present invention rod bacillus gene such as excellent bacillus lactate dehydrogenase gene).

Be included in the scope of the invention is the nucleic acid molecule of bacterial origin or nucleic acid molecule or gene (for example nucleic acid molecule or the gene in Corynebacterium glutamicum source), for example inventor's institute's genes identified such as excellent bacillus or the Corynebacterium glutamicum lactate dehydrogenase gene in gene and/or excellent bacillus source.In addition, be included in the scope of the invention is to be different from the bacterial origin nucleic acid molecule of naturally occurring bacterium and/or excellent bacillus nucleic acid molecule or gene (for example Corynebacterium glutamicum nucleic acid molecule or gene) or gene and/or excellent bacillus to come source nucleic acid molecule or gene (for example nucleic acid molecule or the gene in Corynebacterium glutamicum source) (for example Corynebacterium glutamicum nucleic acid molecule or gene), for example have alternative, insert or disappearance but coding and natural gene product similar substantially proteinic nucleic acid molecule or the gene of existing of the present invention.In one embodiment, isolated nucleic acid molecule comprises the listed nucleotide sequence as SEQ ID NO:1, the listed aminoacid sequence of SEQ ID NO:2 of perhaps encoding.

In another embodiment, isolated nucleic acid molecule of the present invention comprise with the listed nucleotide sequence of SEQ ID NO:1 at least about 60-65%, preferably at least about 70-75%, be more preferably at least about 80-85% and even be more preferably nucleotide sequence at least about 90-95% or higher identity.In another embodiment, isolated nucleic acid molecule under stringent condition with have the nucleic acid molecule of the listed nucleotide sequence hybridization of SEQ ID NO:1.This type of stringent condition is well known by persons skilled in the art and can be at Current Protocols in Molecular Biology, John Wiley ﹠amp; Sons, N.Y. (1989) finds among the 6.3.1-6.3.6.The preferred non-limiting example of strict (for example high strictness) hybridization conditions is: in about 45 ℃ of hybridization, then at 0.2XSSC, wash one or many in 50-65 ℃ among the 0.1%SDS in 6X sodium chloride/sodium citrate (SSC).Preferably, corresponding with the isolated nucleic acid molecule of the present invention of sequence SEQ ID NO:1 hybridization naturally occurring nucleic acid molecule under stringent condition.Such as in the literary composition use, " natural existence " nucleic acid molecule is meant RNA or the dna molecular with the existing nucleotide sequence of nature.

Use the sequence information that is provided in standard molecular biological technique and the literary composition can separate nucleic acid molecule of the present invention (nucleic acid molecule that for example has nucleotide sequence SEQ ID NO:1).For example, can the use standard hybridization and clone technology (for example be described in Sambrook, J., Fritsh, E.F. and Maniatis, T.Molecular Cloning:A Laboratory Manual. the 2nd edition, ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY, those in 1989) isolated nucleic acid molecule, perhaps by using polymerase chain reaction isolated nucleic acid molecule based on the designed synthetic oligonucleotide primer thing of sequence SEQ ID NO:1.Use cDNA, mRNA or alternatively use genomic dna as template and use suitable Oligonucleolide primers, according to the Standard PC R amplification technique nucleic acid of the present invention that can increase.In another preferred embodiment, isolated nucleic acid molecule of the present invention comprises the nucleic acid molecule of nucleotide sequence complement shown in the SEQ ID NO:1.

In another embodiment, isolated nucleic acid molecule is lactate dehydrogenase gene or its part or fragment or comprises lactate dehydrogenase gene or its part or fragment.In one embodiment, isolating serum lactic dehydrogenase nucleic acid molecule or gene comprise the listed nucleotide sequence of SEQ ID NO:1 (for example comprising Corynebacterium glutamicum serum lactic dehydrogenase nucleotide sequence).In another embodiment, isolating serum lactic dehydrogenase nucleic acid molecule or gene comprise the nucleotide sequence of coding as the listed aminoacid sequence of SEQ ID NO:2 (the Corynebacterium glutamicum lactic dehydrogenase enzyme amino acid sequence of for example encoding).In another embodiment, isolating serum lactic dehydrogenase nucleic acid molecule or genes encoding have the proteinic homologue of serum lactic dehydrogenase of aminoacid sequence SEQ IDNO:2.Such as in the literary composition use, term " homologue " comprise with wild-type protein described in the literary composition or amino acid sequence of polypeptide have at least about 30-35%, choosing ground at least about 35-40%, more preferably at least about 40-50% and even more preferably at least about 60%, 70%, 80%, 90% or higher identity, and have and described wild-type protein or the polypeptide functionally active of equivalence or the protein or the polypeptide of biologic activity basically.For example, serum lactic dehydrogenase homologue and the protein with the listed aminoacid sequence of SEQ ID NO:2 have at least about 30-35%, preferably at least about 35-40%, more preferably at least about 40-50% and even more preferably at least about 60%, 70%, 80%, 90% or higher identity, and have and have (promptly as the first-class substantially effect functionally active of the protein of the listed aminoacid sequence of SEQ ID NO:2 or biologic activity, be functional equivalent) (for example, having equivalent basically pantothenate kinase activity).In preferred embodiments, isolating serum lactic dehydrogenase nucleic acid molecule or gene comprise the nucleotide sequence of coding as the listed polypeptide of SEQ ID NO:2.In another embodiment, isolating serum lactic dehydrogenase nucleic acid molecule with have all or part of making nucleic acid molecular hybridization of listed nucleotide sequence among the SEQ ID NO:1, perhaps with have coding and have all or part of making nucleic acid molecular hybridization of nucleotide sequence of the polypeptide of aminoacid sequence SEQ ID NO:2.This type of hybridization conditions is well known by persons skilled in the art and at Current Protocols in MolecularBiology, people such as Ausubel compile John Wiley ﹠amp; Sons, Inc. (1995) can find in the 2nd, 4 and 6 parts.Other stringent condition can be at Molecular Cloning:A LaboratoryManual, people such as Sambrook, and Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) finds in the 7th, 9 and 11 chapters.The preferred non-limiting example of stringent hybridization condition comprises: in about 65-70 ℃ of hybridization (perhaps adding in 50% methane amide in about 42-50 ℃ hybridization at 4X SSC), wash one or many in about 65-70 ℃ subsequently in 1X SSC in 4X sodium chloride/sodium citrate (SSC).The preferred non-limiting example of high stringent hybridization condition comprises: in about 65-70 ℃ of hybridization (perhaps adding in 50% methane amide in about 42-50 ℃ hybridization at 1X SSC), wash one or many in about 65-70 ℃ subsequently in 0.3X SSC in 1X SSC.The preferred non-limiting example of the stringent hybridization condition that reduces comprises: in about 50-60 ℃ of hybridization (perhaps alternatively adding in 50% methane amide in about 40-45 ℃ hybridization at 6X SSC), wash one or many in about 50-60 ℃ subsequently in 2X SSC in 4X SSC.Scope between above-mentioned numerical value is for example at 65-70 ℃ or also be intended to be included among the present invention at 42-50 ℃.In hybridization and lavation buffer solution, (1XSSPE is 0.15M NaCl, 10mM NaH to SSPE ₂PO ₄With 1.25mM EDTA, pH 7.4) can replace SSC (1X SSC is 0.15M NaCl and 15mM Trisodium Citrate); Each hybridization was finished after scouring 15 minutes.For expecting that length is less than the heterozygote of 50bp, hybridization temperature should be than the melting temperature(Tm) (T of heterozygote _m) low 5-10 ℃, T wherein _mDetermine according to following formula.Be less than the heterozygote of 18bp, T for length _m(℃)=2 (A+T base number)+4 (G+C base number).For length 18 and 49bp between heterozygote, T _m(℃)=81.5+16.6 (log ₁₀[Na ⁺])+0.41 (%G+C)-(600/N), wherein N is the base number in the heterozygote, and [Na ⁺] be that the concentration of sodium ion in the hybridization buffer is (for 1X SSC, [Na ⁺]=0.165M).Skilled work personnel also will appreciate that, also can add additional agents so that reduce the non-specific hybridization of nucleic acid molecule and film (as nitrocellulose filter or nylon membrane) in hybridization and/or lavation buffer solution, this type of additional agents includes, but is not limited to encapsulant (for example BSA or salmon or herring sperm carrier DNA), stain remover (for example SDS), sequestrant (for example EDTA), Ficoll, PVP or the like.Particularly, when using nylon membrane, the in addition preferred non-limiting example of stringent hybridization condition is: at 0.25-0.5M NaH ₂PO ₄, among the 7%SDS in about 65 ℃ of hybridization, subsequently at 0.02M NaH ₂PO ₄, among the 1%SDS in 65 ℃ of washing one or many, see for example Church and Gilbert (1984) Proc.Natl.Acad.Sci.USA 81:1991-1995 (perhaps, alternatively being 0.2X SSC, 1%SDS).In another preferred embodiment, isolated nucleic acid molecule comprise with literary composition in listed serum lactic dehydrogenase nucleotide sequence complementary nucleotide sequence (for example, the complete complement of the listed nucleotide sequence of SEQ ID NO:1).

Use the sequence information that is provided in standard molecular biological technique and the literary composition can separate nucleic acid molecule of the present invention (for example serum lactic dehydrogenase nucleic acid molecule or gene).For example, hybridization of use standard and clone technology are (for example, as Sambrook, J., Fritsh, E.F. and Maniatis, T.Molecular Cloning:A Laboratory Manual. second edition, Cold Spring Harbor Laboratory, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY, described in 1989) can isolated nucleic acid molecule, perhaps use based on the designed synthetic oligonucleotide primer thing isolated nucleic acid molecule of listed serum lactic dehydrogenase nucleotide sequence or its flanking sequence in the literary composition by the polymerase chain reaction.Use cDNA, mRNA or alternatively use chromosomal DNA as template and the suitable Oligonucleolide primers of use, according to Standard PC R amplification technique, the nucleic acid of the present invention that can increase (for example, serum lactic dehydrogenase nucleic acid molecule or gene).

Another embodiment of the invention is characterised in that mutant serum lactic dehydrogenase nucleic acid molecule or gene.Such as in the literary composition use, term " mutant nucleic acid molecule " or " mutant gene " comprise nucleic acid molecule or the gene with the nucleotide sequence that comprises at least a change (for example substitute, insert, disappearance), wherein change to make and presented and polypeptide or the different activity of protein by wild-type nucleic acid molecule or coded by said gene by the polypeptide of described mutant code or protein.Preferably, polypeptide or protein that mutant nucleic acid molecule or mutant gene (for example mutant lactate dehydrogenase gene) are coded in simulated condition for example when measuring down (for example measuring in being incubated at the microorganism of uniform temp) compares the activity (lactate dehydrogenase activity that for example, has increase) with increase with the polypeptide or the protein of wild-type nucleic acid molecule or coded by said gene.Mutant gene can also make the wild type peptide production level reduce.

Such as in the literary composition use, " activity of reduction " or " enzymic activity of reduction " is than wild-type nucleic acid molecule or coded by said gene polypeptide or low by at least 5%, the preferred 5-10% at least of activity of proteins, more preferably 10-25% and even the more preferably activity of 25-50%, 50-75% or 75-100% at least at least.Scope between above-mentioned numerical value for example 75-85%, 85-90%, 90-95% also is intended to be included among the present invention.Such as in the literary composition use, " activity of reduction " or " enzymic activity of reduction " also comprises the activity activity of wild-type nucleic acid molecule or coded by said gene polypeptide or activity of proteins low about 100% (for example, than) of deleted or " knocking out ".Any analytical method according to the measurement specific purpose protein active of accepting extensively can be determined activity.Activity can directly be measured or analyze, and for example measures from the activity of proteins of cellular segregation or purifying.Alternatively, can measure or analyze activity in cell or in the substratum of extracellular.

Those skilled in the art should understand that, even single the substituting in nucleic acid or the gene order (for example, being coded in the amino acid whose base that changes in the corresponding aminoacid sequence substitutes) can both remarkably influenced be compared its encoded polypeptide and activity of proteins with corresponding wild type peptide with protein.Such as in the literary composition definition, mutant nucleic acid or mutant gene are (for example, encoding mutant body polypeptide or protein) easily with encode that the nucleic acid or the gene of protein homologs are distinguished mutually as mentioned above, because mutant nucleic acid or mutant gene coding has the active protein of change or polypeptide, randomly can observe in the microorganism (being mutant microorganism) of described mutein or polypeptide and express wild type gene or nucleic acid or produce the different or diacritic phenotype of corresponding microorganism of described mutein or polypeptide expressing described mutant gene or nucleic acid or produce.On the contrary, protein homologs has identical or similar substantially activity, randomly, compares on phenotype and can not distinguish with the corresponding microorganism of expressing wild type gene or nucleic acid when producing in microorganism.Therefore, for example the degree of the sequence identity between nucleic acid molecule, gene, protein or the polypeptide can not be used to distinguish homologue and mutant, but the activity of coded protein and polypeptide is used to distinguish homologue and mutant: homologue has for example low sequence identity (for example 30-50% sequence identity) and has the functionally active of basic equivalence, and mutant for example has 99% sequence identity and has the functionally active of remarkable difference or change.

V. recombinant nucleic acid molecules and carrier

Further aspect of the present invention is recombinant nucleic acid molecules (for example recombinant DNA molecules), it comprises nucleic acid molecule described in the literary composition and/or gene (for example isolated nucleic acid molecule and/or gene), be preferably excellent bacillus gene, be more preferably Corynebacterium glutamicum gene, even be more preferably the Corynebacterium glutamicum lactate dehydrogenase gene.

Further aspect of the present invention is carrier (for example recombinant vectors), and it comprises the nucleic acid molecule described in the literary composition (for example isolating or reorganization nucleic acid molecule and/or gene).Particularly, recombinant vectors is characterised in that and comprises coding bacterial gene product as described herein, preferred excellent bacillus gene product, the more preferably nucleotide sequence of Corynebacterium glutamicum gene product (for example pentose phosphate enzyme such as serum lactic dehydrogenase).

Term " recombinant nucleic acid molecules " comprise change, modification or engineered so that its nucleotides sequence list the intrinsic or natural acid molecule of originating with recombinant nucleic acid molecules different (for example, by add, disappearance or substitute one or more Nucleotide) nucleic acid molecule (for example dna molecular).Preferably, recombinant nucleic acid molecules (for example recombinant DNA molecules) comprises and regulates isolated nucleic acid molecule of the present invention or the gene (for example isolating lactate dehydrogenase gene) that sequence effectively is connected.

Term " recombinant vectors " comprise change, modification or engineered carrier (for example nucleic acid carrier of plasmid, phage, phasmid, virus, clay or other purifying) so that these carriers comprise is more in the intrinsic or natural acid molecule of originating than the reorganization carrier, still less or different nucleotide sequences.Preferably, recombinant vectors comprises lactate dehydrogenase gene or comprises the recombinant nucleic acid molecules that effectively is connected to this lactate dehydrogenase gene of regulating sequence such as promoter sequence, terminator sequence and/or artificial ribosome bind site (RBS).

The nucleotide sequence that phrase " effectively be connected to the regulate sequence " meaning is meant purpose nucleic acid molecule or gene is connected to the adjusting sequence in the mode that allows nucleotide sequence to express (for example strengthen and express, increase expression, constitutive expression, basal expression, weaken expressions, reduce expression or be suppressed expression), is preferably the expression (for example when recombinant nucleic acid molecules is included in the carrier as defined above and imports microorganism) of the coded gene product of nucleotide sequence.

Term " adjusting sequence " comprises the nucleotide sequence that other nucleotide sequence of influence (for example adjust or regulate) is expressed.In one embodiment, regulate sequence and be included in recombinant nucleic acid molecules or recombinant vectors with position and/or the direction similar or identical with respect to the specific purpose gene, as for regulate sequence and goal gene nature viewed, for example natural place and/or direction.For example, recombinant nucleic acid molecules or recombinant vectors comprise with in natural biological with goal gene together and the goal gene of the adjacent effective ways of connecting of adjusting sequence (for example effectively be connected to " natural " and regulate sequence) as on " natural " promotor.Alternatively, recombinant nucleic acid molecules or recombinant vectors comprise and accompany and the adjacent effective purpose of connecting gene of adjusting sequence with another gene (for example different genes) in natural biological.Alternatively, recombinant nucleic acid molecules or recombinant vectors comprise with from the effective purpose of connecting gene of adjusting sequence of another biology.For example, the adjusting sequence (for example other bacterium is regulated sequence, phage is regulated sequence or the like) from microorganism effectively is connected with the specific purpose gene.

In one embodiment, regulate sequence and be the sequence that extrinsic or non-natural exist (sequence of for example modifying, suddenly change, substitute, derive, lacking comprises the sequence of chemosynthesis).The preferred sequence of regulating comprises that promotor, enhanser, termination signal, antitermination signal and other express controlling elements (for example repressor in the mRNA that transcribes or inductor bonded sequence and/or transcribe and/or translate and regulate the combination of proteins site).For example, this type of regulates sequence description in Sambrook, J., Fritsh, E.F. and Maniatis, T.Molecular Cloning:ALaboratory Manual. the 2nd edition, Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY, 1989.Regulate sequence comprise those that instruct that nucleotides sequence is listed in constitutive expression in the microorganism regulate sequences (for example constitutive promoter and strong constitutive promoter), instruct that nucleotides sequence is listed in abduction delivering in the microorganism those regulate sequences (for example inducible promoters such as wood sugar evoked promoter) and weaken or suppress nucleotides sequence and be listed in those adjusting sequences (for example deamplification or repressor sequence) of expressing in the microorganism.By removing or deletion is regulated expression that sequence regulates goal gene also within the scope of the invention.For example, remove participating in producing the transcriptional regulatory sequences that increase is transcribed or composing type is transcribed, thereby the expression of goal gene is reduced.

In one embodiment, recombinant nucleic acid molecules of the present invention or recombinant vectors comprise nucleotide sequence or gene at least a and promotor or the effective coding bacterial gene product (for example, pentose phosphate biosynthetic enzyme such as serum lactic dehydrogenase) that is connected of promoter sequence.The preferred promotor of the present invention comprises excellent bacillus promotor and/or phage promoter (for example infecting the phage of excellent bacillus).In one embodiment, promotor is excellent bacillus promotor, be preferably strong excellent bacillus promotor (for example, the promotor relevant with biological chemistry housekeeping gene in the excellent bacillus or with excellent bacillus in the promotor of glycolytic pathway gene-correlation).In another embodiment, promotor is a phage promoter.

In another embodiment, recombinant nucleic acid molecules of the present invention or recombinant vectors comprise a terminator sequence or a plurality of terminator sequence (for example Transcription Termination subsequence).Term " terminator sequence " comprises and is used for the adjusting sequence that terminator is transcribed.Terminator sequence (or series connection transcription terminator) can be further used for stable mRNA (for example by add structure in mRNA), for example nuclease-resistant.

In another embodiment, recombinant nucleic acid molecules of the present invention or recombinant vectors comprise allow to detect the carrier that comprises described sequence sequence (promptly, but can detect and/or selection markers), for example overcome sequence such as ura3 or ilvE, fluorescent marker and/or the colorimetry marker (for example lacZ/ beta-galactosidase enzymes) of auxotrophic mutation, and/or antibiotics resistance gene (for example amp or tet).

In another embodiment, recombinant vectors of the present invention comprises antibiotics resistance gene.Term " antibiotics resistance gene " comprises promotion or gives host living beings (for example bacillus) resistance antibiotic sequence.In one embodiment, antibiotics resistance gene is selected from: cat (chloramphenicol resistance) gene, tet (to tetracyclin resistance) gene, erm (to the erythromycin resistance) gene, neo (to neomycin resistance) gene and spec (to the spectinomycin resistance) gene.Recombinant vectors of the present invention can also comprise homologous recombination sequence (for example be designed for and allow the goal gene reorganization to enter the chromosomal sequence of host living beings).For example, the amyE sequence can be used as the homology target that reorganization enters host chromosome.

Those skilled in the art be also to be understood that and depend on such as to the selection of the microorganism of pending genetically engineered transformation, desired gene product expression level or the like factor, carrier is carried out particular design.

VI. isolating protein

Another aspect of the present invention is characterised in that isolating protein (for example, isolating pentose phosphate biosynthetic enzyme such as isolating serum lactic dehydrogenase).In one embodiment, protein (for example, isolating pentose phosphate enzyme such as isolating serum lactic dehydrogenase) be produce by recombinant DNA technology and can use standard protein purification technique to separate from microorganism of the present invention to obtain by suitable purification scheme.In another embodiment, protein utilizes the chemosynthesis of standard peptide synthetic technology.

" isolating " or " purifying " protein biosynthetic enzyme of purifying (for example isolating or) is substantially free of from protein originate microbial cell material or other contaminating protein matter, perhaps is substantially free of precursor or other chemical reagent when for chemosynthesis.In one embodiment, protein isolating or purifying contains contaminating protein matter or the chemical reagent that is less than about 30% (with dry weight basis), be more preferably and be less than about 20% contaminating protein matter or chemical reagent, still be more preferably and be less than about 10% contaminating protein matter or chemical reagent, and most preferably be less than about 5% contaminating protein matter or chemical reagent.

In preferred embodiments, protein or gene product are from excellent bacillus (being that excellent bacillus is originated for example).Term " from excellent bacillus " or " excellent bacillus source " comprise by excellent bacillus gene coded protein or gene product.Preferably, gene product is from being selected from following microorganism: Corynebacterium glutamicum, acetylglutamate rod bacillus, Corynebacterium acctoacidophlum or heat are produced ammonia rod bacillus.In particularly preferred embodiments, protein or gene product are from Corynebacterium glutamicum (for example, being the Corynebacterium glutamicum source).Term " from Corynebacterium glutamicum " or " the Corynebacterium glutamicum source " comprise by Corynebacterium glutamicum gene coded protein or gene product.In another preferred embodiment, protein or gene product by excellent bacillus gene homologue (for example are, from the species that are different from excellent bacillus but the gene that has remarkable homology with excellent bacillus gene of the present invention, for example excellent bacillus lactate dehydrogenase gene) coding.

Be included in protein or gene product (for example gene product in Corynebacterium glutamicum source) within the scope of the invention for the protein of bacterial origin or gene product and/or excellent bacillus source, they are coded by naturally occurring bacterium and/or excellent bacillus gene (for example Corynebacterium glutamicum gene), for example present inventor's institute's genes identified such as excellent bacillus or Corynebacterium glutamicum lactate dehydrogenase gene.Further be included in protein or gene product (for example gene product in Corynebacterium glutamicum source) within the scope of the invention for the protein of bacterial origin or gene product and/or excellent bacillus source, they are coded by bacterium that is different from naturally occurring bacterium and/or excellent bacillus gene (for example Corynebacterium glutamicum gene) and/or excellent bacillus gene (for example Corynebacterium glutamicum gene), for example, has the nucleic acid that suddenlys change, insert or delete but a coding proteinic gene similar substantially to the naturally occurring gene product of the present invention.For example, well-known those skilled in the art can be with nucleic acid mutation (for example substituting), owing to the degeneracy of genetic code, the nucleic acid encoding of this sudden change and the natural identical amino acid of coded by said gene amino acid that exists.And well-known those skilled in the art can be with nucleic acid mutation (for example substituting), and it causes conserved amino acid to substitute.Well-known those skilled in the art can compare the function that does not influence gene product basically with amino acid replacement, adding or disappearance with naturally occurring gene product to a certain extent, and wherein each situation is intended to be included within the scope of the invention.

In preferred embodiments, the isolating protein of the present invention (for example, isolating pentose phosphate biosynthetic enzyme is as isolating serum lactic dehydrogenase) has aminoacid sequence shown in the SEQ ID NO:2.In other embodiments, the isolating protein of the present invention be as the listed proteinic homologue of SEQ ID NO:2 (for example comprise with aminoacid sequence SEQ ID NO:2 at least about 30-40%, preferably about 40-50%, be more preferably about 50-60% and even be more preferably the aminoacid sequence of about 60-70%, 70-80%, 80-90%, 90-95% or higher identity, and have and the similar substantially activity of the coded protein active of aminoacid sequence SEQ ID NO:2).

In order to determine the homology percentage ratio of 2 aminoacid sequences or 2 nucleotide sequences, sequence alignment is compared purpose (for example, breach can be introduced article one amino acid or nucleotide sequence compares to reach with the best of second amino acid or nucleotide sequence) to reach the best.When a position in article one sequence by with the second sequence in when identical amino-acid residue or Nucleotide occupy on the corresponding position, then molecule is identical in this position.Identity percentage ratio between the two sequences be two sequences the function (that is, identity percentage ratio=same position number/total positional number * 100) of shared same position number, preferably consider to produce best necessary breach number of comparison and described breach size.

Use mathematical algorithm can realize between the two sequences sequence relatively and identity percentage ratio definite.The preferred non-limiting example that is used for the mathematical algorithm of sequence comparison is as Karlin that makes amendment at Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-77 and the algorithm ((1990) Proc.Natl.Acad.Sci.USA 87:2264-68) of Altschul.This kind algorithm is integrated into people such as Altschul, the NBLAST of (1990) J.Mol.Biol.215:403-10 and XBLAST program (version 2 .0).Use NBLAST program (score value=100, word length=12) to carry out the retrieval of BLAST Nucleotide, to obtain and nucleic acid molecule homologous nucleotide sequence of the present invention.Use XBLAST program (score value=50, word length=3) can carry out the retrieval of BLAST protein, to obtain and protein molecule homologous aminoacid sequence of the present invention.In order to obtain to be used for the comparison jaggy of comparison purpose, as people such as Altschul, (1997) Nucleic Acids Research25 (17): use breach BLAST (Gapped BLAST) described in the 3389-3402.When using BLAST and breach blast program, can use the default parameter of program (for example XBLAST and NBLAST) separately.See http://www.ncbi.nlm.nih.gov.Another the preferred non-limiting example that is used for the mathematical algorithm of sequence comparison is the algorithm of Myers and Miller (1988) ComputAppl Biosci.4:11-17.This kind algorithm is integrated into the ALIGN program, and this program can or obtain on ISREC server (http://www.ch.embnet.org) at for example the GENESTREAM webserver, IGH Montpellier, FRANCE (http://vega.igh.cnrs.fr).When utilizing the ALIGN program to carry out aminoacid sequence relatively the time, can use the remaining table of PAM120 weighting (weight residue table), notch length point penalty be 12 and the breach point penalty be 4.

In another preferred embodiment, utilize the GAP program (can obtain) in the GCG software package at http://www.gcg.com, use Blossom 62 matrixes or PAM250 matrix, and breach be weighted to 12,10,8,6 or 4 and length be weighted to 2,3 or 4, can determine two homology percentage ratios between the aminoacid sequence.In another preferred embodiment, utilize the GAP program (can obtain) in the GCG software package at http://www.gcg.com, use breach be weighted to 50 and length be weighted to 3, can determine two homology percentage ratios between the nucleotide sequence.

The present invention further illustrates by the following embodiment that is not construed as limiting.Whole reference of being quoted in the whole application, patent, sequence table, figure and the content of published quote as a reference in the text.

Embodiment

General method is learned:

Bacterial strain

Corynebacterium glutamicum ATCC 21526 obtains from American type culture collection (Manassas, the U.S.).Because collaborative E.C. 2.7.2.4. bypass suppresses, this kind homoserine auxotrophic strain is secreted Methionin between L-Threonine restricted period.Pre-culture grown in contain 5g L ^-1In the complex medium of fructose or glucose.For agar plate, the extra 12g L that adds in the complex medium ^-1Agar.In order to produce, used additional 1mg ml as the cell that is used for chase experiment and tracer study inoculum ^-1The minimum medium of calcium pantothenate HCl (Wittmann, C. and E.Heinzle.2002.Appl.Environ.Microbiol.68:5843-5859).Following pointed, the concentration of carbon source glucose or fructose, indispensable amino acid Threonine, methionine(Met) and leucic concentration in this substratum, and the concentration of Citrate trianion changes.

Cultivate

Pre-cultivation was made up of three steps, comprise that (i) uses the cell from agar plate to carry out initial cultivation as inoculum in complex medium, (ii) be used to adapt to the Short-term Culture of minimum medium and the (iii) long-term cultivation on the minimum medium of indispensable amino acid concentration raising.Grow overnight from the 10ml complex medium of pre-culture 100ml band flask with indentation of agar plate inoculation.By centrifugal (8800g, 2 minutes, 30 ℃) harvested cell, inoculation is gone into minimum medium and grown to optical density(OD) is 2 then, to obtain to adapt to the index growthing cell of minimum medium.Then by centrifugal (8800g, 30 ℃, 2 minutes) harvested cell, comprising step with aseptic 0.9%NaCl washing.Then they are inoculated in the 6ml minimum medium in the 50ml band flask with indentation, wherein containing starting point concentration is 0.30g L ^-1Threonine, 0.08g L ^-1Methionine(Met), 0.20g L ^-1Leucine and 0.57g L ^-1Citrate trianion.Add 70mM glucose or 80mM fructose respectively as carbon source.The cell growth is up to ruing out of indispensable amino acid, and this can pass through HPLC analytical review.Harvested cell also washed with aseptic NaCl (0.9%) when finished vegetative period.Subsequently cell transfer is gone in the basic spike substratum of 4ml in the 25ml band flask with indentation, so that under the condition that produces Methionin, carry out the metabolic flux analysis.The spike substratum does not contain any Threonine, methionine(Met), leucine or Citrate trianion.For every kind of carbon source, inoculate two parallel bottles that shake, wherein comprise (i) 40mM[1-respectively ¹³C] substrate and the (ii) 20mM[of mark ¹³C ₆] substrate of mark adds the native labelled substrate of 20mM.Whole cultivations are upward changeed per minutes in 30 ℃ and 150 at rotary shaker (Inova 4230, New Brunswick, Edison, NJ, the U.S.) and are carried out.

Chemical reagent

99%[1- ¹³C] glucose, 99%[1- ¹³C] fructose, 99%[ ¹³C ₆] glucose and 99%[ ¹³C ₆] fructose is available from Campro Scientific (Veenendaal, Holland).Yeast extract and Tryptones obtain from Difco Laboratories (Detroit, Michigan, the U.S.).All other applied chemistry reagent are respectively from Sigma (St.Louis, MI, the U.S.), Merck (Darmstadt, Germany) or Fluka (Buchs, Switzerland), and are AG.

Substrate and product analysis

Measure the cell density (OD at 660nm place by using photometer (Marsha Pharmacia biotech, Freiburg, Germany) _660nm) or determine cell concn by gravimetry.Gravimetry is to determine by the 10ml cell that nutrient solution 3700g under room temperature was gathered in the crops in centrifugal 10 minutes, comprises the step that washes with water in the results process.Washed cell 80 ℃ of dryings up to constant weight.Through determining the cell dry mass and the OD that do _660nmBetween correlation factor (g biomass/OD _660nm) be 0.353.

Extracellular substrate and production concentration determine that in culture supernatant this supernatant liquor obtained by 16000g in centrifugal 3 minutes.After deriving, fructose, glucose, sucrose and trehalose are carried out quantitatively by GC to the TMS 9 oxime derivate.For this purpose, used and had HP 5MS post (5% phenyl-methyl-siloxanes-biphenyl dimethyl polysiloxane, 30m * 250 μ m, HewlettPackard, Paolo Alto, CA, the USA U.S.) HP 6890 gas chromatographs (HewlettPackard, Palo Alto, the U.S.) and have four utmost point mass selective detector (Agilent Technologies of electron impact ionization under 70eV, Waldbronn, Germany).Specimen preparation comprise culture supernatant lyophilize, be dissolved in pyridine and use azanol subsequently and (TMS) trifluoroacetamide (BSTFA) (Macherey ﹠amp; Nagel, D ü ren, Germany) (13,14) carry out two step derivatizations of sugar.β-D-ribose is as quantitative internal standard.The injected sample volume is 0.2 μ l.The time-program(me) that GC analyzes is as follows: 150 ℃ (0-5 minute), 8 ℃ of min ^-1(5-25 minute), 310 ℃ (25-35 minute).Helium is used for carrier gas, and flow is 1.5l min ^-1Air inlet temperature is that 310 ℃ and detector temperature are 320 ℃.By utilizing Aminex-HPX-87H Biorad post (300 * 7.8mm, Hercules, CA, the U.S.) and using flow velocity to be 0.8ml min ^-14mM sulfuric acid carry out the HPLC that UV detects as moving phase and at the 210nm place and measure acetate, lactic acid, pyruvic acid, 2-oxoglutaric acid and otan.By the quantitative glycerine of enzymatic determination method (Boehringer, Mannheim, Germany).Have the online derivatize of automatization (o-phthalaldehyde(OPA)+3-thiohydracrylic acid) by utilization, flow velocity is 2ml min ^-1Zorbax Eclypse-AAA post (150 * 4.6mm, 5 μ m, Agilent Technologies, Waldbronn, Germany) and HPLC (Agilent Technologies, Waldbronn, the Germany) analysis of amino acid of fluoroscopic examination.Provided detail in the Guide Book.Butyrine salt is as quantitative internal standard.

¹³The C labeled analysis

By GC-MS the marking mode of Methionin in the culture supernatant and trehalose is carried out quantitatively.Thereby determine single quality isotope-isomerism thing fraction.In present work, they are defined as M ₀(relative quantity of non-marked quality isotope-isomerism thing fraction), M ₁(relative quantity of single labelled quality isotope-isomerism thing fraction) and be used for the more corresponding term of protrude mark.It is as discussed previously that (Rubino F.M.1989.J.Chromatogr.473:125-133), is transformed into the tertiary butyl-dimetylsilyl (TBDMS) derivative with Methionin and analyzes with GC-MS afterwards.To quantitatively carrying out under selection ion monitoring (SIM) pattern of ionic group m/z 431-437 being used for that quality isotope-isomerism thing distributes.The corresponding fragmention of this ionic group, its loss by the tertiary butyl of derivatization residue forms, and therefore comprises whole carbon skeletons (Wittmann, C., M.Hans and E.Heinzle.2002.AnalyticalBiochem.307:379-382) of Methionin.(H.M.Kim and E.Heinzle.2003.Metabolic analysis at miniaturized scale. submit to for Wittmann, C.) as discussed previously determines the marking mode of trehalose from trimethyl silyl (TMS) derivative of trehalose.The marking mode of trehalose is by judging that at the ionic group at m/z 361-367 place this ionic group correspondence comprises the fragmention of the whole monomer unit of trehalose, and should this carbon skeleton equals the carbon skeleton of glucose 6-phosphoric acid.All samples are at first measured with scan pattern, get rid of the product analyzed and the same amount dystopy element between other sample composition thus and disturb.Whole measurements of being undertaken by SIM repeat in duplicate.The testing error of single quality isotope-isomerism thing fraction is respectively in the fructose chase experiment: for [1- ¹³C] fructose is that the Methionin of substrate is 0.85% (M ₀), 0.16% (M ₁), 0.27% (M ₂), 0.35% (M ₃), 0.45% (M ₄), for [1- ¹³C] fructose is that the trehalose of substrate is 0.87% (M ₀), 0.19% (M ₁), 0.44% (M ₂), 0.45% (M ₃), 0.88% (M ₄), and for 50%[ ¹³C ₆] fructose is that the trehalose of substrate is 0.44% (M ₀), 0.54% (M ₁), 0.34% (M ₂), 0.34% (M ₃), 0.19% (M ₄), 0.14% (M ₅) and 0.52% (M ₆).The testing error that MS measures in the glucose chase experiment is respectively: for [1- ¹³C] glucose is that the Methionin of substrate is 0.47% (M ₀), 0.44% (M ₁), 0.21% (M ₂), 0.26% (M ₃), 0.77% (M ₄), for [1- ¹³C] glucose is that the trehalose of substrate is 0.71% (M ₀), 0.85% (M ₁), 0.17% (M ₂), 0.32% (M ₃), 0.46% (M ₄), and for 50%[ ¹³C ₆] glucose is that the trehalose of substrate is 1.29% (M ₀), 0.50% (M ₁), 0.83% (M ₂), 0.84% (M ₃), 1.71% (M ₄), 1.84% (M ₅) and 0.58% (M ₆).

Metabolic model and parameter estirmation

All the metabolism simulation is carried out on Personal Computer.The metabolism network application of the Corynebacterium glutamicum of product Methionin is in Matlab 6.1 and Simulink 3.0 (Mathworks, Inc., Natick, MA, the U.S.).Software application comprises that isotope-isomerism object model in Simulink is so that in the computational grid ¹³The distribution of C mark.For the estimation of parameter, with among isotope-isomerism object model and the Matlab repeatedly optimization algorithm combine.Detail about applied PC Tools provides (Wittmann, C. and E.Heinzle.2002.Appl.Environ.Microbiol.68:5843-5859) by Wittmann and Heinzle.

The metabolism network is based on previous work and comprise that glycolysis-, pentose-phosphate pathway (PPP), tricarboxylic acid (TCA) circulate, the biosynthesizing of covering carboxylation, Methionin and other secretory product (table 1) of pyruvic acid, and the anabolism flux from the intermediate precursor to biomass.In addition, select using glucose and fructose absorption system.The absorption of glucose relates to the phosphorylation (Ohnishi to glucose 6-phosphoric acid by PTS, J., S.Mitsuhashi, M.Hayashi, S.Ando, H.Yokoi, K.Ochiai and M.A.Ikeda.2002.Appl.Microbiol.Biotechnol.58:217-223).For fructose, thinking has two kinds of absorption systems, is respectively: (i) pass through PTS _FructoseAbsorb and change into fructose 1,6-bisphosphate and (ii) pass through PTS via fructose-1-phosphate _SeminoseAbsorption causes forming fructose 6-phosphoric acid (Dominguez, H., C.Rollin, A.Guyonvarch, J.L.Guerquin-Kern, M.Cocaign-Bousquet and N.D.Lindley.1998.Eur.J.Biochem.254:96-102).In addition, fructose-1 is applied in the model so that allow to exist in the glycolysis-top carbon flux of both direction.Related reversible reaction is transaldolase and the transketolase among the PPP.In addition, for the experiment of glucose, think that glucose 6-phosphoric acid isomerase also is a reversible, thereby the trehalose mark has reflected the reversibility of this enzyme sensitively.On the contrary, for the fructose experiment, can not determine the reversibility of glucose 6-phosphoric acid isomerase.In the cell with the fructose growth, glucose 6-phosphoric acid is formed by fructose 6-phosphoric acid uniquely, causes two marking modes that the storehouse is identical.Therefore, change mutually by reversible glucose 6-phosphoric acid isomerase between glucose 6-phosphoric acid and the fructose 6-phosphoric acid, and do not cause to judge the reversible marked difference of glucose 6-phosphoric acid isomerase.But the measuring mark of Methionin and trehalose is for the reversibility of flux between (i) phosphoenolpyruvic acid/pyruvic acid and the oxysuccinic acid/oxaloacetic acid lump storehouse and (ii) the malate dehydrogenase (malic acid dehydrogenase) in the TCA circulation and the reversibility of fumarate hydratase are insensitive.Therefore these reactions are considered to be irreversible.In this research, can not obtain from natural mark and [ ¹³C ₆] the L-Ala mark of labeled substrate mixture, described L-Ala mark is responsive for these traffic parameter.Based on previous result, the oxoethanoic acid approach is assumed that it is inactive (Wittmann, C. and E.Heinzle.2002.Appl.Environ.Microbiol.68:5843-5859).

Be used from the distribution of calculating metabolic flux about the stoichiometry data of Corynebacterium glutamicum growth, product formation and biomass composition and the mass spectrum flag data one of secreted Methionin and trehalose.Can provide the experiment (M of Methionin and trehalose in two groups of parallel tests _{I, exp}) and simulation (M _{I, calc}) the flux group of minimum deviation is considered to the best evaluation of Flux Distribution in the pair cell between the quality isotope-isomerism thing fraction.As described in appendix, all determine at two networks of the cell of growing on the glucose and the cell of on fructose, growing.Therefore method of least squares is fine.As error criterion, used the weighted sum of method of least squares (SLS), here S _I.expIt is the standard deviation of measuring (equation 1).

SLS = \underset{i}{Σ} \frac{{(M_{i, \exp} - M_{i, calc})}^{2}}{{S_{i, \exp}}^{2}}

(equation 1)

Use multiple parameter initialization so that whether the Flux Distribution that institute obtains has represented overall optimum value.For all strains examined, in the Methionin production process glucose absorption flux be set to 100% and network in other flux be given as the glucose absorption flux carried out normalized relative molar flux.

Statistical evaluation

(Wittmann, C. and E.Heinzle.2002.Appl.Environ.Microbiol.68:5843-5859) carries out statistical study to the metabolic flux that carries out that is obtained by previous described Monte-Carlo method.For each bacterial strain, statistical analysis is undertaken by the 100 subparameters estimation distances of swimming (parameterestimation runs), is different statistically to the experimental data that comprises measured quality isotope-isomerism thing ratio and measured flux thus.From the data computation that obtained 90% confidence limit of single parameter.

Example I: the Methionin production of being undertaken by the Corynebacterium glutamicum that grows on fructose and the glucose

In comparable glucose and fructose batch culture, analyzed the metabolic flux that produces the Corynebacterium glutamicum of Methionin.For this purpose, the cell transfer of growth is in advance gone into the spike substratum and cultivated about 5 hours.When chase experiment begins and finish, the analysis of substrate and product has been disclosed greatest differences between two kinds of carbon sources.Bacterium in the glucose growth produced 11.1mM Methionin altogether, and the bacterium that grows only produces the lower concentration Methionin of 8.6mM on fructose.Between 5 hours incubation period, cell concn is from 3.9g L ^-1Increase to 6.0g L ^-1(glucose) and from 3.5g L ^-1Increase to 4.4g L ^-1(fructose).Owing to do not have Threonine and methionine(Met) in the substratum, cell may utilize inner source synthesising biological amount.Average special sugared specific absorption (1.71mmol g with glucose ^-1h ^-1) compare average special sugared specific absorption (the 1.93mmol g of fructose ^-1h ^-1) higher.Described in table 1, Corynebacterium glutamicum ATCC 21526 turnout that obtained are significantly different between fructose and glucose.This relates to primary product Methionin and multiple byproduct.About Methionin, the turnout on fructose is 244mmol mol ^-1And therefore than (the 281mmol mol of the turnout on glucose ^-1) lower.In addition, carbon source has tremendous influence to the turnout of biomass, and compares on glucose, and the turnout of biomass almost reduces by 50% on fructose.Observed carbon source at Protosol, glycerine and lactic acid byproduct has been formed remarkable influence.On fructose, the accumulation of these byproducts greatly increases raising.The turnout of glycerine improves 10 times, and Protosol and lactic acid secretion increase by 6 times.Protosol is the main byproduct that produces on fructose.Because lower biomass production amount, so the cell of growing on fructose significantly reduces (table 2) to the demand of synthesizing metabolic precursor thereof.

Table 1: at the Methionin of Corynebacterium glutamicum ATCC 21526 biomass and the meta-bolites from glucose (left side) and fructose (right side) in the production phase.The experiment turnout is at (i) 40mM[1- ¹³C] labeled substrate and (ii) 20mM[ ¹³C ₆] labeled substrate adds the mean value of twice parallel cultivation on the natural labeled substrate of 20mM and the corresponding deviation between twice cultivation.Except the turnout of biomass is with (the mg number of dry biomass) (mmol) ^-1Beyond providing, the full scale production amount with (product mmol number) (mol) ^-1Provide.

Turnout	Methionin production on glucose	Methionin production on fructose
Turnout	Methionin production on glucose	Methionin production on fructose	Biomass Methionin	54.1±0.8 281.0±2.0	28.5±0.0 244.4±23.3

Valine alanine glycine dihydroxyacetone glycerine trehalose KG acetic acid pyruvic acid lactic acid

0.1±0.0 0.1±0.0 6.6±0.0 26.3±15.3 3.8±2.4 3.3±0.5 1.6±0.4 45.1±0.3 1.2±0.4 7.1±1.7

0.0±0.0 0.4±0.1 7.1±0.4 156.6±25.8 38.4±3.9 0.9±0.1 6.5±0.3 36.2±5.7 2.1±0.5 38.3±3.5

Table 2: Corynebacterium glutamicum ATCC 21526 Methionin in the production phase to anabolism demand from glucose (left side) and fructose (right side).Experimental data is at (i) [1- ¹³C] labeled substrate and (ii) 1: 1 natural labeled substrate with [ ¹³C ₆] mean value of twice parallel cultivation on the mixture of labeled substrate and the deviation between twice cultivation.

The precursor demand ^*Mmol (mol glucose) ^-1	Methionin production on glucose	Methionin production on fructose
The precursor demand ^*Mmol (mol glucose) ^-1	Methionin production on glucose	Methionin production on fructose	Glucose fructose-1, 6-diphosphate 6-phosphopentose 5-phosphoric acid erythrose 4-glyceraldehyde phosphate 3-phosphoric acid 3-phoshoglyceric acid pyruvic acid/phosphoenolpyruvate KG oxaloacetic acid acetyl coenzyme A diaminopimelic acid+lysine^**	11.09±0.16 3.84±0.06 47.50±0.70 14.50±0.22 6.98±0.10 59.95±0.89 107.80±1.60 92.51±1.37 48.91±0.72 135.30±2.00 18.83±0.28	5.84±0.05 2.02±0.02 25.05±0.21 7.64±0.06 3.68±0.03 36.85±0.31 56.80±0.48 48.73±0.41 45.76±0.38 71.25±0.60 9.92±0.08

^*) estimation of precursor demand is based on the turnout (table 1) of the experiment biomass that each bacterial strain is obtained and the before measured biomass composition (Marx for Corynebacterium glutamicum, A., A.A.de Graaf, W.Wiechert, L.Eggeling and H.Sahm.1996.Biotechnol.Bioeng.49:111-129).

^*) diaminopimelic acid and Methionin is considered to independent anabolism precursor.This be since from pyruvic acid and oxaloacetic acid to the anabolism flux of diaminopimelic acid (cell walls) and Methionin (protein), except helping Methionin excretory flux, also help total flux by the Methionin biosynthetic pathway.

Example II: in the chase experiment ¹³The hand inspection of C marking mode

The relative mass isotope-isomerism thing fraction of secreted Methionin and trehalose is quantitative by GC-MS.These quality isotope-isomerism thing fractions are responsive and finger printing that therefore show the biosystem fluxome that studies for flux in the cell.As shown in Figure 2, the marking mode of secreted Methionin and trehalose is significantly different between the Corynebacterium glutamicum cell of growing on glucose and the fructose.For two kinds the radioactive labelling thing of using and two kinds of measured products all found these differences.This shows that depending on applied carbon source exists essential distinction in the carbon flux mode.As indicated previously, at [1- ¹³C] and [ ¹³C ₆] the quality isotope-isomerism thing fraction (Wittmann much at one that Corynebacterium glutamicum carried out twice parallel cultivation on the glucose mixture, C., H.M.Kim and E.Heinzle.2003.Metabolic flux analysis at miniaturized scale. submit to).Therefore, viewed difference may be relevant with the substrate specificity obvious difference in metabolic flux.

EXAMPLE III: the estimation of flux in the cell

The central issue of being studied is the comparative study of flux in the Methionin production period Corynebacterium glutamicum cell when being carbon source with glucose and fructose respectively.For this reason, the experimental data that obtains from chase experiment is used to use the metabolic flux distribution of above-mentioned flux estimation computed in software for every kind of substrate.Parameter estirmation by will test and calculated mass isotope-isomerism thing fraction between minimum deviation realize.In each step of optimizing, the method for being carried out is utilized the metabolite balance.This comprises (i) product excretory stoichiometry data (table 2) and (ii) for the anabolism demand stoichiometry data (table 3) of biomass precursor.The interior flux group (set) of cell that provides minimum deviation between experiment and the analog mark pattern is used as the optimum estimate of Flux Distribution in the pair cell.For two kinds of situations, use a plurality of initialization values to obtain identical Flux Distribution, show to have identified overall minimum quantity.Clearly, between the quality isotope-isomerism thing ratio of testing definite quality isotope-isomerism thing and calculating, has good consistence (table 4).

Table 3: the relative mass isotope-isomerism thing fraction that is incubated at the Corynebacterium glutamicum ATCC 21526 secreted Methionins and the trehalose of the product Methionin on glucose and the fructose respectively.For two kinds of carbon sources, at (i) [1- ¹³C] mark spike substrate and (ii) natural ¹³C mark spike substrate and [ ¹³C ₆] carried out two parallel chase experiments on 1: 1 mixture of mark spike substrate.The predicted numerical value (calc) of the corresponding optimization flux group that has shown experiment GC/MS data (exp) and made by mathematical model.M ₀The relative quantity of expression non-marked quality isotope-isomerism thing fraction, M ₁The relative quantity of representing single labelled quality isotope-isomerism thing fraction, and the more relative quantity of protrude mark quality isotope-isomerism thing fraction represented in corresponding term.

Methionin is (at [1- ¹³C] on the labeled substrate)					Trehalose is (at [1- ¹³C] on the labeled substrate)					Trehalose is (at 50%[ ¹²C ₆] on the labeled substrate)
Methionin is (at [1- ¹³C] on the labeled substrate)					Trehalose is (at [1- ¹³C] on the labeled substrate)					Trehalose is (at 50%[ ¹²C ₆] on the labeled substrate)							M ₀	M ₁	M ₂	M ₃	M ₄	M ₀	M ₁	M ₂	M ₃	M ₄	M ₀	M ₁	M ₂	M ₃	M ₄	M ₅	M ₆
Glucose exp calc fructose exp calc	0.234 0.242 0.133 0.139	0.360 0.355 0.316 0.321	0.247 0.245 0.304 0.298	0.110 0.110 0.162 0.159	0.037 0.037 0.062 0.061	0.110 0.114 0.212 0.195	0.551 0.549 0.412 0.419	0.216 0.212 0.244 0.254	0.094 0.094 0.092 0.094	0.023 0.023 0.030 0.030	0.271 0.268 0.141 0.144	0.114 0.113 0.103 0.103	0.087 0.085 0.104 0.102	0.115 0.113 0.250 0.245	0.069 0.068 0.133 0.131	0.066 0.064 0.110 0.111	M ₀	M ₁	M ₂	M ₃	M ₄	M ₀	M ₁	M ₂	M ₃	M ₄	M ₀	M ₁	M ₂	M ₃	M ₄	M ₅	M ₆	0.279 0.289 0.159 0.164

EXAMPLE IV: for fructose and glucose, at the metabolic flux of Methionin production period

Be shown in figure (4,5) for Flux Distribution in the cell that Corynebacterium glutamicum obtained of the product Methionin of on glucose and fructose, growing.Clearly, flux can depend on applied carbon source and very big difference is arranged in the cell.On glucose, 62% carbon flux current is to PPP, and the 36% glycolysis-chain (Fig. 4) of flowing through only.Because this high relatively amount, 124% NADPH produces by glucose 6-phosphate dehydrogenase among the PPP and 6-Phosphogluconic dehydrogenase.For fructose, situation fully different (Fig. 5).The flux distribution of being carried out has disclosed two kinds of activity in vivo that PTS absorbs for fructose, and 92.3% fructose is taken in by the special PTS fructose of fructose thus.By contrast, small portion fructose (7.7%) is taken in by the PTS seminose.Therefore, most fructose are at fructose 1, and 6-bisphosphate level enters glycolysis-, and only sub-fraction enters the glycolysis-chain in fructose 6-phosphoric acid upstream.Compare with the cell of growing on glucose, PPP presents the activity of rapid reduction, and only 14.4%.Glucose 6-phosphoric acid isomerase plays opposite effect to two kinds of carbon sources.In the cell of growing on glucose, 36.2% net flux flows to fructose 6-phosphoric acid from glucose 6-phosphoric acid, then observes 15.2% reverse net flux for fructose.

On fructose, the flux of flow through glucose 6-phosphoric acid isomerase and PPP is the about twice of flux of PTS seminose of flowing through.Yet this is not that gluconeogenesis provides extra carbon flux to PPP because of the gluconeogenesis flux of carbon from fructose 1,6 bisphosphate to fructose 6-phosphoric acid.In fact, the fructose 1 of flowing through, the flux of 6-diphosphatase institute this reaction of catalysis are zero.The metabolic reaction of being responsible for flowing to the extra flux of PPP is irreversible enzyme transaldolase and the transketolase among the PPP.The extra flux of this of about 3.5% is provided by transketolase 2, and this makes the carbon that is derived from PPP circulate again and enters this approach.In addition, 4.2% flux has flowed to fructose 6-phosphoric acid and PPP by the effect of transaldolase.

Depend on carbon source, around the pyruvic acid node, can also observe the diverse flux mode (Fig. 4,5) in the Corynebacterium glutamicum that produces Methionin.For glucose, the flux that enters the Methionin approach is 30.0%, and is 25.4% to reduce for the flux that fructose enters the Methionin approach.Compare with fructose, the Methionin turnout that improves for glucose is the major cause of this kind flux difference, and causes also having facilitated this difference to being used for cell walls synthetic diaminopimelic acid with the more high-biomass turnout of the higher demand of the Methionin that is used for protein synthesis.Therefore for glucose, the covering flux is 44.5% and is significantly higher than the flux for fructose (33.5%) of comparing.This mainly is because to being used for the higher demand of the oxaloacetic acid that Methionin produces, but also be since on glucose to the higher anabolism demand of oxaloacetic acid and 2-oxoglutaric acid.On the other hand, compare, for the flow through flux lower basically (70.9%) of pyruvic oxidase of glucose with fructose (95.2%).Flux current in the flux that carbon flux that this TCA of entering round-robin reduces has caused having reduced for glucose more than 30% is through TCA cyclophorase (Fig. 3,4).

By the Monte-Carlo method flux that is obtained is carried out statistical evaluation, with calculate 90% fiducial interval of definite traffic parameter.Multiple crucial flux as shown in table 5, fiducial interval is narrower usually.For example, flow through the fiducial interval of flux of glucose 6-phosphate dehydrogenase for only being 1.2% at the cell of growing on the glucose and only being 3.5% for the cell of on fructose, growing.Therefore institute's choosing method allows accurately to estimate flux.Can reach a conclusion: cause by applied carbon source beyond doubt for glucose and the viewed flux difference of fructose.

Have been noted that for the average specific substrate of fructose and take in (1.93mmol g ^-1h ¹) take in (1.77mmol g a little more than average specific substrate for glucose ^-1h ^-1).Thus, compare with relative flux discussed above, for glucose with mmol g ^-1h ^-1Flux is little in the absolute cell of expression an increase.Yet the Corynebacterium glutamicum Flux Distribution of the product Methionin of growing on fructose and glucose is so fully different respectively, so that all comparisons that draw from above are also effective for absolute carbon flux.

Table 4: by using mass spectrum and meta-bolites equilibrated ¹³The determined statistical evaluation that grows in Corynebacterium glutamicum ATCC 21526 metabolic fluxs of the product Methionin on fructose (left side) and the glucose (right side) of C tracer study: 90% fiducial interval of main traffic parameter obtains by the Monte-Carlo method, comprises that each substrate with significant difference experimental data is carried out 100 subparameters estimates the distance of swimming.

Traffic parameter	Glucose	Fructose
Traffic parameter	Glucose	Fructose	Net flux passes through PTS _FrcFructose take in and to pass through PTS _ManFructose take in glucose 6-phosphoric acid isomerase	- - [35.7 36.8]	[90.0 96.1] [3.9 10.0] [13.4 16.9]

Phosphofructokinase fructose 1, the 6-diphosphatase ^*Fructose 1,6-diphosphatase aldolase G 6 PD transaldolase transketolase 1 transketolase 2 glyceraldehyde 3 phosphate dehydrogenase pyruvate kinase pyruvic dehydrogenase pyruvate carboxylase citrate synthase isocitric dehydrogenase ketoglutaric dehydrogenase aspartokinase flux invertibitys^**Glucose 6-phosphoric acid isomerase transaldolase transketolase 1 transketolase 2

[35.7 36.8] - [73.7 73.8] [62.5 63.7] [19.4 19.8] [19.4 19.8] [17.9 18.3] [158.1 164.5] [156.2 167.4] [69.5 72.5] [43.7 44.8] [51.2 54.8] [51.2 54.8] [41.6 45.6] [29.6 30.3] [4.5 5.1] [4.3 4.9] [0.0 0.0] [0.4 0.6]

- [-2.1 3.4] [91.7 92.9] [12.6 16.1] [3.6 4.1] [3.6 4.1] [2.9 4.0] [163.3 174.6] [158.9 168.2] [87.1 102.3] [29.9 37.3] [76.5 91.5] [76.5 91.5] [70.9 86.0] [21.8 29.2] - [14.5 18.2] [0.0 0.1] [0.0 0.1]

^*The negative flux of low confidence region equals the positive flux (phosphofructokinase of flowing through) on the reverse direction.

^*The flux reversibility is defined as the ratio of opposite flux and net flux.

The discussion of example I-IV:

A. substrate specificity cultural characteristic

Produce the Corynebacterium glutamicum announcement of the growth on fructose and glucose respectively of Methionin, growth and product form and depend on applied carbon source strongly.Before also reported, another bacterial strain for Corynebacterium glutamicum, growth has significantly reduced Methionin turnout and biomass on fructose, wherein compare with glucose, Methionin and biomass production amount reduce by 30% and 20% (Kiefer respectively, P., E.Heinzle and C.Wittmann.2002.J.Ind.Microbiol.Biotechnol.28:338-43).Compare with glucose, on fructose, cultivate Corynebacterium glutamicum and corynebacterium melassecola (the C.melassecola) (Dominguez that is associated with higher carbon dioxide production rate, H., C.Rollin, A.Guyonvarch, J.L.Guerquin-Kern, M.Cocaign-Bousquet and N.D.Lindley.1998.Eur.J.Biochem.254:96-102; Kiefer, P., E.Heinzle and C.Wittmann.2002.J.Ind.Microbiol.Biotechnol.28:338-43).This is consistent with the flux that raises for the viewed TCA round-robin of flowing through of this carbon source in this research.Also observe substrate specificity difference for byproduct.Compare with glucose, lower for the formation of fructose trehalose.This enters glucolytic inlet point difference relevant (Kiefer, P., E.Heinzle and C.Wittmann.2002.J.Ind.Microbiol.Biotechnol.28:338-43) with glucose and fructose.Consider the shooting system in the Corynebacterium glutamicum, the utilization of glucose causes the formation of trehalose precursor glucose 6-phosphoric acid, and fructose is transformed into fructose 1, therefore the 6-bisphosphate also enters the center metabolism (Dominguez in glucose 6-phosphoric acid downstream, H., C.Rollin, A.Guyonvarch, J.L.Guerquin-Kern, M.Cocaign-Bousquet and N.D.Lindley.1998.Eur.J.Biochem.254:96-102).When fructose used as carbon source, other byproduct such as Protosol, glycerine and lactic acid significantly increased.From the angle of Methionin production, this is not desired, forms byproduct because quite most carbon withdraws from from the metabolism of center.For fructose, specific substrate is taken in (1.93mmol g ^-1h ^-1) be higher than glucose (1.77mmol g ^-1h ^-1).This result is different from the previous research (Dominguez that the corynebacterium melassecola ATCC 17965 of exponential growth is carried out, H., C.Rollin, A.Guyonvarch, J.L.Guerquin-Kern, M.Cocaign-Bousquet and N.D.Lindley.1998.Eur.J.Biochem.254:96-102), wherein formerly observe similar special uptake rate for fructose and glucose in the research.Viewed in our research may be because the bacterial strain difference of being studied for the higher uptake rate of fructose.Corynebacterium melassecola is relevant species with Corynebacterium glutamicum, but may be different on some metabolic characteristic.The bacterial strain of being studied in this work is before to obtain by classical bacterial strain optimization.This may import influences the sudden change that substrate is taken in.The another kind of explanation is the difference of culture condition.Under the condition of limiting growth and Methionin production, fructose can more effectively be utilized.

B. metabolic flux distributes

Shown greatest differences for Flux Distribution in the cell of the Corynebacterium glutamicum of the product Methionin that glucose and fructose obtained.Statistical evaluation to the flux that obtained has disclosed 90% narrow fiducial interval, so that viewed flux difference undoubtedly can be owing to applied carbon source.The flux that relates between glycolysis-and the PPP of one of the most significant difference distributes.On glucose, 62.3% the carbon PPP approach of flowing through.Observed (Marx in the research that the PPP advantage of the Corynebacterium glutamicum of product Methionin is formerly different on this substrate, A., A.A.de Graaf, W.Wiechert, L.Eggeling and H.Sahm.1996.Biotechnol.Bioeng.49:111-129; Wittmann, C. and E.Heinzle.2001.Eur.J.Biochem.268:2441-2455; Wittmann, C. and E.Heinzle.2002.Appl.E nviron.Microbiol.68:5843-5859).On fructose, the flux that enters PPP is reduced to 14.4%.As pass through the metabolic flux that carried out analyze evaluation, this mainly be owing to fructose at fructose 1,6-bisphosphate level enter with fructose 1,6 diphosphatase inactivation between unfavorable combination.Viewed fructose 1, the inactivation of 6 diphosphatases and the very consistent (Dominguez of zymetology measuring result of the corynebacterium melassecola ATCC 17965 during exponential growth on fructose and the glucose respectively, H., C.Rollin, A.Guyonvarch, J.L.Guerquin-Kern, M.Cocaign-Bousquet and N.D.Lindley.1998.Eur.J.Biochem.254:96-102).

Surprisingly, when Corynebacterium glutamicum was cultivated on fructose, the flux of flow through glucose 6-phosphoric acid isomerase and PPP was the PTS that flows through _SeminoseAbout twice of flux.Because the inactivation of fructose 1,6 diphosphatase, so this is not caused by the gluconeogenesis flux.In fact, Corynebacterium glutamicum has the effective metabolic cycles through fructose 6-phosphoric acid, glucose 6-phosphoric acid and ribose 5-phosphoric acid.The extra flux that enters PPP is that the effect by transketolase 2 and transaldolase provides, and wherein transketolase 2 will circulate again from the carbon of PPP and return this approach, and transaldolase makes Glycerose 3-phosphoric acid return PPP, therefore walks around Gluconeogenesis.The NADPH restriction that this circulation is active can help cell to overcome to be substrate with fructose the time.Flux for the very big reduction that arrives glucose 6-phosphoric acid in the Corynebacterium glutamicum that on fructose, grows, can also explain the reduction (Kiefer that trehalose forms on this substrate, P., E.Heinzle and C.Wittmann.2002.J.Ind.Microbiol.Biotechnol.28:338-43).Glucose 6-phosphoric acid isomerase can depend on carbon source and work in the opposite direction.When growing on glucose, net flux directly flows to fructose 6-phosphoric acid from glucose 6-phosphoric acid, and observes opposite net flux on fructose.This has emphasized the importance of the reversibility of this enzyme for metabolism handiness in the Corynebacterium glutamicum.

The C.NADPH metabolism

Below calculating provides the metabolic comparison of NADPH of producing the Corynebacterium glutamicum of Methionin on fructose and glucose.Whole supplies of having calculated NADPH from the fluxmeter of estimated flow through glucose 6-phosphate dehydrogenase, 6-Phosphogluconic dehydrogenase and isocitric enzyme.On glucose, glucose 6-phosphate dehydrogenase (62.0%) and glucose 6-phosphate dehydrogenase (62.0%) provide most NADPH among the PPP.The degree that isocitric enzyme (52.9%) provides is less.It is different fully to the contribution that NADPH supplies with the TCA circulation to observe PPP on fructose, and wherein isocitric enzyme (83.3%) is the main source of NADPH.On fructose, glucose 6-phosphate dehydrogenase (14.4%) and glucose 6-phosphate dehydrogenase (14.4%) produce considerably less NADPH.NADPH is that growth and Methionin form necessary.Grow required NADPH from 11.51mmol NAPDH (g biomass) ^-1The stoichiometric requirement amount is calculated, suppose that this is for growth (Dominguez on glucose and fructose, H., C.Rollin, A.Guyonvarch, J.L.Guerquin-Kern, M.Cocaign-Bousquet and N.D.Lindley.1998.Eur.J.Biochem.254:96-102) be identical with testing biomass production amount (table 1) for this institute.For the biomass production on glucose, Corynebacterium glutamicum consumes 62.3% NADPH, and this is than higher as carbon source (32.8%) with fructose.The amount of the NADPH that product is synthetic required is from estimated flux (table 1) that enters Methionin and 4mol (mol Methionin) ^-1Corresponding chemical metering NADPH demand determine.For being 112.4% and be 97.6% from glucose production Methionin for producing Methionin from fructose.All NADPH supplies with (176.9%) and is significantly higher than fructose (112.1%) on glucose, and this may mainly be because the increase of PPP flux on glucose.On glucose, the NADPH balance is almost closed.On the contrary, on fructose, observe significant NADPH (18.3%) and lack.This causes such query, and promptly except glucose 6-phosphate dehydrogenase above-mentioned, 6-Phosphogluconic dehydrogenase and isocitric enzyme, can the enzyme of catalysis metabolic reaction provide NADPH.The malic enzyme of possible candidate NADPH dependence seemingly.Before, compare with the cell of on glucose, growing, detect the activity specific (Dominguez that this enzyme increases in the corynebacterium melassecola of on fructose, growing, H., C.Rollin, A.Guyonvarch, J.L.Guerquin-Kern, M.Cocaign-Bousquet and N.D.Lindley.1998.Eur.J.Biochem.254:96-102).Yet the flux of this certain enzyme of flowing through can not solve in this research by experiment.Suppose that malic enzyme is the enzyme of the generation NADPH of shortage, 18.3% flux will be enough to replenish the obvious shortage of NADPH.Studies show that as the detailed flux of the Corynebacterium glutamicum of carbon source malic enzyme does not have remarkable activity (Petersen with glucose, S., A.A.deGraaf, L.Eggeling, M.M  llney, W.Wiechert and H.Sahm.2000.J.Biol.Chem.75:35932-35941).Yet the situation on fructose can be associated the activity in vivo that this enzyme improves.

The D.NADH metabolism

On fructose, Corynebacterium glutamicum shows that NADH forms enzymic activity and increases.On fructose, 421.2% NADH is formed by Glycerose 3-phosphate dehydrogenase, pyruvic oxidase, 2-oxoglutaric acid desaturase and malate dehydrogenase (malic acid dehydrogenase).On glucose, the generation of NADH only is 322.4%.In addition, significantly be lower than demand on glucose in anabolic NADH demand on the fructose.Significantly the NADH that increases produces with the metabolic demand amount that reduces and causes the NADH/NAD ratio that increases.For corynebacterium melassecola, before showed, compare growth causes increasing on fructose NADH/NAD with glucose than (Dominguez, H., C.Rollin, A.Guyonvarch, J.L.Guerquin-Kern, M.Cocaign-Bousquet and N.D.Lindley.1998.Eur.J.Biochem.254:96-102).This causes the query to Methionin production period NADH regenerative system on fructose.The cell of growing on fructose presents enhanced Protosol, glycerine and lactic acid secretion.It may be because higher NADH/NAD ratio that Protosol that increases and glycerine form.Showed before that NADH can suppress glyceraldehyde dehydrogenase, so that the surplus of Protosol and glycerine may be relevant with the reduction of this enzyme throughput.Protosol also may be because the influence of high NADH/NAD ratio to the minimizing of glycerine in addition, and therefore helps the regeneration of excessive NADH.Form the required NADH of lactic acid from pyruvic acid and may have similar background to the generation of glycerine.Compare with exponential growth, under the Methionin working condition, superfluous even may be higher with high relatively TCA circulation biomass production amount active and that the reduce NADH that is feature.

E. be used on fructose, optimizing effective target spot of the Corynebacterium glutamicum that produces Methionin

Based on the flux mode that is obtained, illustrated several being used for the Corynebacterium glutamicum that grows on fructose has been carried out effective target spot that Methionin production is optimized.Central point is that NADPH supplies with.Fructose 1,6 diphosphatase is to be used to increase the target spot that NADPH supplies with.Go to regulate (as its increased activity) and cause the more high-throughput of PPP of flowing through, thereby cause NADPH regeneration that increases and the Methionin turnout that increases.By fructose 1, the enhancing of 6-diphosphatase make the flux of the PPP that flows through increase production for aromatic amino acid also be favourable (Ikeda, M.2003.Adv.Biochem.Eng.Biotechnol.79:1-36).From the viewpoint of Methionin production, growing period fructose 1 on fructose, the inactivation of 6-diphosphatase is deleterious, but not wondrous, because this gluconeogenesis enzyme growing period on sugar is unwanted and is suppressed probably.In prokaryotic cell prokaryocyte, by for example fructose 1,6-diphosphatase, fructose-2,6 diphosphatase, metal ion and AMP make this enzyme be in effective metabolism control (Skrypal, I.G. and O.V.Iastrebova.2002.Mikrobiol are Z.64:82-94) down.Known Corynebacterium glutamicum can grow on acetate (Wendisch, V.F., A.A.de Graaf, H.SahmH. and B.Eikmans.2000.J.Bacteriol.182:3088-3096), and this enzyme is essential for keeping gluconeogenesis therein.Another the effective target spot that increases the PPP flux of flowing through is to be used for the PTS that fructose is taken in.To PTS _FructoseAnd PTS _SeminoseBetween the modification that distributes of flux can produce more a high proportion of fructose (it enters in fructose 6-phosphoric acid level), and therefore also cause the PPP flux that increases.In addition, the enhancing that may obviously help the malic enzyme that the NADPH on fructose supplies with may be the purpose target spot.

Another bottleneck comprises the strong secretion of Protosol, glycerine and lactic acid.The formation of Protosol and glycerine can be by going to regulate (for example disappearance of corresponding enzyme) blocking-up.Dihydroxyacetone phosphate to the conversion of Protosol may be by corresponding phosphatase catalytic.Yet, in Corynebacterium glutamicum still not to dihydroxyacetone phosphatase carry out note (see the taxonomy website of state-run biotechnology information center (NCBI): Http:// www3.ncbi.nlm.nih.gov/Taxonomy/).This reaction can also be by kinases such as lactic dehydrogenase enzyme catalysis.At present, in the Corynebacterium glutamicum gene group database two clauses and subclauses relate to the Protosol kinases (see state-run biotechnology information center (NCBI) taxonomy website: Http:// www3.ncbi.nlm.nih.gov/Taxonomy/).

In one embodiment, one or more genes in the above combination gene are gone to regulate the production that is beneficial to fine chemicals such as Methionin.

Lactic acid secretion can also be regulated by going, for example knocking out of serum lactic dehydrogenase and eliminating.Regeneration may be important for NADH although glycerine and lactic acid form, in any case its negative interaction to the organism overall performance can not be got rid of.As previous supposition, be subjected to (Dominguez under the situation of Glycerose 3-phosphate dehydrogenase capabilities limits at the carbon flux than lower part glycolysis-chain of flowing through, H., C.Rollin, A.Guyonvarch, J.L.Guerquin-Kern, M.Cocaign-Bousquet and N.D.Lindley.1998.Eur.J.Biochem.254:96-102), the restraining effect of Protosol and glycerine production may finally cause the changing its course of carbon flux of the activation of fructose 1,6 diphosphatase and the PPP that flows through.Should be noted that, Protosol is not utilized between the Corynebacterium glutamicum incubation period again, and therefore as for the synthetic waste that then shows carbon of product, yet for lactic acid situation really not so (Cocaign-Bousquet, M. and N.D.Lindley.1995.Enz.Microbiol.Technol.17:260-267).

In addition, produce for the Methionin by Corynebacterium glutamicum, sucrose also is useful carbon source, for example unites use with the inventive method.Sucrose is the main carbon source in the molasses.As shown in before, the fructose units in the sucrose is at fructose 1, and 6-diphosphatase level enters glycolysis-(Dominguez, H. and N.D.Lindley.1996.Appl.Environ.Microbiol.62:3878-3880).Therefore, this part sucrose molecules-supposition fructose 1,6-diphosphatase inactivation-may not enter PPP, thus the NADPH supply in the bacterial strain that produces Methionin is restricted.

EXAMPLE V: the structure of plasmid PCIS LYSC

In the first step of strain construction, require in Corynebacterium glutamicum ATCC 13032, to carry out the allelotrope exchange of lysC wild type gene.Under this kind situation, in the lysC gene, carry out the Nucleotide exchange so that the amino acid Thr in resulting protein on the position 311 is replaced by Ile.From and use Oligonucleolide primers SEQ ID NO:3 and SEQ ID NO:4 initial, by Pfu Turbo PCR system (Stratagene, the U.S.) and according to product description amplification lysC as ATCC 13032 chromosomal DNAs of PCR reaction template.The chromosomal DNA for preparing Corynebacterium glutamicum ATCC 13032 according to the method for people (1994) Microbiology 140:1817-1828 such as people such as Tauch (1995) Plasmid33:168-179 or Eikmanns.5 ' end at amplified fragments is that SalI restriction enzyme site and its 3 ' end are the MluI restriction enzyme site.Before the clone, digest the fragment that is increased and use GFX by these two kinds of Restriction Enzymes ^TMPCR DNA and gel strips zone purification test kit (Amersham Pharmacia, Freiburg) purifying.

SEQ ID NO：3：

5’-GAGAGAGAGACGCGTCCCAGTGGCTGAGACGCATC-3’

SEQ ID NO：4：

5’-CTCTCTCTGTCGACGAATTCAATCTTACGGCCTG-3’

By SalI and MluI restriction site resulting polynucleotide are cloned among the pCLIK5MCS (being called pCIS (SEQ ID NO:5) hereinafter) of the SacB with integration, and conversion enters among the intestinal bacteria XL-1blue.Upward the cell that contains plasmid is screened by coating the LB agar (Lennox, 1955, Virology, 1: 190) that contains kantlex (20 μ g/ml).Separation quality grain also confirms to expect nucleotide sequence by checking order.Method by Qiagen company is also used material preparation plasmid DNA from Qiagen company.According to the described sequencing reaction that carries out of people such as Sanger (1977) Proceedings ofthe National Academy of Sciences USA 74:5463-5467.(PE Applied Biosystems Weiterstadt) separates sequencing reaction and analyzes to use ABI prism 377.Classify resulting plasmid pCIS lysC as SEQ ID NO:6.

Example VI: from the mutagenesis of the lysC gene of Corynebacterium glutamicum

Use QuickChange test kit (company: according to product description the lysC gene of Corynebacterium glutamicum is carried out directed mutagenesis the Stratagene/ U.S.).In plasmid pCIS lysC (SEQ ID NO:6), carry out mutagenesis.Synthetic following Oligonucleolide primers is used for by QuickChange method (Stratagene) thr 311 being replaced to 311ile:

SEQ ID NO：7：

5’-CGGCACCACCGACATCATCTTCACCTGCCCTCGTTCCG-3’

SEQ ID NO：8：

5’-CGGAACGAGGGCAGGTGAAGATGATGTCGGTGGTGCCG-3’

The use of these Oligonucleolide primers in the QuickChange reaction causes taking place on 932 Nucleotide exchange (becoming T by C) in lysC genes of SEQ ID NO:9.Enter among the intestinal bacteria XL1-blue and after preparing plasmid in conversion, confirmed amino acid exchange Thr311Ile in the lysC gene by [a] sequencing reaction.Plasmid called after pCIS lysC thr311ile is listed in SEQ IDNO:10.

As described in people such as Liebl (1989) FEMS Microbiology Letters 53:299-303, plasmid pCIS lysC thr311ile is transformed among the Corynebacterium glutamicum ATCC 13032 by electroporation.Modification to this scheme is described among the DE 10046870.Use as people such as Sambrook (1989), Molecular cloning.A Laboratory Manual, standard method described in the Cold Spring Harbor checks that by Southern trace and hybridization the karyomit(e) on the lysC locus of each transformant arranges.This measure guarantees that transformant integrated the plasmid that is transformed by homologous recombination on the lysC locus.This type of bacterium colony after the grow overnight, is coated sucrose CM nutrient agar (10% sucrose) with cell and is gone up and cultivated 24 hours under 30 ℃ in not containing antibiotic substratum.Because existing sacB gene is a toxic products with sucrose inversion among the carrier pCISlysC thr311ile, therefore the bacterium colony that can grow only by by wild-type lysC gene and second homologous recombination step between the sudden change lysC thr311ile gene lack those bacterium colonies of sacB gene.In the homologous recombination process, or the gene of wild type gene or sudden change lacks with the sacB gene.If SacB gene and wild type gene come along remove, then cause producing the transformant of sudden change.

Picking growth bacterium colony is also checked its kantlex sensitivity phenotype.The bacterium colony of disappearance SacB gene must demonstrate kantlex susceptibility growth behavior simultaneously.This type of kantlex susceptibility of research clone's Methionin productivity (seeing embodiment 6) in shaking bottle.For relatively, choose undressed Corynebacterium glutamicum ATCC 13032.The bacterium colony that Methionin production improves is compared in selection with control group, reclaim chromosomal DNA, by the respective area and the order-checking of PCR reaction amplification lysC gene.To have the Methionin composite character of increase and on 932 of lysC, have this kind clone who suddenlys change that detects and be called ATCC13032lysCfbr.

Example VII A: the preparation of plasmid PK19MOB SACB δ serum lactic dehydrogenase

According to people such as Tauch, people such as (1995) Plasmid 33:168-179 or Eikmanns, the method for (1994) Microbiology 140:1817-1828 prepares the chromosomal DNA of Corynebacterium glutamicum ATCC 13032.According to as people such as Innis, (1990) PCR Protocols.A Guide to Methodsand Applications, the described standard method of Academic Press is used Oligonucleolide primers SEQ ID NO:11 and 12 by polymerase chain reaction (PCR), (company: Stratagene) amplification has the lactate dehydrogenase gene of flank region as the chromosomal DNA of template and Pfu Turbo polysaccharase.

SEQ ID NO：11：

5’-CTAGCTAGCCATTGTCCTTCTGGCAGT-3’

SEQ ID NO：12：

5’-CTAGTCTAGACGCTCGTGTTCCTTTAGA-3’

Use GFX ^TM(Amersham Pharmacia is Freiburg) according to the dna fragmentation of the resulting about 2.0kb size of product description purifying for PCR DNA and gel strips zone purification test kit.Afterwards, (Roche Diagnostics is Mannheim) with its fracture and use GFX to use restriction enzyme NheI and XbaI ^TMPCR DNA and gel strips zone purification test kit purifying DNA fragment.

Plasmid pK19mob sacB, SEQ ID NO:13 carries out with restriction enzyme NheI and XbaI also that enzyme is cut and use GFX after electrophoretic separation ^TMPCR DNA separates the fragment of 5.5kb size with gel strips zone purification test kit.

Use and connect test kit (Roche Diagnostics fast, Mannheim) according to product description carrier segments is connected together with the PCR fragment, and according to as people such as Sambrook, (Molecular Cloning.A Laboratory Manual, Cold Spring Harbor, (1989)) described standard method will connect product and be transformed into competence intestinal bacteria XL-1Blue (Stratagene, La Jolla, the U.S.).Go up the screening that the cell of carrier is carried in realization by coating the LB agar (Lennox, 1955, Virology, 1: 190) that contains kantlex (20 μ g/mL).

According to the method for Qiagen company and use the material of Qiagen company to carry out the preparation of plasmid DNA.Sequencing reaction is according to people such as Sanger, and (1977) Proceedings of the NationalAcademy of Sciences USA 74:5463-5467 carries out.(PEApplied Biosystems, Weiterstadt) method is separated the sequencing reaction thing and is analyzed by ABI Prism 377.

Resulting called after pK19 serum lactic dehydrogenase (SEQ ID NO:14).

Use restriction enzyme EcoRI and BglI (Roche Diagnostics, Mannheim) digested plasmid pK19 serum lactic dehydrogenase and after electrophoretic separation, use GFX subsequently ^TMPCR DNA separates the fragment of 6.7kb size with gel strips zone purification test kit.Use the Klenow enzyme to handle after this fragment according to product description, (Roche Diagnostics Mannheim) connects once more according to product description to use rapid DNA to connect test kit.According to people such as Sambrook, the standard method that (Molecular Cloning.A Laboratory Manual, Cold Spring Harbor, (1989)) are described will connect product and be transformed into competence intestinal bacteria XL-1Blue (Stratagene, La Jolla, the U.S.).Go up realization to carrying the screening of carrier cell by coating the LB agar (Lennox, 1955, Virology, 1: 190) that contains kantlex (20 μ g/mL).

According to the method for Qiagen company and use the material of Qiagen company to carry out the preparation of plasmid DNA.Sequencing reaction is according to people such as Sanger, and (1977) Proceedings of the NationalAcademy of Sciences USA 74:5463-5467 carries out.(PEApplied Biosystems, Weiterstadt) method is separated the sequencing reaction thing and is analyzed by ABI Prism 377.Resulting plasmid pK19 δ serum lactic dehydrogenase is listed in SEQ ID NO:15.

Example VII A I: Methionin production

As people such as Liebl, (1989) FEMS Microbiology Letters 53:299-303 is described, uses electroporation method that plasmid pK19 δ serum lactic dehydrogenase is transformed into Corynebacterium glutamicum ATCC13032lysC ^FbrModification to this scheme is described among the DE 10046870.Use as people such as Sambrook (1989), Molecular cloning.A Laboratory Manual, standard method described in the ColdSpring Harbor checks that by Southern trace and hybridization the karyomit(e) on each transformant lactate dehydrogenase gene seat arranges.This measure guarantees that transformant integrated the plasmid that is transformed by homologous recombination on the lactate dehydrogenase gene seat.This type of bacterium colony after the grow overnight, is coated sucrose CM nutrient agar (10% sucrose) with cell and is gone up and cultivated 24 hours under 30 ℃ in not containing antibiotic substratum.

Because existing sacB gene is a toxic products with sucrose inversion in the carrier pK19 δ serum lactic dehydrogenase, therefore the bacterium colony that can grow is only for lacking those bacterium colonies of sacB gene by second homologous recombination step between wild-type lactate dehydrogenase gene and the institute's truncated gene.In the homologous recombination process, or the gene of wild type gene or brachymemma lacks with the sacB gene.If the SacB gene is removed with wild type gene, then cause producing the transformant of sudden change.

Picking growth bacterium colony is also checked its kantlex sensitivity phenotype.The bacterium colony of disappearance SacB gene must demonstrate kantlex susceptibility growth behavior simultaneously.As people such as Innis, (1990) PCRProtocols.A Guide to Methods and Applications, Academic Press describes, and utilizes polymerase chain reaction (PCR) to check according to standard method whether the expection replacement of truncated gene to natural gene has taken place.Analyze for this, separated initial strain and resultant clone's chromosomal DNA.For this target, use toothpick to take out each clone and be suspended from 100 μ LH from agar plate ₂Among the O, boiled 10 minutes at 95 ℃.In each case, get the resulting solution of 10 μ L as the template among the PCR.The primer that uses is oligonucleotide CK360 and CK 361.Because the selection of oligonucleotide can be expected with the resulting PCR product of initial strain DNA than bigger with the resulting PCR product of truncated gene.Positive colony called after ATCC13032 Psod lysC ^FbrThe δ serum lactic dehydrogenase.

In order to study the effect that δ serum lactic dehydrogenase construct is produced Methionin, under 30 ℃ with strains A TCC13032, ATCC13032 lysC ^FbrWith ATCC13032 lysC ^FbrThe δ serum lactic dehydrogenase is gone up in CM flat board (10.0g/l D-glucose, 2.5g/l NaCl, 2.0g/l urea, 10.0g/l bacto-tryptone (Difco), 5.0g/l yeast extract (Difco), 5.0g/l extractum carnis (Difco), 22.0g/l agar (Difco) are through autoclaving (121 ℃ following 20 minutes)) and was cultivated 2 days.Subsequently, cell is scraped and is suspended in the salt solution from flat board.For main culture, with 10ml substratum I and 0.5g through autoclaved CaCO ₃(Riedel de Haen) places 100ml Erlenmeyer flask, inoculates until OD with cell suspending liquid ₆₀₀Be 1.5, go up in 220 rev/mins at Infors AJ118 type shaking table (company: Infors, Bottmingen, Switzerland) and cultivated 39 hours.Determine to secrete the lysine concentration to the substratum then.

Substratum I:

40g/l sucrose

60g/l molasses (with 100% sugared cubage)

10g/l (NH ₄) ₂SO ₄

0.4g/l MgSO ₄·7H ₂O

0.6g/l KH ₂PO ₄

0.3mg/l thiamines HCl

(from the 1mg/ml storage solutions, this storage solutions is by filtration sterilization and use NH for the 1mg/l vitamin H ₄OH is adjusted to pH 8.0)

2mg/l FeSO ₄

2mg/l MnSO ₄

Use NH ₄OH transfers to pH 7.8, autoclaving (121 ℃, 20 minutes).

In addition, adding vitamin B12 (hydroxocobalamine, Sigma Chemicals) storage solutions (200 μ g/ml are by filtration sterilization) is 100 μ g/l until the vitamin B12 final concentration.

On the Agilent 1100 serial LC HPLC of system, measure amino acid concentration by the Agilent high pressure liquid chromatography (HPLC).Carry out the pre-column derivatization effect with o-phthalaldehyde(OPA) and allow formed amino acid is carried out quantitatively, and upward aminoacid mixture is separated at Hypersil AA post (Agilent).

And, utilize the zymetology method of inspection to determine concentration of lactic acid.

Equivalent

Those skilled in the art should be realized that or can only utilize normal experiment to determine many equivalents of the particular of the present invention described in the literary composition.Following claim is intended to comprise this type of equivalent.

Sequence table

＜110〉Bath good fortune (BASF AKTIENGESELLSCHAFT)

＜120〉method by the fermentative preparation fine chemicals

<130>BGI-160PC2

<150>PCT/IB2003/006435

<151>2003-12-18

<160>15

＜170〉be used for the FastSEQ edition 4 .0 of Windows

<210>1

<211>1660

<212>DNA

＜213〉Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(301)...(1563)

<400>1

tcggcatcct ctggggtagc gtcaacgcaa tcctcggaac cgtcatcgca gaaaacttcg 60

cacctgaggt ccgctacacc ggcgctaccc tgggttacca agtcggagca gcactcttcg 120

gcggtaccgc acccattatc gcagcatggc tgttcgaaat ctccggcgga caatggtggc 180

caatcgccgt ctacgtcgct gcatgttgcc ttctctctgt gatcgcctcg ttcttcatcc 240

aacgcgtcgc gcaccaagag aactaaaatc taagtaaaac ccctccgaaa ggaaccaccc 300

atg gtg aaa cgt caa ctg ccc aac ccc gca gaa cta ctc gaa ctc atg 348

Met Val Lys Arg Gln Leu Pro Asn Pro Ala Glu Leu Leu Glu Leu Met

1 5 10 15

aag ttc aaa aag cca gag ctc aac ggc aag aaa cga cgc cta gac tcc 396

Lys Phe Lys Lys Pro Glu Leu Asn Gly Lys Lys Arg Arg Leu Asp Ser

20 25 30

gcg ctc acc atc tac gac ctg cgt aaa att gct aaa cga cgc acc cca 444

Ala Leu Thr Ile Tyr Asp Leu Arg Lys Ile Ala Lys Arg Arg Thr Pro

35 40 45

gct gcc gcg ttc gac tac acc gac ggc gca gcc gag gcc gaa ctc tca 492

Ala Ala Ala Phe Asp Tyr Thr Asp Gly Ala Ala Glu Ala Glu Leu Ser

50 55 60

atc aca cgc gca cgt gaa gca ttc gaa aac atc gaa ttc cac cca gac 540

Ile Thr Arg Ala Arg Glu Ala Phe Glu Asn Ile Glu Phe His Pro Asp

65 70 75 80

atc ctc aag cct gca gaa cac gta gac acc acc acc caa atc ctg ggc 588

Ile Leu Lys Pro Ala Glu His Val Asp Thr Thr Thr Gln Ile Leu Gly

85 90 95

gga acc tcc tcc atg cca ttc ggc atc gca cca acc ggc ttc acc cgc 636

Gly Thr Ser Ser Met Pro Phe Gly Ile Ala Pro Thr Gly Phe Thr Arg

100 105 110

ctc atg cag acc gaa ggt gaa atc gca ggt gcc gga gct gca ggc gct 684

Leu Met Gln Thr Glu Gly Glu Ile Ala Gly Ala Gly Ala Ala Gly Ala

115 120 125

gca gga att cct ttc acc ctg tcc acc ctg ggc act acc tcc atc gaa 732

Ala Gly Ile Pro Phe Thr Leu Ser Thr Leu Gly Thr Thr Ser Ile Glu

130 135 140

gac gtc aag gcc acc aac ccc aac ggc cga aac tgg ttc cag ctc tac 780

Asp Val Lys Ala Thr Asn Pro Asn Gly Arg Asn Trp Phe Gln Leu Tyr

145 150 155 160

gtc atg cgc gac cgc gaa atc tcc tac ggc ctc gtc gaa cgc gca gcc 828

Val Met Arg Asp Arg Glu Ile Ser Tyr Gly Leu Val Glu Arg Ala Ala

165 170 175

aaa gca gga ttc gac acc ctg atg ttc acc gtg gat acc ccc atc gcc 876

Lys Ala Gly Phe Asp Thr Leu Met Phe Thr Val Asp Thr Pro Ile Ala

180 185 190

ggc tac cgc atc cgc gat tcc cgc aac gga ttc tcc atc ccg cca cag 924

Gly Tyr Arg Ile Arg Asp Ser Arg Asn Gly Phe Ser Ile Pro Pro Gln

195 200 205

ctg acc cca tcc acc gtg ctc aat gca atc cca cgc cca tgg tgg tgg 972

Leu Thr Pro Ser Thr Val Leu Asn Ala Ile Pro Arg Pro Trp Trp Trp

210 215 220

atc gac ttc ctg acc acc cca acc ctt gag ttc gca tcc ctt tcc tcg 1020

Ile Asp Phe Leu Thr Thr Pro Thr Leu Glu Phe Ala Ser Leu Ser Ser

225 230 235 240

acc ggc gga acc gtg ggc gac ctc ctc aac tcc gcg atg gat ccc acc 1068

Thr Gly Gly Thr Val Gly Asp Leu Leu Asn Ser Ala Met Asp Pro Thr

245 250 255

att tct tac gaa gac ctc aag gtc atc cgt gaa atg tgg cca ggc aag 1116

Ile Ser Tyr Glu Asp Leu Lys Val Ile Arg Glu Met Trp Pro Gly Lys

260 265 270

ctc gta gtc aag ggt gtc cag aac gtt gaa gac tcc gtc aaa ctc ctc 1164

Leu Val Val Lys Gly Val Gln Asn Val Glu Asp Ser Val Lys Leu Leu

275 280 285

gac caa ggc gtc gac ggc ctc atc ctc tcc aac cac ggt ggc cgt caa 1212

Asp Gln Gly Val Asp Gly Leu Ile Leu Ser Asn His Gly Gly Arg Gln

290 295 300

ctc gac cgc gca cca gtc cca ttc cac ctc ctg cca cag gta cgc aag 1260

Leu Asp Arg Ala Pro Val Pro Phe His Leu Leu Pro Gln Val Arg Lys

305 310 315 320

gaa gtc gga tct gaa cca acc atc atg atc gac acc ggc atc atg aac 1308

Glu Val Gly Ser Glu Pro Thr Ile Met Ile Asp Thr Gly Ile Met Asn

325 330 335

ggc gcc gac atc gtc gca gcc gta gcc atg ggc gct gac ttc acc ctc 1356

Gly Ala Asp Ile Val Ala Ala Val Ala Met Gly Ala Asp Phe Thr Leu

340 345 350

atc ggt cgt gcc tac ctc tac gga ctc atg gcc gga ggc cgc gaa ggc 1404

Ile Gly Arg Ala Tyr Leu Tyr Gly Leu Met Ala Gly Gly Arg Glu Gly

355 360 365

gtc gac cgc acc atc gcc att ctc cgc agc gag atc acc cgc acc atg 1452

Val Asp Arg Thr Ile Ala Ile Leu Arg Ser Glu Ile Thr Arg Thr Met

370 375 380

gct ctc ctc ggt gtt tcc tcc ctc gaa gaa ctc gag cca cgc cac gtc 1500

Ala Leu Leu Gly Val Ser Ser Leu Glu Glu Leu Glu Pro Arg His Val

385 390 395 400

acc cag ctg gcc aag atg gtt cca gtt tct gac gca act cgt tct gca 1548

Thr Gln Leu Ala Lys Met Val Pro Val Ser Asp Ala Thr Arg Ser Ala

405 410 415

gcg gcg gag att taa aagtttctct ccttagctat taaaaggtgc ccatccgttt 1603

Ala Ala Glu Ile *

420

ggatgggcac cttctcgttt cttgcaatcg gcatattcag tcaaaaaatg ttgaaat 1660

<210>2

<211>420

<212>PRT

＜213〉Corynebacterium glutamicum

<400>2

Met Val Lys Arg Gln Leu Pro Asn Pro Ala Glu Leu Leu Glu Leu Met

1 5 10 15

Lys Phe Lys Lys Pro Glu Leu Asn Gly Lys Lys Arg Arg Leu Asp Ser

20 25 30

Ala Leu Thr Ile Tyr Asp Leu Arg Lys Ile Ala Lys Arg Arg Thr Pro

35 40 45

Ala Ala Ala Phe Asp Tyr Thr Asp Gly Ala Ala Glu Ala Glu Leu Ser

50 55 60

Ile Thr Arg Ala Arg Glu Ala Phe Glu Asn Ile Glu Phe His Pro Asp

65 70 75 80

Ile Leu Lys Pro Ala Glu His Val Asp Thr Thr Thr Gln Ile Leu Gly

85 90 95

Gly Thr Ser Ser Met Pro Phe Gly Ile Ala Pro Thr Gly Phe Thr Arg

100 105 110

Leu Met Gln Thr Glu Gly Glu Ile Ala Gly Ala Gly Ala Ala Gly Ala

115 120 125

Ala Gly Ile Pro Phe Thr Leu Ser Thr Leu Gly Thr Thr Ser Ile Glu

130 135 140

Asp Val Lys Ala Thr Asn Pro Asn Gly Arg Asn Trp Phe Gln Leu Tyr

145 150 155 160

Val Met Arg Asp Arg Glu Ile Ser Tyr Gly Leu Val Glu Arg Ala Ala

165 170 175

Lys Ala Gly Phe Asp Thr Leu Met Phe Thr Val Asp Thr Pro Ile Ala

180 185 190

Gly Tyr Arg Ile Arg Asp Ser Arg Asn Gly Phe Ser Ile Pro Pro Gln

195 200 205

Leu Thr Pro Ser Thr Val Leu Asn Ala Ile Pro Arg Pro Trp Trp Trp

210 215 220

Ile Asp Phe Leu Thr Thr Pro Thr Leu Glu Phe Ala Ser Leu Ser Ser

225 230 235 240

Thr Gly Gly Thr Val Gly Asp Leu Leu Asn Ser Ala Met Asp Pro Thr

245 250 255

Ile Ser Tyr Glu Asp Leu Lys Val Ile Arg Glu Met Trp Pro Gly Lys

260 265 270

Leu Val Val Lys Gly Val Gln Asn Val Glu Asp Ser Val Lys Leu Leu

275 280 285

Asp Gln Gly Val Asp Gly Leu Ile Leu Ser Asn His Gly Gly Arg Gln

290 295 300

Leu Asp Arg Ala Pro Val Pro Phe His Leu Leu Pro Gln Val Arg Lys

305 310 315 320

Glu Val Gly Ser Glu Pro Thr Ile Met Ile Asp Thr Gly Ile Met Asn

325 330 335

Gly Ala Asp Ile Val Ala Ala Val Ala Met Gly Ala Asp Phe Thr Leu

340 345 350

Ile Gly Arg Ala Tyr Leu Tyr Gly Leu Met Ala Gly Gly Arg Glu Gly

355 360 365

Val Asp Arg Thr Ile Ala Ile Leu Arg Ser Glu Ile Thr Arg Thr Met

370 375 380

Ala Leu Leu Gly Val Ser Ser Leu Glu Glu Leu Glu Pro Arg His Val

385 390 395 400

Thr Gln Leu Ala Lys Met Val Pro Val Ser Asp Ala Thr Arg Ser Ala

405 410 415

Ala Ala Glu Ile

420

<210>3

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>3

gagagagaga cgcgtcccag tggctgagac gcatc 35

<210>4

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>3

ctctctctgt cgacgaattc aatcttacgg cctg 34

<210>5

<211>4323

<212>DNA

＜213〉Corynebacterium glutamicum

<400>5

tcgagaggcc tgacgtcggg cccggtacca cgcgtcatat gactagttcg gacctaggga 60

tatcgtcgac atcgatgctc ttctgcgtta attaacaatt gggatcctct agacccggga 120

tttaaatcgc tagcgggctg ctaaaggaag cggaacacgt agaaagccag tccgcagaaa 180

cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc 240

gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt 300

ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag 360

ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca 420

agatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac 480

gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca 540

atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt 600

gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg 660

tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga 720

agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct 780

cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg 840

gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg 900

gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc 960

gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat 1020

ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac 1080

tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt 1140

gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct 1200

cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc 1260

tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca 1320

ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga 1380

tcctccagcg cggggatctc atgctggagt tcttcgccca cgctagcggc gcgccggccg 1440

gcccggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc 1500

gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 1560

cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 1620

tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 1680

cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 1740

aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 1800

cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 1860

gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 1920

ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 1980

cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 2040

aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 2100

tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 2160

ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 2220

tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 2280

ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 2340

agattatcaa aaaggatctt cacctagatc cttttaaagg ccggccgcgg ccgccatcgg 2400

cattttcttt tgcgttttta tttgttaact gttaattgtc cttgttcaag gatgctgtct 2460

ttgacaacag atgttttctt gcctttgatg ttcagcagga agctcggcgc aaacgttgat 2520

tgtttgtctg cgtagaatcc tctgtttgtc atatagcttg taatcacgac attgtttcct 2580

ttcgcttgag gtacagcgaa gtgtgagtaa gtaaaggtta catcgttagg atcaagatcc 2640

atttttaaca caaggccagt tttgttcagc ggcttgtatg ggccagttaa agaattagaa 2700

acataaccaa gcatgtaaat atcgttagac gtaatgccgt caatcgtcat ttttgatccg 2760

cgggagtcag tgaacaggta ccatttgccg ttcattttaa agacgttcgc gcgttcaatt 2820

tcatctgtta ctgtgttaga tgcaatcagc ggtttcatca cttttttcag tgtgtaatca 2880

tcgtttagct caatcatacc gagagcgccg tttgctaact cagccgtgcg ttttttatcg 2940

ctttgcagaa gtttttgact ttcttgacgg aagaatgatg tgcttttgcc atagtatgct 3000

ttgttaaata aagattcttc gccttggtag ccatcttcag ttccagtgtt tgcttcaaat 3060

actaagtatt tgtggccttt atcttctacg tagtgaggat ctctcagcgt atggttgtcg 3120

cctgagctgt agttgccttc atcgatgaac tgctgtacat tttgatacgt ttttccgtca 3180

ccgtcaaaga ttgatttata atcctctaca ccgttgatgt tcaaagagct gtctgatgct 3240

gatacgttaa cttgtgcagt tgtcagtgtt tgtttgccgt aatgtttacc ggagaaatca 3300

gtgtagaata aacggatttt tccgtcagat gtaaatgtgg ctgaacctga ccattcttgt 3360

gtttggtctt ttaggataga atcatttgca tcgaatttgt cgctgtcttt aaagacgcgg 3420

ccagcgtttt tccagctgtc aatagaagtt tcgccgactt tttgatagaa catgtaaatc 3480

gatgtgtcat ccgcattttt aggatctccg gctaatgcaa agacgatgtg gtagccgtga 3540

tagtttgcga cagtgccgtc agcgttttgt aatggccagc tgtcccaaac gtccaggcct 3600

tttgcagaag agatattttt aattgtggac gaatcaaatt cagaaacttg atatttttca 3660

tttttttgct gttcagggat ttgcagcata tcatggcgtg taatatggga aatgccgtat 3720

gtttccttat atggcttttg gttcgtttct ttcgcaaacg cttgagttgc gcctcctgcc 3780

agcagtgcgg tagtaaaggt taatactgtt gcttgttttg caaacttttt gatgttcatc 3840

gttcatgtct ccttttttat gtactgtgtt agcggtctgc ttcttccagc cctcctgttt 3900

gaagatggca agttagttac gcacaataaa aaaagaccta aaatatgtaa ggggtgacgc 3960

caaagtatac actttgccct ttacacattt taggtcttgc ctgctttatc agtaacaaac 4020

ccgcgcgatt tacttttcga cctcattcta ttagactctc gtttggattg caactggtct 4080

attttcctct tttgtttgat agaaaatcat aaaaggattt gcagactacg ggcctaaaga 4140

actaaaaaat ctatctgttt cttttcattc tctgtatttt ttatagtttc tgttgcatgg 4200

gcataaagtt gcctttttaa tcacaattca gaaaatatca taatatctca tttcactaaa 4260

taatagtgaa cggcaggtat atgtgatggg ttaaaaagga tcggcggccg ctcgatttaa 4320

atc 4323

<210>6

<211>5860

<212>DNA

＜213〉Corynebacterium glutamicum

<400>6

cccggtacca cgcgtcccag tggctgagac gcatccgcta aagccccagg aaccctgtgc 60

agaaagaaaa cactcctctg gctaggtaga cacagtttat aaaggtagag ttgagcgggt 120

aactgtcagc acgtagatcg aaaggtgcac aaaggtggcc ctggtcgtac agaaatatgg 180

cggttcctcg cttgagagtg cggaacgcat tagaaacgtc gctgaacgga tcgttgccac 240

caagaaggct ggaaatgatg tcgtggttgt ctgctccgca atgggagaca ccacggatga 300

acttctagaa cttgcagcgg cagtgaatcc cgttccgcca gctcgtgaaa tggatatgct 360

cctgactgct ggtgagcgta tttctaacgc tctcgtcgcc atggctattg agtcccttgg 420

cgcagaagcc caatctttca cgggctctca ggctggtgtg ctcaccaccg agcgccacgg 480

aaacgcacgc attgttgatg tcactccagg tcgtgtgcgt gaagcactcg atgagggcaa 540

gatctgcatt gttgctggtt tccagggtgt taataaagaa acccgcgatg tcaccacgtt 600

gggtcgtggt ggttctgaca ccactgcagt tgcgttggca gctgctttga acgctgatgt 660

gtgtgagatt tactcggacg ttgacggtgt gtataccgct gacccgcgca tcgttcctaa 720

tgcacagaag ctggaaaagc tcagcttcga agaaatgctg gaacttgctg ctgttggctc 780

caagattttg gtgctgcgca gtgttgaata cgctcgtgca ttcaatgtgc cacttcgcgt 840

acgctcgtct tatagtaatg atcccggcac tttgattgcc ggctctatgg aggatattcc 900

tgtggaagaa gcagtcctta ccggtgtcgc aaccgacaag tccgaagcca aagtaaccgt 960

tctgggtatt tccgataagc caggcgaggc tgcgaaggtt ttccgtgcgt tggctgatgc 1020

agaaatcaac attgacatgg ttctgcagaa cgtctcttct gtagaagacg gcaccaccga 1080

catcaccttc acctgccctc gttccgacgg ccgccgcgcg atggagatct tgaagaagct 1140

tcaggttcag ggcaactgga ccaatgtgct ttacgacgac caggtcggca aagtctccct 1200

cgtgggtgct ggcatgaagt ctcacccagg tgttaccgca gagttcatgg aagctctgcg 1260

cgatgtcaac gtgaacatcg aattgatttc cacctctgag attcgtattt ccgtgctgat 1320

ccgtgaagat gatctggatg ctgctgcacg tgcattgcat gagcagttcc agctgggcgg 1380

cgaagacgaa gccgtcgttt atgcaggcac cggacgctaa agttttaaag gagtagtttt 1440

acaatgacca ccatcgcagt tgttggtgca accggccagg tcggccaggt tatgcgcacc 1500

cttttggaag agcgcaattt cccagctgac actgttcgtt tctttgcttc cccacgttcc 1560

gcaggccgta agattgaatt cgtcgacatc gatgctcttc tgcgttaatt aacaattggg 1620

atcctctaga cccgggattt aaatcgctag cgggctgcta aaggaagcgg aacacgtaga 1680

aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga 1740

caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat 1800

agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct 1860

ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct 1920

gatggcgcag gggatcaaga tctgatcaag agacaggatg aggatcgttt cgcatgattg 1980

aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 2040

actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 2100

ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg 2160

aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 2220

ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 2280

tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 2340

tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 2400

gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 2460

aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg 2520

atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 2580

tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 2640

tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 2700

tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 2760

tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc 2820

acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg 2880

ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccacgc 2940

tagcggcgcg ccggccggcc cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 3000

gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 3060

ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 3120

acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 3180

cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 3240

caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 3300

gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 3360

tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 3420

aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 3480

ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 3540

cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 3600

tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 3660

tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 3720

ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 3780

aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 3840

aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaaggccg 3900

gccgcggccg ccatcggcat tttcttttgc gtttttattt gttaactgtt aattgtcctt 3960

gttcaaggat gctgtctttg acaacagatg ttttcttgcc tttgatgttc agcaggaagc 4020

tcggcgcaaa cgttgattgt ttgtctgcgt agaatcctct gtttgtcata tagcttgtaa 4080

tcacgacatt gtttcctttc gcttgaggta cagcgaagtg tgagtaagta aaggttacat 4140

cgttaggatc aagatccatt tttaacacaa ggccagtttt gttcagcggc ttgtatgggc 4200

cagttaaaga attagaaaca taaccaagca tgtaaatatc gttagacgta atgccgtcaa 4260

tcgtcatttt tgatccgcgg gagtcagtga acaggtacca tttgccgttc attttaaaga 4320

cgttcgcgcg ttcaatttca tctgttactg tgttagatgc aatcagcggt ttcatcactt 4380

ttttcagtgt gtaatcatcg tttagctcaa tcataccgag agcgccgttt gctaactcag 4440

ccgtgcgttt tttatcgctt tgcagaagtt tttgactttc ttgacggaag aatgatgtgc 4500

ttttgccata gtatgctttg ttaaataaag attcttcgcc ttggtagcca tcttcagttc 4560

cagtgtttgc ttcaaatact aagtatttgt ggcctttatc ttctacgtag tgaggatctc 4620

tcagcgtatg gttgtcgcct gagctgtagt tgccttcatc gatgaactgc tgtacatttt 4680

gatacgtttt tccgtcaccg tcaaagattg atttataatc ctctacaccg ttgatgttca 4740

aagagctgtc tgatgctgat acgttaactt gtgcagttgt cagtgtttgt ttgccgtaat 4800

gtttaccgga gaaatcagtg tagaataaac ggatttttcc gtcagatgta aatgtggctg 4860

aacctgacca ttcttgtgtt tggtctttta ggatagaatc atttgcatcg aatttgtcgc 4920

tgtctttaaa gacgcggcca gcgtttttcc agctgtcaat agaagtttcg ccgacttttt 4980

gatagaacat gtaaatcgat gtgtcatccg catttttagg atctccggct aatgcaaaga 5040

cgatgtggta gccgtgatag tttgcgacag tgccgtcagc gttttgtaat ggccagctgt 5100

cccaaacgtc caggcctttt gcagaagaga tatttttaat tgtggacgaa tcaaattcag 5160

aaacttgata tttttcattt ttttgctgtt cagggatttg cagcatatca tggcgtgtaa 5220

tatgggaaat gccgtatgtt tccttatatg gcttttggtt cgtttctttc gcaaacgctt 5280

gagttgcgcc tcctgccagc agtgcggtag taaaggttaa tactgttgct tgttttgcaa 5340

actttttgat gttcatcgtt catgtctcct tttttatgta ctgtgttagc ggtctgcttc 5400

ttccagccct cctgtttgaa gatggcaagt tagttacgca caataaaaaa agacctaaaa 5460

tatgtaaggg gtgacgccaa agtatacact ttgcccttta cacattttag gtcttgcctg 5520

ctttatcagt aacaaacccg cgcgatttac ttttcgacct cattctatta gactctcgtt 5580

tggattgcaa ctggtctatt ttcctctttt gtttgataga aaatcataaa aggatttgca 5640

gactacgggc ctaaagaact aaaaaatcta tctgtttctt ttcattctct gtatttttta 5700

tagtttctgt tgcatgggca taaagttgcc tttttaatca caattcagaa aatatcataa 5760

tatctcattt cactaaataa tagtgaacgg caggtatatg tgatgggtta aaaaggatcg 5820

gcggccgctc gatttaaatc tcgagaggcc tgacgtcggg 5860

<210>7

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>7

cggcaccacc gacatcatct tcacctgccc tcgttccg 38

<210>8

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>8

cggaacgagg gcaggtgaag atgatgtcgg tggtgccg 38

<210>9

<211>1263

<212>DNA

＜213〉Corynebacterium glutamicum

<400>9

gtggccctgg tcgtacagaa atatggcggt tcctcgcttg agagtgcgga acgcattaga 60

aacgtcgctg aacggatcgt tgccaccaag aaggctggaa atgatgtcgt ggttgtctgc 120

tccgcaatgg gagacaccac ggatgaactt ctagaacttg cagcggcagt gaatcccgtt 180

ccgccagctc gtgaaatgga tatgctcctg actgctggtg agcgtatttc taacgctctc 240

gtcgccatgg ctattgagtc ccttggcgca gaagcccaat ctttcacggg ctctcaggct 300

ggtgtgctca ccaccgagcg ccacggaaac gcacgcattg ttgatgtcac tccaggtcgt 360

gtgcgtgaag cactcgatga gggcaagatc tgcattgttg ctggtttcca gggtgttaat 420

aaagaaaccc gcgatgtcac cacgttgggt cgtggtggtt ctgacaccac tgcagttgcg 480

ttggcagctg ctttgaacgc tgatgtgtgt gagatttact cggacgttga cggtgtgtat 540

accgctgacc cgcgcatcgt tcctaatgca cagaagctgg aaaagctcag cttcgaagaa 600

atgctggaac ttgctgctgt tggctccaag attttggtgc tgcgcagtgt tgaatacgct 660

cgtgcattca atgtgccact tcgcgtacgc tcgtcttata gtaatgatcc cggcactttg 720

attgccggct ctatggagga tattcctgtg gaagaagcag tccttaccgg tgtcgcaacc 780

gacaagtccg aagccaaagt aaccgttctg ggtatttccg ataagccagg cgaggctgcg 840

aaggttttcc gtgcgttggc tgatgcagaa atcaacattg acatggttct gcagaacgtc 900

tcttctgtag aagacggcac caccgacatc accttcacct gccctcgttc cgacggccgc 960

cgcgcgatgg agatcttgaa gaagcttcag gttcagggca actggaccaa tgtgctttac 1020

gacgaccagg tcggcaaagt ctccctcgtg ggtgctggca tgaagtctca cccaggtgtt 1080

accgcagagt tcatggaagc tctgcgcgat gtcaacgtga acatcgaatt gatttccacc 1140

tctgagattc gtatttccgt gctgatccgt gaagatgatc tggatgctgc tgcacgtgca 1200

ttgcatgagc agttccagct gggcggcgaa gacgaagccg tcgtttatgc aggcaccgga 1260

cgc 1263

<210>10

<211>5860

<212>DNA

＜213〉Corynebacterium glutamicum

<400>10

cccggtacca cgcgtcccag tggctgagac gcatccgcta aagccccagg aaccctgtgc 60

agaaagaaaa cactcctctg gctaggtaga cacagtttat aaaggtagag ttgagcgggt 120

aactgtcagc acgtagatcg aaaggtgcac aaaggtggcc ctggtcgtac agaaatatgg 180

cggttcctcg cttgagagtg cggaacgcat tagaaacgtc gctgaacgga tcgttgccac 240

caagaaggct ggaaatgatg tcgtggttgt ctgctccgca atgggagaca ccacggatga 300

acttctagaa cttgcagcgg cagtgaatcc cgttccgcca gctcgtgaaa tggatatgct 360

cctgactgct ggtgagcgta tttctaacgc tctcgtcgcc atggctattg agtcccttgg 420

cgcagaagcc caatctttca cgggctctca ggctggtgtg ctcaccaccg agcgccacgg 480

aaacgcacgc attgttgatg tcactccagg tcgtgtgcgt gaagcactcg atgagggcaa 540

gatctgcatt gttgctggtt tccagggtgt taataaagaa acccgcgatg tcaccacgtt 600

gggtcgtggt ggttctgaca ccactgcagt tgcgttggca gctgctttga acgctgatgt 660

gtgtgagatt tactcggacg ttgacggtgt gtataccgct gacccgcgca tcgttcctaa 720

tgcacagaag ctggaaaagc tcagcttcga agaaatgctg gaacttgctg ctgttggctc 780

caagattttg gtgctgcgca gtgttgaata cgctcgtgca ttcaatgtgc cacttcgcgt 840

acgctcgtct tatagtaatg atcccggcac tttgattgcc ggctctatgg aggatattcc 900

tgtggaagaa gcagtcctta ccggtgtcgc aaccgacaag tccgaagcca aagtaaccgt 960

tctgggtatt tccgataagc caggcgaggc tgcgaaggtt ttccgtgcgt tggctgatgc 1020

agaaatcaac attgacatgg ttctgcagaa cgtctcttct gtagaagacg gcaccaccga 1080

catcatcttc acctgccctc gttccgacgg ccgccgcgcg atggagatct tgaagaagct 1140

tcaggttcag ggcaactgga ccaatgtgct ttacgacgac caggtcggca aagtctccct 1200

cgtgggtgct ggcatgaagt ctcacccagg tgttaccgca gagttcatgg aagctctgcg 1260

cgatgtcaac gtgaacatcg aattgatttc cacctctgag attcgtattt ccgtgctgat 1320

ccgtgaagat gatctggatg ctgctgcacg tgcattgcat gagcagttcc agctgggcgg 1380

cgaagacgaa gccgtcgttt atgcaggcac cggacgctaa agttttaaag gagtagtttt 1440

acaatgacca ccatcgcagt tgttggtgca accggccagg tcggccaggt tatgcgcacc 1500

cttttggaag agcgcaattt cccagctgac actgttcgtt tctttgcttc cccacgttcc 1560

gcaggccgta agattgaatt cgtcgacatc gatgctcttc tgcgttaatt aacaattggg 1620

atcctctaga cccgggattt aaatcgctag cgggctgcta aaggaagcgg aacacgtaga 1680

aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga 1740

caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat 1800

agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct 1860

ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct 1920

gatggcgcag gggatcaaga tctgatcaag agacaggatg aggatcgttt cgcatgattg 1980

aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 2040

actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 2100

ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg 2160

aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 2220

ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 2280

tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 2340

tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 2400

gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 2460

aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg 2520

atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 2580

tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 2640

tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 2700

tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 2760

tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc 2820

acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg 2880

ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccacgc 2940

tagcggcgcg ccggccggcc cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 3000

gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 3060

ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 3120

acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 3180

cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 3240

caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 3300

gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 3360

tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 3420

aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 3480

ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 3540

cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 3600

tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 3660

tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 3720

ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 3780

aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 3840

aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaaggccg 3900

gccgcggccg ccatcggcat tttcttttgc gtttttattt gttaactgtt aattgtcctt 3960

gttcaaggat gctgtctttg acaacagatg ttttcttgcc tttgatgttc agcaggaagc 4020

tcggcgcaaa cgttgattgt ttgtctgcgt agaatcctct gtttgtcata tagcttgtaa 4080

tcacgacatt gtttcctttc gcttgaggta cagcgaagtg tgagtaagta aaggttacat 4140

cgttaggatc aagatccatt tttaacacaa ggccagtttt gttcagcggc ttgtatgggc 4200

cagttaaaga attagaaaca taaccaagca tgtaaatatc gttagacgta atgccgtcaa 4260

tcgtcatttt tgatccgcgg gagtcagtga acaggtacca tttgccgttc attttaaaga 4320

cgttcgcgcg ttcaatttca tctgttactg tgttagatgc aatcagcggt ttcatcactt 4380

ttttcagtgt gtaatcatcg tttagctcaa tcataccgag agcgccgttt gctaactcag 4440

ccgtgcgttt tttatcgctt tgcagaagtt tttgactttc ttgacggaag aatgatgtgc 4500

ttttgccata gtatgctttg ttaaataaag attcttcgcc ttggtagcca tcttcagttc 4560

cagtgtttgc ttcaaatact aagtatttgt ggcctttatc ttctacgtag tgaggatctc 4620

tcagcgtatg gttgtcgcct gagctgtagt tgccttcatc gatgaactgc tgtacatttt 4680

gatacgtttt tccgtcaccg tcaaagattg atttataatc ctctacaccg ttgatgttca 4740

aagagctgtc tgatgctgat acgttaactt gtgcagttgt cagtgtttgt ttgccgtaat 4800

gtttaccgga gaaatcagtg tagaataaac ggatttttcc gtcagatgta aatgtggctg 4860

aacctgacca ttcttgtgtt tggtctttta ggatagaatc atttgcatcg aatttgtcgc 4920

tgtctttaaa gacgcggcca gcgtttttcc agctgtcaat agaagtttcg ccgacttttt 4980

gatagaacat gtaaatcgat gtgtcatccg catttttagg atctccggct aatgcaaaga 5040

cgatgtggta gccgtgatag tttgcgacag tgccgtcagc gttttgtaat ggccagctgt 5100

cccaaacgtc caggcctttt gcagaagaga tatttttaat tgtggacgaa tcaaattcag 5160

aaacttgata tttttcattt ttttgctgtt cagggatttg cagcatatca tggcgtgtaa 5220

tatgggaaat gccgtatgtt tccttatatg gcttttggtt cgtttctttc gcaaacgctt 5280

gagttgcgcc tcctgccagc agtgcggtag taaaggttaa tactgttgct tgttttgcaa 5340

actttttgat gttcatcgtt catgtctcct tttttatgta ctgtgttagc ggtctgcttc 5400

ttccagccct cctgtttgaa gatggcaagt tagttacgca caataaaaaa agacctaaaa 5460

tatgtaaggg gtgacgccaa agtatacact ttgcccttta cacattttag gtcttgcctg 5520

ctttatcagt aacaaacccg cgcgatttac ttttcgacct cattctatta gactctcgtt 5580

tggattgcaa ctggtctatt ttcctctttt gtttgataga aaatcataaa aggatttgca 5640

gactacgggc ctaaagaact aaaaaatcta tctgtttctt ttcattctct gtatttttta 5700

tagtttctgt tgcatgggca taaagttgcc tttttaatca caattcagaa aatatcataa 5760

tatctcattt cactaaataa tagtgaacgg caggtatatg tgatgggtta aaaaggatcg 5820

gcggccgctc gatttaaatc tcgagaggcc tgacgtcggg 5860

<210>11

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>11

ctagctagcc attgtccttc tggcagt 27

<210>12

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>12

ctagtctaga cgctcgtgtt cctttaga 28

<210>13

<211>5720

<212>DNA

＜213〉Corynebacterium glutamicum

<400>13

ggtcgactct agaggatccc cgggtaccga gctcgaattc actggccgtc gttttacaac 60

gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt 120

tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca 180

gcctgaatgg cgaatggcga taagctagct tcacgctgcc gcaagcactc agggcgcaag 240

ggctgctaaa ggaagcggaa cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg 300

atgaatgtca gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag 360

gtagcttgca gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc 420

gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac 480

tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagatc tgatcaagag 540

acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc 600

gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat 660

gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg 720

tccggtgccc tgaatgaact ccaagacgag gcagcgcggc tatcgtggct ggccacgacg 780

ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta 840

ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta 900

tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc 960

gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc 1020

gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg 1080

ctcaaggcgc ggatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg 1140

ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt 1200

gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc 1260

ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc 1320

atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgctagag 1380

gatcgatcct ttttaaccca tcacatatac ctgccgttca ctattattta gtgaaatgag 1440

atattatgat attttctgaa ttgtgattaa aaaggcaact ttatgcccat gcaacagaaa 1500

ctataaaaaa tacagagaat gaaaagaaac agatagattt tttagttctt taggcccgta 1560

gtctgcaaat ccttttatga ttttctatca aacaaaagag gaaaatagac cagttgcaat 1620

ccaaacgaga gtctaataga atgaggtcga aaagtaaatc gcgcgggttt gttactgata 1680

aagcaggcaa gacctaaaat gtgtaaaggg caaagtgtat actttggcgt caccccttac 1740

atattttagg tcttttttta ttgtgcgtaa ctaacttgcc atcttcaaac aggagggctg 1800

gaagaagcag accgctaaca cagtacataa aaaaggagac atgaacgatg aacatcaaaa 1860

agtttgcaaa acaagcaaca gtattaacct ttactaccgc actgctggca ggaggcgcaa 1920

ctcaagcgtt tgcgaaagaa acgaaccaaa agccatataa ggaaacatac ggcatttccc 1980

atattacacg ccatgatatg ctgcaaatcc ctgaacagca aaaaaatgaa aaatatcaag 2040

tttctgaatt tgattcgtcc acaattaaaa atatctcttc tgcaaaaggc ctggacgttt 2100

gggacagctg gccattacaa aacgctgacg gcactgtcgc aaactatcac ggctaccaca 2160

tcgtctttgc attagccgga gatcctaaaa atgcggatga cacatcgatt tacatgttct 2220

atcaaaaagt cggcgaaact tctattgaca gctggaaaaa cgctggccgc gtctttaaag 2280

acagcgacaa attcgatgca aatgattcta tcctaaaaga ccaaacacaa gaatggtcag 2340

gttcagccac atttacatct gacggaaaaa tccgtttatt ctacactgat ttctccggta 2400

aacattacgg caaacaaaca ctgacaactg cacaagttaa cgtatcagca tcagacagct 2460

ctttgaacat caacggtgta gaggattata aatcaatctt tgacggtgac ggaaaaacgt 2520

atcaaaatgt acagcagttc atcgatgaag gcaactacag ctcaggcgac aaccatacgc 2580

tgagagatcc tcactacgta gaagataaag gccacaaata cttagtattt gaagcaaaca 2640

ctggaactga agatggctac caaggcgaag aatctttatt taacaaagca tactatggca 2700

aaagcacatc attcttccgt caagaaagtc aaaaacttct gcaaagcgat aaaaaacgca 2760

cggctgagtt agcaaacggc gctctcggta tgattgagct aaacgatgat tacacactga 2820

aaaaagtgat gaaaccgctg attgcatcta acacagtaac agatgaaatt gaacgcgcga 2880

acgtctttaa aatgaacggc aaatggtacc tgttcactga ctcccgcgga tcaaaaatga 2940

cgattgacgg cattacgtct aacgatattt acatgcttgg ttatgtttct aattctttaa 3000

ctggcccata caagccgctg aacaaaactg gccttgtgtt aaaaatggat cttgatccta 3060

acgatgtaac ctttacttac tcacacttcg ctgtacctca agcgaaagga aacaatgtcg 3120

tgattacaag ctatatgaca aacagaggat tctacgcaga caaacaatca acgtttgcgc 3180

cgagcttcct gctgaacatc aaaggcaaga aaacatctgt tgtcaaagac agcatccttg 3240

aacaaggaca attaacagtt aacaaataaa aacgcaaaag aaaatgccga tgggtaccga 3300

gcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg 3360

ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccctcgcg gacgtgctca 3420

tagtccacga cgcccgtgat tttgtagccc tggccgacgg ccagcaggta ggccgacagg 3480

ctcatgccgg ccgccgccgc cttttcctca atcgctcttc gttcgtctgg aaggcagtac 3540

accttgatag gtgggctgcc cttcctggtt ggcttggttt catcagccat ccgcttgccc 3600

tcatctgtta cgccggcggt agccggccag cctcgcagag caggattccc gttgagcacc 3660

gccaggtgcg aataagggac agtgaagaag gaacacccgc tcgcgggtgg gcctacttca 3720

cctatcctgc ccggctgacg ccgttggata caccaaggaa agtctacacg aaccctttgg 3780

caaaatcctg tatatcgtgc gaaaaaggat ggatataccg aaaaaatcgc tataatgacc 3840

ccgaagcagg gttatgcagc ggaaaagcgc tgcttccctg ctgttttgtg gaatatctac 3900

cgactggaaa caggcaaatg caggaaatta ctgaactgag gggacaggcg agagacgatg 3960

ccaaagagct cctgaaaatc tcgataactc aaaaaatacg cccggtagtg atcttatttc 4020

attatggtga aagttggaac ctcttacgtg ccgatcaacg tctcattttc gccaaaagtt 4080

ggcccagggc ttcccggtat caacagggac accaggattt atttattctg cgaagtgatc 4140

ttccgtcaca ggtatttatt cggcgcaaag tgcgtcgggt gatgctgcca acttactgat 4200

ttagtgtatg atggtgtttt tgaggtgctc cagtggcttc tgtttctatc agctcctgaa 4260

aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg 4320

gaacctctta cgtgccgatc aacgtctcat tttcgccaaa agttggccca gggcttcccg 4380

gtatcaacag ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt 4440

tattcggcgc aaagtgcgtc gggtgatgct gccaacttac tgatttagtg tatgatggtg 4500

tttttgaggt gctccagtgg cttctgtttc tatcagggct ggatgatcct ccagcgcggg 4560

gatctcatgc tggagttctt cgcccacccc aaaaggatct aggtgaagat cctttttgat 4620

aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 4680

gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 4740

acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 4800

tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag 4860

ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 4920

atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 4980

agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 5040

cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 5100

agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 5160

acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 5220

gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 5280

ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 5340

gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 5400

gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 5460

gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa 5520

tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat 5580

gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg 5640

ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac 5700

gccaagcttg catgcctgca 5720

<210>14

<211>6693

<212>DNA

＜213〉Corynebacterium glutamicum

<400>14

accatttccg ttcatttaaa gacgttcgcg cgtcaatttc atctgtactg tgtagatgca 60

tcagcggttt catcactttt ttcagtgtga atcatcgttt agctcaatca taccgagagc 120

gccgtttgct aactcaaccg tgcgtttttt atcgctttgc agaagttttt gactttcttg 180

acggaagaat gatgtgcttt tgccatagta tgctttgtta aataaagatt cttcgccttg 240

gtagccatct tcagttccag tgtttgcttc aaatactaag tatttgtggc ctttatcttc 300

tacgtagtga ggatctctca gcgtatggtt gtcgcctgag ctgtagttgc cttcatcgat 360

gaactgctgt acattttgat acgtttttcc gtcaccgtca aagattgatt tataatcctc 420

tacaccgttg atgttcaaag agctgtctga tgctgatacg ttaacttgtg cagttgtcag 480

tgtttgtttg ccgtaatgtt taccggagaa atcagtgtag aataaacgga tttttccgtc 540

agatgtaaat gtggctgaac ctgaccattc ttgtgtttgg tcttttagga tagaatcatt 600

tgcatcgaat ttgtcgctgt ctttaaagac gcggccagcg tttttccagc tgtcaataga 660

agtttcgccg actttttgat agaacatgta aatcgatgtg tcatccgcat ttttaggatc 720

tccggctaat gcaaagacga tgtggtagcc gtgatagttt gcgacagtgc cgtcagcgtt 780

ttgtaatggc cagctgtccc aaacgtccag gccttttgca gaagagatat ttttaattgt 840

ggacgaatca aattcagaaa cttgatattt ttcatttttt tgctgttcag ggatttgcag 900

catatcatgg cgtgtaatat gggaaatgcc gtatgtttcc ttatatggct tttggttcgt 960

ttctttcgca aacgcttgag ttgcgcctcc tgccagcagt gcggtagtaa aggttaatac 1020

tgttgcttgt tttgcaaact ttttgatgtt catcgttcat gtctcctttt ttatgtactg 1080

tgttagcggt ctgcttcttc cagccctcct gtttgaagat ggcaagttag ttacgcacaa 1140

taaaaaaaga cctaaaatat gtaaggggtg acgccaaagt atacactttg ccctttacac 1200

attttaggtc ttgcctgctt tatcagtaac aaacccgcgc gatttacttt tcgacctcat 1260

tctattagac tctcgtttgg attgcaactg gtctattttc ctcttttgtt tgatagaaaa 1320

tcataaaagg atttgcagac tacgggccta aagaactaaa aaatctatct gtttcttttc 1380

attctctgta ttttttatag tttctgttgc atgggcataa agttgccttt ttaatcacaa 1440

ttcagaaaat atcataatat ctcatttcac taaataatag tgaacggcag gtatatgtga 1500

tgggttaaaa aggatcgatc ctctagcgaa ccccagagtc ccgctcagaa gaactcgtca 1560

agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg 1620

aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg 1680

tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca 1740

ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg 1800

tcgggcatcc gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct 1860

tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg 1920

cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc 1980

attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc 2040

tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc 2100

acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcttgg 2160

agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct 2220

gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg 2280

aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg 2340

cgaaacgatc ctcatcctgt ctcttgatca gatcttgatc ccctgcgcca tcagatcctt 2400

ggcggcaaga aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc 2460

ccagctggca attccggttc gcttgctgtc cataaaaccg cccagtctag ctatcgccat 2520

gtaagcccac tgcaagctac ctgctttctc tttgcgcttg cgttttccct tgtccagata 2580

gcccagtagc tgacattcat ccggggtcag caccgtttct gcggactggc tttctacgtg 2640

ttccgcttcc tttagcagcc cttgcgccct gagtgcttgc ggcagcgtga agctagccat 2700

tgtccttctg gcagttgctt gcgccgccct cgttgccacc atctggatgc cactgttcgg 2760

atccttctcc gaccgcgtca accgtgcagt gctctacagg atctgtgcat ccgcaaccat 2820

cgtgctgatt gtcccttact acttggtcct caacaccggc gaaatttggg cactgtttat 2880

cactaccgtg attggcttcg gcatcctctg gggtagcgtc aacgcaatcc tcggaaccgt 2940

catcgcagaa aacttcgcac ctgaggtccg ctacaccggc gctaccctgg gttaccaagt 3000

cggagcagca ctcttcggcg gtaccgcacc cattatcgca gcatggctgt tcgaaatctc 3060

cggcggacaa tggtggccaa tcgccgtcta cgtcgctgca tgttgccttc tctctgtgat 3120

cgcctcgttc ttcatccaac gcgtcgcgca ccaagagaac taaaatctaa gtaaaacccc 3180

tccgaaagga accacccatg gtgaaacgtc aactgcccaa ccccgcagaa ctactcgaac 3240

tcatgaagtt caaaaagcca gagctcaacg gcaagaaacg acgcctagac tccgcgctca 3300

ccatctacga cctgcgtaaa attgctaaac gacgcacccc agctgccgcg ttcgactaca 3360

ccgacggcgc agccgaggcc gaactctcaa tcacacgcgc acgtgaagca ttcgaaaaca 3420

tcgaagcgaa ggcgtcgacc gcaccatcgc cattctccgc agcgagatca cccgcaccat 3480

ggctctcctc ggtgtttcct ccctcgaaga actcgagcca cgccacgtca cccagctggc 3540

caagatggtt ccagtttctg acgcaactcg ttctgcagcg gcggagattt aaaagtttct 3600

ctccttagct attaaaaggt gcccatccgt ttggatgggc accttctcgt ttcttgcaat 3660

cggcatattc agtcaaaaaa tgttgaaatc agcactttca atttgggaca tctactctta 3720

ggagaaaagc cacaaacctt tcccacccca caaccgtgtg ttctgcagtc gacccagttt 3780

agaggaaaca tgagtgactt cacggaaaat acttggactg tccactacga cgaagatggt 3840

gatttcccaa aattcttcaa ctctctaaag gaacacgagc gtctagagtc gacctgcagg 3900

catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 3960

acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 4020

gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 4080

tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 4140

cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 4200

gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 4260

aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 4320

gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 4380

aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 4440

gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 4500

ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 4560

cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 4620

ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 4680

actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 4740

tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 4800

gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 4860

ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 4920

cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 4980

ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttggggtg ggcgaagaac 5040

tccagcatga gatccccgcg ctggaggatc atccagccct gatagaaaca gaagccactg 5100

gagcacctca aaaacaccat catacactaa atcagtaagt tggcagcatc acccgacgca 5160

ctttgcgccg aataaatacc tgtgacggaa gatcacttcg cagaataaat aaatcctggt 5220

gtccctgttg ataccgggaa gccctgggcc aacttttggc gaaaatgaga cgttgatcgg 5280

cacgtaagag gttccaactt tcaccataat gaaataagat cactaccggg cgtatttttt 5340

gagttatcga gattttcagg agctgataga aacagaagcc actggagcac ctcaaaaaca 5400

ccatcataca ctaaatcagt aagttggcag catcacccga cgcactttgc gccgaataaa 5460

tacctgtgac ggaagatcac ttcgcagaat aaataaatcc tggtgtccct gttgataccg 5520

ggaagccctg ggccaacttt tggcgaaaat gagacgttga tcggcacgta agaggttcca 5580

actttcacca taatgaaata agatcactac cgggcgtatt ttttgagtta tcgagatttt 5640

caggagctct ttggcatcgt ctctcgcctg tcccctcagt tcagtaattt cctgcatttg 5700

cctgtttcca gtcggtagat attccacaaa acagcaggga agcagcgctt ttccgctgca 5760

taaccctgct tcggggtcat tatagcgatt ttttcggtat atccatcctt tttcgcacga 5820

tatacaggat tttgccaaag ggttcgtgta gactttcctt ggtgtatcca acggcgtcag 5880

ccgggcagga taggtgaagt aggcccaccc gcgagcgggt gttccttctt cactgtccct 5940

tattcgcacc tggcggtgct caacgggaat cctgctctgc gaggctggcc ggctaccgcc 6000

ggcgtaacag atgagggcaa gcggatggct gatgaaacca agccaaccag gaagggcagc 6060

ccacctatca aggtgtactg ccttccagac gaacgaagag cgattgagga aaaggcggcg 6120

gcggccggca tgagcctgtc ggcctacctg ctggccgtcg gccagggcta caaaatcacg 6180

ggcgtcgtgg actatgagca cgtccgcgag ggcgtcccgg aaaacgattc cgaagcccaa 6240

cctttcatag aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg gcgtcgcttg 6300

gtcggtcatt tcgctcggta cccatcggca ttttcttttg cgtttttatt tgttaactgt 6360

taattgtcct tgttcaagga tgctgtcttt gacaacagat gttttcttgc ctttgatgtt 6420

cagcargaag ctcggcgcaa acgttgattg tttgtctgcg tagaatcctc tgtttgtcat 6480

atagcttgta atcacgacat tgtttcctty tcgcttgagg tacagcgaag tgtgagtaag 6540

taaraggtta catcgttagg atcaagatcc attcttaaca caaggccagt tttgttcagc 6600

ggcttgtatg ggccagttaa agaattataa acataaccaa gcatgtaaat atcgttagac 6660

gtaatgccgt caatcgtcat tattgatccg cgg 6693

<210>15

<211>7561

<212>DNA

＜213〉Corynebacterium glutamicum

<400>15

accatttccg ttcatttaaa gacgttcgcg cgtcaatttc atctgtactg tgtagatgca 60

tcagcggttt catcactttt ttcagtgtga atcatcgttt agctcaatca taccgagagc 120

gccgtttgct aactcaaccg tgcgtttttt atcgctttgc agaagttttt gactttcttg 180

acggaagaat gatgtgcttt tgccatagta tgctttgtta aataaagatt cttcgccttg 240

gtagccatct tcagttccag tgtttgcttc aaatactaag tatttgtggc ctttatcttc 300

tacgtagtga ggatctctca gcgtatggtt gtcgcctgag ctgtagttgc cttcatcgat 360

gaactgctgt acattttgat acgtttttcc gtcaccgtca aagattgatt tataatcctc 420

tacaccgttg atgttcaaag agctgtctga tgctgatacg ttaacttgtg cagttgtcag 480

tgtttgtttg ccgtaatgtt taccggagaa atcagtgtag aataaacgga tttttccgtc 540

agatgtaaat gtggctgaac ctgaccattc ttgtgtttgg tcttttagga tagaatcatt 600

tgcatcgaat ttgtcgctgt ctttaaagac gcggccagcg tttttccagc tgtcaataga 660

agtttcgccg actttttgat agaacatgta aatcgatgtg tcatccgcat ttttaggatc 720

tccggctaat gcaaagacga tgtggtagcc gtgatagttt gcgacagtgc cgtcagcgtt 780

ttgtaatggc cagctgtccc aaacgtccag gccttttgca gaagagatat ttttaattgt 840

ggacgaatca aattcagaaa cttgatattt ttcatttttt tgctgttcag ggatttgcag 900

catatcatgg cgtgtaatat gggaaatgcc gtatgtttcc ttatatggct tttggttcgt 960

ttctttcgca aacgcttgag ttgcgcctcc tgccagcagt gcggtagtaa aggttaatac 1020

tgttgcttgt tttgcaaact ttttgatgtt catcgttcat gtctcctttt ttatgtactg 1080

tgttagcggt ctgcttcttc cagccctcct gtttgaagat ggcaagttag ttacgcacaa 1140

taaaaaaaga cctaaaatat gtaaggggtg acgccaaagt atacactttg ccctttacac 1200

attttaggtc ttgcctgctt tatcagtaac aaacccgcgc gatttacttt tcgacctcat 1260

tctattagac tctcgtttgg attgcaactg gtctattttc ctcttttgtt tgatagaaaa 1320

tcataaaagg atttgcagac tacgggccta aagaactaaa aaatctatct gtttcttttc 1380

attctctgta ttttttatag tttctgttgc atgggcataa agttgccttt ttaatcacaa 1440

ttcagaaaat atcataatat ctcatttcac taaataatag tgaacggcag gtatatgtga 1500

tgggttaaaa aggatcgatc ctctagcgaa ccccagagtc ccgctcagaa gaactcgtca 1560

agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg 1620

aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg 1680

tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca 1740

ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg 1800

tcgggcatcc gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct 1860

tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg 1920

cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc 1980

attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc 2040

tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc 2100

acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcttgg 2160

agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct 2220

gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg 2280

aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg 2340

cgaaacgatc ctcatcctgt ctcttgatca gatcttgatc ccctgcgcca tcagatcctt 2400

ggcggcaaga aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc 2460

ccagctggca attccggttc gcttgctgtc cataaaaccg cccagtctag ctatcgccat 2520

gtaagcccac tgcaagctac ctgctttctc tttgcgcttg cgttttccct tgtccagata 2580

gcccagtagc tgacattcat ccggggtcag caccgtttct gcggactggc tttctacgtg 2640

ttccgcttcc tttagcagcc cttgcgccct gagtgcttgc ggcagcgtga agctagccat 2700

tgtccttctg gcagttgctt gcgccgccct cgttgccacc atctggatgc cactgttcgg 2760

atccttctcc gaccgcgtca accgtgcagt gctctacagg atctgtgcat ccgcaaccat 2820

cgtgctgatt gtcccttact acttggtcct caacaccggc gaaatttggg cactgtttat 2880

cactaccgtg attggcttcg gcatcctctg gggtagcgtc aacgcaatcc tcggaaccgt 2940

catcgcagaa aacttcgcac ctgaggtccg ctacaccggc gctaccctgg gttaccaagt 3000

cggagcagca ctcttcggcg gtaccgcacc cattatcgca gcatggctgt tcgaaatctc 3060

cggcggacaa tggtggccaa tcgccgtcta cgtcgctgca tgttgccttc tctctgtgat 3120

cgcctcgttc ttcatccaac gcgtcgcgca ccaagagaac taaaatctaa gtaaaacccc 3180

tccgaaagga accacccatg gtgaaacgtc aactgcccaa ccccgcagaa ctactcgaac 3240

tcatgaagtt caaaaagcca gagctcaacg gcaagaaacg acgcctagac tccgcgctca 3300

ccatctacga cctgcgtaaa attgctaaac gacgcacccc agctgccgcg ttcgactaca 3360

ccgacggcgc agccgaggcc gaactctcaa tcacacgcgc acgtgaagca ttcgaaaaca 3420

tcgaattcca cccagacatc ctcaagcctg cagaacacgt agacaccacc acccaaatcc 3480

tgggcggaac ctcctccatg ccattcggca tcgcaccaac cggcttcacc cgcctcatgc 3540

agaccgaagg tgaaatcgca ggtgccggag ctgcaggcgc tgcaggaatt cctttcaccc 3600

tgtccaccct gggcactacc tccatcgaag acgtcaaggc caccaacccc aacggccgaa 3660

actggttcca gctctacgtc atgcgcgacc gcgaaatctc ctacggcctc gtcgaacgcg 3720

cagccaaagc aggattcgac accctgatgt tcaccgtgga tacccccatc gccggctacc 3780

gcatccgcga ttcccgcaac ggattctcca tcccgccaca gctgacccca tccaccgtgc 3840

tcaatgcaat cccacgccca tggtggtgga tcgacttcct gaccacccca acccttgagt 3900

tcgcatccct ttcctcgacc ggcggaaccg tgggcgacct cctcaactcc gcgatggatc 3960

ccaccatttc ttacgaagac ctcaaggtca tccgtgaaat gtggccaggc aagctcgtag 4020

tcaagggtgt ccagaacgtt gaagactccg tcaaactcct cgaccaaggc gtcgacggcc 4080

tcatcctctc caaccacggt ggccgtcaac tcgaccgcgc accagtccca ttccacctcc 4140

tgccacaggt acgcaaggaa gtcggatctg aaccaaccat catgatcgac accggcatca 4200

tgaacggcgc cgacatcgtc gcagccgtag ccatgggcgc tgacttcacc ctcatcggtc 4260

gtgcctacct ctacggactc atggccggag gccgcgaagg cgtcgaccgc accatcgcca 4320

ttctccgcag cgagatcacc cgcaccatgg ctctcctcgg tgtttcctcc ctcgaagaac 4380

tcgagccacg ccacgtcacc cagctggcca agatggttcc agtttctgac gcaactcgtt 4440

ctgcagcggc ggagatttaa aagtttctct ccttagctat taaaaggtgc ccatccgttt 4500

ggatgggcac cttctcgttt cttgcaatcg gcatattcag tcaaaaaatg ttgaaatcag 4560

cactttcaat ttgggacatc tactcttagg agaaaagcca caaacctttc ccaccccaca 4620

accgtgtgtt ctgcagtcga cccagtttag aggaaacatg agtgacttca cggaaaatac 4680

ttggactgtc cactacgacg aagatggtga tttcccaaaa ttcttcaact ctctaaagga 4740

acacgagcgt ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct 4800

gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat 4860

aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc 4920

actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg 4980

cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct 5040

gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 5100

atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 5160

caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 5220

gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 5280

ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 5340

cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 5400

taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 5460

cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 5520

acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 5580

aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt 5640

atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 5700

atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 5760

gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 5820

gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 5880

ctagatcctt ttggggtggg cgaagaactc cagcatgaga tccccgcgct ggaggatcat 5940

ccagccctga tagaaacaga agccactgga gcacctcaaa aacaccatca tacactaaat 6000

cagtaagttg gcagcatcac ccgacgcact ttgcgccgaa taaatacctg tgacggaaga 6060

tcacttcgca gaataaataa atcctggtgt ccctgttgat accgggaagc cctgggccaa 6120

cttttggcga aaatgagacg ttgatcggca cgtaagaggt tccaactttc accataatga 6180

aataagatca ctaccgggcg tattttttga gttatcgaga ttttcaggag ctgatagaaa 6240

cagaagccac tggagcacct caaaaacacc atcatacact aaatcagtaa gttggcagca 6300

tcacccgacg cactttgcgc cgaataaata cctgtgacgg aagatcactt cgcagaataa 6360

ataaatcctg gtgtccctgt tgataccggg aagccctggg ccaacttttg gcgaaaatga 6420

gacgttgatc ggcacgtaag aggttccaac tttcaccata atgaaataag atcactaccg 6480

ggcgtatttt ttgagttatc gagattttca ggagctcttt ggcatcgtct ctcgcctgtc 6540

ccctcagttc agtaatttcc tgcatttgcc tgtttccagt cggtagatat tccacaaaac 6600

agcagggaag cagcgctttt ccgctgcata accctgcttc ggggtcatta tagcgatttt 6660

ttcggtatat ccatcctttt tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga 6720

ctttccttgg tgtatccaac ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc 6780

gagcgggtgt tccttcttca ctgtccctta ttcgcacctg gcggtgctca acgggaatcc 6840

tgctctgcga ggctggccgg ctaccgccgg cgtaacagat gagggcaagc ggatggctga 6900

tgaaaccaag ccaaccagga agggcagccc acctatcaag gtgtactgcc ttccagacga 6960

acgaagagcg attgaggaaa aggcggcggc ggccggcatg agcctgtcgg cctacctgct 7020

ggccgtcggc cagggctaca aaatcacggg cgtcgtggac tatgagcacg tccgcgaggg 7080

cgtcccggaa aacgattccg aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat 7140

ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc gctcggtacc catcggcatt 7200

ttcttttgcg tttttatttg ttaactgtta attgtccttg ttcaaggatg ctgtctttga 7260

caacagatgt tttcttgcct ttgatgttca gcargaagct cggcgcaaac gttgattgtt 7320

tgtctgcgta gaatcctctg tttgtcatat agcttgtaat cacgacattg tttccttytc 7380

gcttgaggta cagcgaagtg tgagtaagta araggttaca tcgttaggat caagatccat 7440

tcttaacaca aggccagttt tgttcagcgg cttgtatggg ccagttaaag aattataaac 7500

ataaccaagc atgtaaatat cgttagacgt aatgccgtca atcgtcatta ttgatccgcg 7560

g 7561

Claims

1. one kind is used for increasing the flow through method of metabolic flux of pentose-phosphate pathway of microorganism, is included in to make the culturing micro-organisms under the condition that the metabolic flux of pentose-phosphate pathway increases of flowing through, wherein said microorganism comprise to be gone the gene of regulating.

2. the described method of claim 1, wherein fructose or sucrose are as carbon source.

3. the described method of claim 1, wherein fructose is as carbon source.

4. the described method of claim 1, wherein gene is a serum lactic dehydrogenase.

5. the described method of claim 4, wherein lactate dehydrogenase gene is from Corynebacterium (Corynebacterium).

6. the described method of claim 4, wherein lactate dehydrogenase gene be express insufficient.

7. the described method of claim 1, wherein genes encoding serum lactic dehydrogenase.

8. the described method of claim 7, wherein serum lactic dehydrogenase has the activity of reduction.

9. the described method of claim 1, wherein microorganism is a gram-positive microorganism.

10. the described method of claim 1, wherein microorganism belongs to Corynebacterium.

11. the described method of claim 10, wherein microorganism is Corynebacterium glutamicum (Corynebacterium glutamicum).

12. the described method of claim 1, wherein with microbial fermentation to produce fine chemicals.

13. the described method of claim 1, wherein microorganism also comprises one or more extra regulatory gene of going.

14. the described method of claim 13, the regulatory gene of going that wherein one or more are extra is selected from ask gene, dapA gene, asd gene, dapB gene, ddh gene, lysA gene, lysE gene, pycA gene, zwf gene, pepCL gene, gap gene, zwa1 gene, tkt gene, tad gene, mqo gene, tpi gene, pgk gene and sigC gene.

15. the described method of claim 14, the regulatory gene of going that wherein one or more are extra was expression.

16. the described method of claim 13, the regulatory gene coding that goes that wherein one or more are extra is selected from following protein: feedback opposing E.C. 2.7.2.4., the dihydrodipicolinate synthase, aspartate-semialdehyde dehydrogenase, the dihydrodipicolinate reductase, diaminopimelate dehydrogenase, the diaminopimelic acid epimerase, Methionin output, pyruvate carboxylase, glucose-6-phosphate dehydrogenase (G6PD), Phosphoenolpyruvate carboxylase, glyceraldehyde-3-phosphate dehydrogenase, the RPF protein precursor, transketolase, transaldolase, the menaquinone oxydo-reductase, triose-phosphate isomerase, glycerol 3-phosphate acid kinase and RNA polymerase Sigma Factors sigC.

17. the described method of claim 16, wherein protein has the activity of increase.

18. the described method of claim 13, the regulatory gene of going that wherein one or more are extra is selected from: pepCK gene, malE gene, glgA gene, pgi gene, dead gene, menE gene, citE gene, mikE17 gene, poxB gene, zwa2 gene and sucC gene.

19. the described method of claim 18, the regulatory gene of going that wherein one or more are extra is that weaken, that reduce or downtrod.

20. the described method of claim 13, the regulatory gene coding that goes that wherein one or more are extra is selected from following protein: rna helicase enzyme, o-succinyl-phenylformic acid-CoA ligase enzyme, citrate lyase β chain, transcriptional, pyruvic oxidase, RPF protein precursor and succinyl CoA synthetase that phosphoenolpyruvate carboxykinase, malic enzyme, Glycogensynthase, G-6-P isomerase, ATP rely on.

21. the described method of claim 20, wherein protein has the activity of reduction.

22. a method that is used to produce fine chemicals comprises:

A) cultivate serum lactic dehydrogenase wherein and gone the microorganism regulated; With

B) accumulate fine chemicals in substratum or in the microorganism cells, produce fine chemicals thus.

23. a method that is used to produce fine chemicals is included in and makes culturing micro-organisms under the condition that fine chemicals produces, wherein at least a pentose phosphate biosynthetic pathway gene or enzyme are gone to regulate in described microorganism.

24. the described method of claim 23, wherein said biosynthesis gene is a serum lactic dehydrogenase.

25. the described method of claim 23, wherein said biosynthetic enzyme is a serum lactic dehydrogenase.

26. claim 22 or 24 described methods, wherein the lactic dehydrogenase expression of enzymes reduces.

27. claim 22 or 25 described methods, wherein lactate dehydrogenase activity reduces.

28. the described method of claim 22, it also comprises the recovery fine chemicals.

29. claim 22 or 23 described methods, wherein one or more extra genes go to regulate.

30. the described method of claim 29, the regulatory gene of going that wherein one or more are extra is selected from: ask gene, dapA gene, asd gene, dapB gene, ddh gene, lysA gene, lysE gene, pycA gene, zwf gene, pepCL gene, gap gene, zwa1 gene, tkt gene, tad gene, mqo gene, tpi gene, pgk gene and sigC gene.

31. the described method of claim 30, the regulatory gene of going that wherein one or more are extra was expression.

32. the described method of claim 29, the regulatory gene coding that goes that wherein one or more are extra is selected from following protein: feedback opposing E.C. 2.7.2.4., the dihydrodipicolinate synthase, aspartate-semialdehyde dehydrogenase, the dihydrodipicolinate reductase, diaminopimelate dehydrogenase, the diaminopimelic acid epimerase, Methionin output, pyruvate carboxylase, glucose-6-phosphate dehydrogenase (G6PD), Phosphoenolpyruvate carboxylase, glyceraldehyde-3-phosphate dehydrogenase, the RPF protein precursor, transketolase, transaldolase, the menaquinone oxydo-reductase, triose-phosphate isomerase, glycerol 3-phosphate acid kinase and RNA polymerase Sigma Factors sigC.

33. the described method of claim 32, wherein protein has the activity of increase.

34. the described method of claim 29, the regulatory gene of going that wherein one or more are extra is selected from: pepCK gene, malE gene, glgA gene, pgi gene, dead gene, menE gene, citE gene, mikE17 gene, poxB gene, zwa2 gene and sucC gene.

35. the described method of claim 34, the regulatory gene of going that wherein one or more are extra is that weaken, that reduce or downtrod.

36. the described method of claim 29, the regulatory gene coding that goes that wherein one or more are extra is selected from following protein: rna helicase enzyme, o-succinyl-phenylformic acid-CoA ligase enzyme, citrate lyase β chain, transcriptional, pyruvic oxidase, RPF protein precursor and succinyl CoA synthetase that phosphoenolpyruvate carboxykinase, malic enzyme, Glycogensynthase, G-6-P isomerase, ATP rely on.

37. the described method of claim 36, wherein protein has the activity of reduction.

38. claim 22 or 23 described methods, wherein microorganism is a gram-positive microorganism.

39. claim 22 or 23 described methods, wherein microorganism belongs to Corynebacterium.

40. the described method of claim 39, wherein microorganism is a Corynebacterium glutamicum.

41. claim 22 or 23 described methods, wherein fine chemicals is a Methionin.

42. the described method of claim 41, wherein Methionin is to produce with the turnout of 100g/L at least.

43. the described method of claim 41, wherein Methionin is to produce with the turnout of 150g/L at least.

44. claim 22 or 23 described methods, wherein fructose or sucrose are as carbon source.

45. claim 22 or 23 described methods, wherein fructose is as carbon source.

46. claim 22 or 24 described methods, wherein serum lactic dehydrogenase comprises the nucleotide sequence of SEQ IDNO:1.

47. claim 22 or 24 described methods, wherein the serum lactic dehydrogenase coding comprises the polypeptide of the aminoacid sequence of SEQ IDNO:2.

48. a recombinant microorganism, it has the pentose phosphate biosynthetic pathway of regulating.

49. a recombinant microorganism, it comprises the pentose phosphate biosynthesis gene of regulating.

50. the described recombinant microorganism of claim 49, the wherein said regulatory gene of going is a serum lactic dehydrogenase.

51. the described recombinant microorganism of claim 50, wherein the lactic dehydrogenase expression of enzymes reduces.

52. the described recombinant microorganism of claim 50, wherein said lactate dehydrogenase gene coding has the active lactic dehydrogenase zymoprotein of reduction.

53. the described recombinant microorganism of claim 49, wherein microorganism belongs to Corynebacterium.

54. the described recombinant microorganism of claim 53, wherein microorganism is a Corynebacterium glutamicum.