CN1694899B

CN1694899B - Methods for the production of methioning

Info

Publication number: CN1694899B
Application number: CN02829972.8A
Authority: CN
Inventors: M·蓬佩尤斯; B·克勒格尔; H·施罗德; O·策尔德尔; G·哈伯豪尔; S·哈夫纳; C·克洛普罗格
Original assignee: Evonik Degussa GmbH
Priority date: 2002-11-29
Filing date: 2002-11-29
Publication date: 2011-07-06
Anticipated expiration: 2022-11-29
Also published as: CN1694899A; BR0215958A; BRPI0215958B1; AU2002368410A1; EP1567549A1; WO2004050694A1; JP2006521083A

Abstract

Nucleic acid molecules, designated MR nucleic acid molecules, which encode novel MR proteins from Corynebacterium glutamicum are described which are involved in biosynthesis of a fine chemical, e.g., methionine biosynthesis. The invention also pro-vides antisense nucleic acid molecules, transgenic expression cassettes and vectors containing MR nucleic acid molecules, and host cells into which the expression cassettes or vectors have been introduced. The invention still further provides methods of producing methionine from microorganisms, e.g., C. glutamicum, which involve culturing recombinant microorganisms which overex press or underexpress at least one MR molecule of the invention under conditions such that methionine is produced. Also featured are methods of producing a fine chemical, e.g., methionine, which involve culturing recombinant microorganisms having se-lected MR genes deleted or mutated under conditions such that the fine chemical, e.g., methionine, is produced.

Description

Produce the method for methionine(Met)

Technical field

At present, produce methionine(Met) by improving the chemical process of setting up with DL-methionine(Met) racemic mixture form.Most of DL-methionine(Met)s are produced by the variation scheme of identical chemical step method, wherein relate to toxicity, danger, parent material inflammable, unstable and highly odorous or intermediate.The parent material that is used for chemosynthesis is: propenal, thiomethyl alcohol and prussic acid.Chemosynthesis comprises thiomethyl alcohol and the acrolein reaction that produces intermediate 3-methyl mercapto propionic aldehyde (MMR).In further step, MMR and prussic acid reaction utilize 2 normal caustic alkali (caustics) such as NaOH and two/monovalent Na then to form 5-(2-methylmercaptoethyl) glycolylurea ₂CO ₃Together hydrolysis it, produce sodium-DL-methionine(Met) salt and 1 equivalent Na ₂CO ₃, 1 equivalent NH ₃With two/monovalent CO ₂In step subsequently, with 1.5 equivalent sulfuric acid and 1 equivalent Na ₂CO ₃In and sodium-DL-methionine(Met) salt to produce DL-methionine(Met) Na ₂SO ₄And CO ₂Obviously, compare with the methionine(Met) amount that produces, this chemical process has produced the useless salt of a large amount of molar excess.This fact has caused economy and ecological problem.

Fermentation process is usually based on culturing micro-organisms on nutrition; described nutrition comprises carbohydrate source (for example, sugar is such as dextrose fructose or sucrose), and nitrogenous source (for example; ammonia) and sulphur source (for example, vitriol or thiosulphate) and other dulbecco minimum essential medium Dulbecco composition.This method only produces natural product L-methionine(Met) and biomass is only arranged as by product.Because fermentation process does not use toxicity, danger, parent material inflammable, unstable and highly odorous, and does not produce salt,, this method synthesizes so being better than chemical methionine(Met).Can in organism such as intestinal bacteria (E.coli) or corynebacterium (Corynebacter), produce methionine(Met) (Kase H., Nakayama K. (1975) Agric.Biol.Chem.39,153-160 page or leaf; Chatterjee etc. (1999) Acta Biotechnol.14, pp199-204 page or leaf; Harma S.Gomes, (2001) J.Eng.Life Sci.1, the pp69-73 page or leaf, JP50031092, DE 2105189).

All viable cell all have many complicated katabolism and anabolism abilities that interconnect approach that have.In order to keep the balance between this extremely complicated metabolism network various piece, cell has utilized the regulation system of fine tuning.By the synthetic and enzymic activity of regulatory enzyme independently or side by side, cell can be controlled the activity of pathways metabolism of fundamental difference with the variation needs of reflection cell.In transcriptional level or translation skill, perhaps at transcriptional level and translation skill inducing or checking of enzymic synthesis can take place.Several mechanism of transcriptional level are regulated the (summary of genetic expression in the prokaryotic organism, referring to for example Lewin, B (1990) Genes IV, Part 3: " Controlling prokaryotic genes by transcription ", Oxford University Press:Oxford, 213-301 page or leaf, reference wherein, and Michal, G. (1999) Biochemical Pathways:An Atlas of Biochemistry and MolecularBiology, John Wiley ﹠amp; Sons).By all these known regulate processes of other gene mediated, this other gene itself produces various external actions and replys (for example, the availability of temperature, nutrient or illumination).The exemplary rho factor that participates in this type adjustment comprises transcription factor.These factors are the protein in conjunction with DNA, and by increasing genetic expression (just regulating) or reducing genetic expression (the negative adjusting).These regulate the transcription factor of expressing itself can be the object of regulating.For example, combine the activity that to regulate them with dna binding protein dna by low-molecular weight compound, stimulate thus or suppress these protein and DNA go up the combining of suitable binding site (referring to, Helmann for example, J.D. and Chamberlin, M.J. (1988) " Structure and function ofbacterial sigma factors. " Ann.Rev.Biochem.57:839-872; Adhya, S. (1995) " The lac and gal operons today " and Boos, W. etc., " The maltose system. ", two pieces of documents exist: Regulation of Gene Expression in Escherichia coli (Lin, E.C.C. and Lynch, A.S., editor .) Chapman; Hall:New York, 181-200 and 201-229 page or leaf; And Moran, C.P. (1993) " RNA polymerase and transcriptionfactors. " in:Bacillus subtilis and other gram-positive bacteria, Sonenshein, A.L. etc. edit .ASM:Washington, D.C., 653-667 page or leaf).

The whole genome of glutamic acid rod rod bacterium (Corynebacterium glutamicum) ATCC 13032 is disclosed under Genbank recording mechanism: AP005283.Yet, do not provide any prompting of methionine(Met) biosynthesizing specificity instrumentality.

The plus or minus that the cell of the various factors such as substrate, katabolic product and end product and extracellular levels can controlling gene be transcribed and translated is regulated.Typically, the expression of gene of the active necessary enzyme of coding particular approach is induced by the high level of the substrate molecule of this approach.Similarly, when having the end product of high-caliber this approach in the cell, tend to check this genetic expression (Snyder, L. and Champness, W. (1997) The Molecular Biology of Bacteria ASM:Washington).Other outside and internal factor such as envrionment conditions (for example, heat, oxidative stress or hunger; Referring to, Lin for example, E.C.C. and Lynch, A.S., editor (1995) Regulation ofGene Expression in Escherichia coli.Chapman ﹠amp; Hall:New York) also can regulatory gene express.

The regulating networks of at large understanding control cell metabolism in the microorganism are most important for producing chemical by the fermentation high yield.Can remove or reduce the negative regulation and control system of pathways metabolism to improve the synthetic of expectation chemical, and similarly, can activate the positive regulation and control system of the pathways metabolism of expectation product with forming, perhaps on activity this Controlling System of optimization (as Hirose, Y. and Okada, H. (1979) " Microbial Production of Amino Acids ", in:Pepper, H.J. and Perlman, D. (editor) Microbial Technology, second edition .1 volume, 7 chapters, AcademicPress:New York is described).

Yet, the methionine(Met) biosynthetic means that comes the fermentative production methionine(Met) based on the modification of methionine(Met) biosynthesizing regulation system was not also described so far.In the method for the fermentative production methionine(Met) of all reports, for economic production, it is too low that productive rate seems.Therefore, need improved method.

Summary of the invention

The invention provides and be used to regulate fine chemicals, preferably the biosynthetic novel method of sulfocompound (resembling, for example methionine(Met)).Have been found that metabolism of the present invention regulate ( mEtabolic rEgulatory) (" MR ") molecule participates in fine chemicals, the preferably biosynthesizing of sulfocompound (resembling, for example methionine(Met)).

Especially, have been found that MR molecule RXA00655 is the methionine(Met) biosynthesizing, the down regulator of halfcystine biosynthesizing and/or sulphur reduction approach, for example transcriptional.Have been found that also MR molecule RXN02910 is the biosynthetic positive regulator of methionine(Met), for example transcriptional.Here the nucleotide sequence of RXA00655 is recorded in SEQ ID NO:1, and the peptide sequence of RXA00655 is recorded in SEQ ID NO:2 here.Here the nucleotide sequence of RXN02910 is recorded in SEQ ID NO:5, and the peptide sequence of RXN02910 is recorded in SEQ ID NO:6 here.Here the homologous protein of RXA00655 is recorded in SEQ ID NO:19,21 and 23.The nucleotide sequence of RXN02910 homologous protein described here is recorded in SEQ ID NO:18,20 and 22.

Can be by regulating MR expression of nucleic acids of the present invention, for example increase the expression of RXN02910 nucleic acid molecule or increase the RXN02910 activity of proteins, suppress the expression of RXA00655 nucleic acid molecule or suppress the RXA00655 activity of proteins, perhaps pass through to modify the sequence of MR nucleic acid molecule of the present invention to change the activity of MR nucleic acid molecule, regulate, for example increase fine chemicals, methionine(Met) for example, halfcystine and/or methionine(Met) biosynthetic pathway, other compound of sulphur reduction approach and/or halfcystine biosynthetic pathway from the production of microorganism (for example, improve fine chemicals, for example other compound of methionine(Met) or methionine(Met) biosynthetic pathway is from the production or the output of corynebacterium (Corynebacterium) or brevibacterium sp (Brevibacterium) species).In another embodiment, can be linked together with increasing RXN02910 expression of nucleic acids or activity of proteins and inhibition RXA00655 expression of nucleic acids or activity of proteins, to regulate, (for example for example increase from microorganism, corynebacterium or brevibacterium sp species) fine chemicals, for example production of other compound of methionine(Met), halfcystine and/or methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway.

The present invention is characterised in that the method for the microorganisms producing of regulating sulfocompound, this method is included in culturing micro-organisms under the condition of the production that can regulate sulfocompound, and wherein this microorganism has and expresses or the active at least a methionine(Met) biosynthesizing instrumentality of being regulated.Preferably, described sulfocompound is selected from methionine(Met), halfcystine, S-adenosylmethionine and homocysteine.And preferably, increase the production of described sulfocompound (for example, methionine(Met)).

The present invention relates to increase the method for producing by the sulfocompound of microorganism, this method is included under the condition of the production that increases sulfocompound and cultivates for example microorganism of RXN02910 of overexpression methionine(Met) biosynthesizing positive regulator.And, the method that the sulfocompound that the invention still further relates to increases microorganism is produced, this method is included in cultivates downward modulation under the condition of the production that increases sulfocompound and expresses (underexpress) methionine(Met) biosynthesizing down regulator, for example microorganism of RXA00655.

Therefore, the present invention relates to produce the method for other compound of methionine(Met) and methionine(Met) biosynthetic pathway.These methods are included under the condition of other compound of producing methionine(Met) or methionine(Met) biosynthetic pathway, cultivate the microorganism of overexpression RXN02910 gene product.This method comprises that also cultivation RXA00655 gene product expression is subjected to the microorganism that presses down, to produce other compound of methionine(Met) or methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway.The present invention also provides the method that is used to produce fine chemicals, and this method comprises that uniting the microorganism of cultivating overexpression RXN02910 gene product and RXA00655 gene product expression is subjected to the microorganism that presses down.

MR protein can for example participate in the following proteinic back adjusting of transcribing, translate or translate, and wherein the normal metabolic function of this protein pair cell is important.Because can obtain being used for the cloning vector (such as following example) of corynebacterium glutamicum (Corynebacterium glutamicum), so can in the genetically engineered of this organism, use nucleic acid molecule of the present invention so that it becomes better or more effective fine chemicals, for example producer of methionine(Met).

Fine chemicals, for example this raising of methionine(Met) on productive rate, production and/or production efficiency can be by operation gene of the present invention, for example due to the direct effect of RXA00655 or RXN02910, perhaps can be owing to due to the indirect action of this operation.Particularly, normally regulate change in the corynebacterium glutamicum MR protein of productive rate, production and/or production efficiency of methionine metabolism approach and may directly influence ultimate production or the throughput rate of producing fine chemicals (for example methionine(Met), other compound of halfcystine and/or methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway) from this organism.Change in the protein of participation methionine metabolism approach also may be to the fine chemicals of expectation, and for example the productive rate of methionine(Met), output and/or production efficiency have remote effect.It must be complicated that metabolism is regulated, and the regulation mechanism of control different approaches can intersect at a plurality of points, like this can the more than one approach of rapid adjustment according to the specific cells incident.This makes the proteinic modification of the adjusting of an approach also can be exerted an influence to the adjusting of many other approach, and the fine chemicals that some approach in these other approach may participate in expecting, for example biosynthesizing of methionine(Met) or degraded.In this indirect mode, will influence the production of the fine chemicals that produces the pathways metabolism that is different from the direct approach of regulating of this MR protein certainly to the adjusting of MR protein effect.

Nucleic acid of the present invention and protein molecule can be used for directly improving output, productive rate and/or the production efficiency of producing methionine(Met) from corynebacterium glutamicum.Utilize recombination engineering known in the art, can operate of the present invention one or more and regulate protein, to adjust its function or expression.For example, for coded amino acid, the gene (for example metY gene) of the required polypeptide of methionine(Met) biosynthesizing for example, participating in the MR protein that this gene transcription checks, for example suddenly change on the RXA00655, it no longer can be checked transcribe, can cause the increase that this amino acid is produced.Similarly, by changing the MR protein active, cause the fine chemicals that participates in expectation, for example the proteinic translation of the corynebacterium glutamicum of the production of methionine(Met) increases or activates this proteinic posttranslational modification, also can increase the production of this chemical.Opposite situation also may be useful: by to corynebacterium glutamicum that participate in to regulate the degradation approach proteinic transcribe or translate to implement to increase check, perhaps, can increase the production of this chemical by negative this protein of modifying in translation back.In each case, all can increase the overall yield or the throughput rate of the fine chemicals of expectation.

This change also may improve fine chemicals by indirect mechanism in protein of the present invention and the nucleic acid molecule, for example the output of methionine(Met), productive rate and/or production efficiency.The metabolism of any compound is inevitable in the cell interweave with other biosynthesizing and degradation pathway, and essential cofactor, intermediate or substrate may be provided or limited by this approach by another approach in approach.Therefore, by regulating one or more modulability activity of proteins of the present invention, may influence the activity or the production efficiency of another fine chemicals biosynthesizing or degradation pathway.And one or more are regulated proteinic operation may increase the overall capacity that (particularly growth conditions may be in the large scale fermentation cultivation of suboptimum) cell is grown and bred in the cultivation.For example, when causing the Nucleotide biosynthesizing, checks (therefore in MR protein of the present invention normally with the extracellular nutrition supplement of replying suboptimum, stop cell fission) time, by this MR protein that suddenlys change, make the aporepressor ability reduce, can increase the biosynthesizing of Nucleotide and increase cell fission possibly.Cause the cell growth that increases in the culture and the change in the splitted MR protein, can be at least since in the culture quantity of the cell of production chemical increase and cause the increase of producing output, life rate and/or the production efficiency of the fine chemicals of one or more expectations by culture.

The invention provides the new transgenic expression cassette that comprises adjusting sequence and nucleic acid molecule, this nucleic acid molecule encoding metabolic pathway proteins matter (MR protein) or its fragment, wherein said adjusting sequence can the preferably expression of the described nucleic acid molecule of mediation in microorganism.Preferably, described adjusting sequence is and the allogenic promotor of described nucleic acid molecule.Preferably, regulating sequence is the promoter sequence that works in corynebacterium glutamicum.

MR albumen mass-energy for example, is carried out the enzymatic step of transcribing, translate or translate the back adjusting that participates in pathways metabolism in the corynebacterium glutamicum.The proteinic nucleic acid molecule of coding MR is called the MR nucleic acid molecule here.In preferred embodiment, MR protein participates in transcribing, translating of one or more pathways metabolisms or translates the back and regulate.

In particularly preferred embodiments, the MR nucleic acid molecule is selected from:

A) comprise NO:1 with SEQ ID, SEQ ID NO:5, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20 or SEQ ID NO:22 nucleotide sequence or its complementary sequence have the nucleic acid molecule of the nucleotide sequence of at least 60% identity;

B) comprise the segmental nucleic acid molecule of at least 30 Nucleotide of nucleic acid, wherein said nucleic acid comprises SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ IDNO:18, nucleotide sequence or its complementary sequence of SEQ ID NO:20 or SEQ ID NO:22;

C) nucleic acid encoding molecule, described polypeptide comprises the NO:2 with SEQ ID, SEQ IDNO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21 or SEQ ID NO:23 aminoacid sequence have the aminoacid sequence at least about 60% identity; With

D) the segmental nucleic acid molecule of coded polypeptide, described polypeptide comprises SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, the aminoacid sequence of SEQ IDNO:21 or SEQ ID NO:23, wherein said fragment comprises SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, at least 10 continuous amino acid residues in the aminoacid sequence of SEQ ID NO:21 or SEQ ID NO:23.

Transgene expression cassette can comprise with respect to the nucleic acid molecule of described promoter sequence at the coding MR of antisense or sense orientation protein (for example RXA00655 or RXA02910).

In the preferred embodiment of the present invention, the nucleic acid molecule encoding polypeptide of coding MR protein (for example RXA00655 or RXA02910), this polypeptide comprises SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, the aminoacid sequence shown in SEQ ID NO:21 or the SEQ ID NO:23.Especially preferred described nucleic acid molecule comprises SEQ ID NO:1, SEQ IDNO:5, SEQ ID NO:16, SEQ ID NO:18, nucleotide sequence shown in SEQ ID NO:20 or the SEQ ID NO:22.Nucleic acid molecule also can coded polypeptide natural allelic variant, wherein said polypeptide comprises SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ IDNO:19, the aminoacid sequence shown in SEQ ID NO:21 or the SEQ ID NO:23.

Another aspect of the present invention relates to the carrier that comprises at least one transgene expression cassette of the present invention.Preferably, this carrier is an expression vector.

Another aspect of the present invention relates to at least one transgene expression cassette of the present invention or at least one carrier of the present invention or expression vector transfection or transformed host cells.Preferably, host cell belongs to corynebacterium (Corynebacterium) or brevibacterium sp (Brevibacterium).In one embodiment, by in appropriate media, cultivating host cell, use this host cell to produce MR protein.Then, can separate this MR protein from substratum or host cell.

Another aspect of the present invention relates to the microorganism of introducing or having changed the hereditary change of MR gene.In one embodiment, by introducing nucleic acid molecule of the present invention, changed the genome of microorganism as genetically modified encoding wild type or sudden change MR sequence.In another embodiment, by with the MR dna homolog reorganization that changes, changed, for example functionally destroyed endogenous MR gene in the microbial genome.In another embodiment, changed the endogenous or MR gene introduced in the microorganism by one or more point mutation, disappearance or inversion, but this gene encoding function MR protein still.In another embodiment, (for example, by disappearance, brachymemma, inversion or point mutation) changed one or more regulatory regions of MR gene in the microorganism (for example, promotor, aporepressor or inductor), to regulate the MR expression of gene.In preferred embodiment, microorganism belongs to corynebacterium or brevibacterium sp, preferred especially corynebacterium glutamicum.In preferred embodiment, microorganism also is used for desired compounds such as amino acid, the production of preferred especially methionine(Met).

Another aspect of the present invention relates to the method for producing fine chemicals.This method comprises cultivates the cell that contains the carrier that instructs MR nucleic acid molecule expression of the present invention, to produce fine chemicals.In preferred embodiment, this method comprises that further acquisition contains the step of the cell of this carrier, wherein with the carrier transfectional cell that instructs the MR expression of nucleic acid.Another preferred embodiment in, this method further comprises from culture and reclaims fine chemicals, for example step of methionine(Met).In particularly preferred embodiments, cell derives from corynebacterium or brevibacterium sp, perhaps is selected from those bacterial strains shown in the table 1.

Another aspect of the present invention relates to the method that the molecule that is used for regulating microorganism is produced.This method comprises with the reagent exposing cell of regulating MR protein active or MR expression of nucleic acid, and the cell related activity is changed with identical specific activity under the situation that does not have this reagent.In preferred embodiment, regulate one or more corynebacterium glutamicum pathways metabolism regulation systems of cell, to improve by this microorganisms producing expectation fine chemicals, for example productive rate of methionine(Met) or throughput rate.The reagent of regulating the MR protein active can be the reagent that stimulates MR protein active or MR expression of nucleic acid.Stimulate the example of the reagent of MR protein active or MR expression of nucleic acid to comprise the proteinic nucleic acid of coding MR, small molecules, the active MR protein of introducing in the cell.The example of the reagent that suppresses the MR activity or express comprises small molecules and antisense MR nucleic acid molecule.

The present invention relates to the method for the productive rate that is used to regulate cells produce expectation compound on the other hand, and this method comprises to be introduced wild-type or sudden change MR gene in the cell, makes this MR gene maintain on the independent plasmid or is incorporated in the genome of host cell.If be incorporated in the genome, this integration can be at random, perhaps can this integration take place by homologous recombination, thereby by the copy displacement natural gene of introducing, makes that the production of expectation compound is regulated in the cell.In preferred embodiment, increase described productive rate.Another preferred embodiment in, described chemical is a fine chemicals.In particularly preferred embodiments, described fine chemicals is an amino acid.In especially preferred embodiment, described amino acid is methionine(Met).

Description of drawings

Fig. 1 has described self the clone technology principle based on homologous recombination, and this technology is used to prepare the corynebacterium glutamicum strain system of methionine(Met) biosynthesizing down regulator (RXA00655 is shown in SEQ ID NO:1) defective.

Detailed description of the Invention

The invention provides metabolism regulate ( mEtabolic rEgulatory) (MR) nucleic acid and protein molecule, for example RXA00655 and RXN02910 nucleic acid and protein molecule, they pass through for example to participating in the gene of methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway, for example the metY gene carries out transcriptional regulatory, and the participation microorganism, for example the metabolism in the corynebacterium glutamicum is regulated, and comprises fine chemicals, for example biosynthetic adjusting of methionine(Met).

Therefore, the present invention relates to based on methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway in the operation microorganism to produce fine chemicals, sulfocompound for example, for example method of other compound of methionine(Met), halfcystine and/or methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway.

Term " methionine(Met) biosynthetic pathway " comprises and relates to methionine(Met) formation or (for example synthesize the middle methionine(Met) biosynthetic enzyme that utilizes, biosynthesizing enzyme coding gene encoded polypeptides), the approach of compound (for example, precursor, substrate, intermediate or product), cofactor or the like.Methionine(Met) is the needed amino acid of human nutrition.Bacterium is synthesized their methionine(Met) (Escherichia Coli and Salmonella:Cellular and MolecularBiology from amino acid and its biosynthesizing intermediate, Neidhardt, Frederick C.Curtiss, Roy III Ingraham, John L. edits, second edition .1996, ASM Press and Hwang BJ.Yeom HJ.Kim Y.Lee HS.Journalof Bacteriology.184 (5): 1277-86,2002).Term " halfcystine biosynthetic pathway " comprises and relates to halfcystine formation or (for example synthesize the middle halfcystine biosynthetic enzyme that utilizes, biosynthesizing enzyme coding gene encoded polypeptides), the approach of compound (for example, precursor, substrate, intermediate or product), cofactor or the like.Comprise term " sulphur reduction approach " approach that relates to following enzyme, this enzyme play the effect of metabolism mineral compound such as sulphur and its derivative.Have been found that RXA00655 is the fine chemicals biosynthesizing, for example the biosynthetic down regulator of methionine(Met), for example transcriptional.Therefore, RXA00655 genetic expression check or the rejecting of inhibition or RXA00655 gene will cause microorganism, for example fine chemicals in the corynebacterium glutamicum, for example the increase production of other compound of methionine(Met), halfcystine and/or methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway.The sudden change of minimizing or inhibition RXA00655 genetic expression or RXA00655 polypeptide active is in case introducing also can cause microorganism, for example fine chemicals in the corynebacterium glutamicum, for example the increase production of other compound of methionine(Met), halfcystine and/or methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway.

Have been found that also RXN02910 is the fine chemicals biosynthesizing, for example the biosynthetic positive regulator of methionine(Met) is for example transcribed positive regulator.Therefore, the overexpression of RXN02910 gene causes microorganism, for example increase production of other compound of methionine(Met) and methionine(Met) biosynthetic pathway in the corynebacterium glutamicum.The sudden change that increases RXN02910 genetic expression or RXN02910 polypeptide active also causes microorganism, for example increase production of other compound of methionine(Met) and methionine(Met) biosynthetic pathway in the corynebacterium glutamicum after introducing.

And, RXA00655 expresses in microorganism prevents or the overexpression of the rejecting associating RXN02910 gene of inhibition or RXA00655 gene also causes fine chemicals in the microorganism, for example increase production of other compound of methionine(Met), halfcystine and/or methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway.And, by having the microorganism that the RXA00655 that prevents or suppress expresses or having of the cultivation of the microorganism of RXA00655 gene knockout with the microorganism of overexpression RXN02910 gene, cause also that from the microorganisms producing fine chemicals for example the production of other compound of methionine(Met), halfcystine and/or methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway increases.

Therefore, one aspect of the present invention is characterised in that the production fine chemicals, the method of other compound of methionine(Met) or methionine(Met) biosynthetic pathway for example, this method is included in can produce fine chemicals, for example cultivates the microorganism of overexpression RXN02910 under the condition of other compound of methionine(Met) or methionine(Met) biosynthetic pathway.The microorganism of overexpression RXN02910 comprises through operation and the microorganism of overexpression RXN02910.

Term " overexpression " or " overexpression " comprise that the expression level of gene product (for example, RXN02910 gene product) is higher than the preoperative expression level of microorganism or is higher than expression level in the corresponding microorganism of not operating.For example, specific gene, for example the overexpression of RXN02910 comprises with this genetic expression of not operating with in the organism of this specific gene of overexpression and comparing, and is high by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% expression.The present invention includes above-mentioned percentage ratio intermediary scope and identity value.In one embodiment, can genetic manipulation (for example, genetic modification) microorganism with the overexpression gene product, make the level of this gene product be higher than before the microorganism operation expression levels or be higher than expression levels in the corresponding microorganism of not operating.Genetic manipulation (for example can include but not limited to, by adding strong promoter, inducible promoter or a plurality of promotor, perhaps regulate sequence and make that to express be composing type by removing) change or modification and relevant adjusting sequence or the site of specific gene expression, change the chromosome position of specific gene, change the contiguous nucleotide sequence of specific gene such as ribosome bind site, increase the copy number of specific gene, the protein that modification participation specific gene is transcribed and/or the specific gene product is translated (for example, regulate protein, supressor, enhanser, transcriptional activator etc.), perhaps any other conventional means (for example including but not limited to that the application of antisense nucleic acid molecule is with the expression of blocking-up repressor) of regulating of the expression of the releasing specific gene of this area routine.

On the other hand, the invention is characterized in the production fine chemicals, the method of other compound of methionine(Met), halfcystine and/or methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway for example, this method is included in can produce fine chemicals, for example under the condition of other compound of methionine(Met), halfcystine and/or methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway, cultivate microorganism with RXA00655 expression of preventing or suppressing.Having microorganism that the RXA00655 that prevents expresses comprises and is operated to such an extent that RXA00655 expresses and is obstructed or the microorganism that suppresses.

Term " preventing of expression ", " inhibition of expression " or " downward modulation is expressed " comprise that the expression level of gene product (for example RXA00655 gene product or RXN02910 gene product) is lower than the preceding expression levels of microorganism operation or is lower than expression levels in the corresponding microorganism of not operating.In one embodiment, to genetic expression prevent or suppress to comprise the operation microorganism make gene no longer express.For example, specific gene, for example the downward modulation of RXA00655 is expressed and to be comprised and do not operate this genetic expression that following mileometer adjustment reaches in the organism of this specific gene and compare low 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% expression.The present invention includes above-mentioned percentage ratio intermediary scope and identity value.In one embodiment, can genetic manipulation (for example, genetic modification) microorganism makes the level of this gene product be lower than the preceding expression levels of microorganism operation or be lower than the gene product level of expressing in the corresponding microorganism of not operating with the expressing gene product.Genetic manipulation can include but not limited to change or modification is expressed relevant adjusting sequence or site with specific gene, change the chromosome position of specific gene, change the contiguous nucleotide sequence of specific gene such as ribosome bind site, reduce the copy number of specific gene, the protein that modification participation specific gene is transcribed and/or the specific gene product is translated (for example, regulate protein, supressor, enhanser, transcriptional activator etc.), the application of antisense nucleic acid molecule, any other conventional means that the releasing of the rejecting of target gene or this area routine is regulated the expression of specific gene.The microorganism of RXA00655 genetic expression or RXN02910 genetic expression defective comprises the microorganism of preventing or suppressed RXA00655 or RXN02910 expression.

On the other hand, the invention is characterized in the production fine chemicals, the method for methionine(Met) for example, this method is included in the production fine chemicals, for example cultivates the active microorganism of RXN02910 with increase under the condition of methionine(Met)." the active microorganism of RXN02910 with increase " comprises and being operated to increase the active microorganism of RXN02910.

On the other hand, the present invention is characterised in that the production fine chemicals, the method of other compound of methionine(Met), halfcystine and/or methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway for example, this method is included in the production fine chemicals, for example under the condition of other compound of methionine(Met), halfcystine and/or methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway, cultivate the active microorganism of RXA00655 with minimizing." the active microorganism of RXA00655 that minimizing is arranged " comprises and being operated to suppress or to prevent the active microorganism of RXA00655.

Term " RXN02910 activity " comprises and causes the fine chemicals biosynthesizing, for example the biosynthetic any activity of methionine(Met).Thereby the RXN02910 activity includes but not limited to just regulate the biosynthesizing that the methionine(Met) biosynthetic pathway causes other compound of methionine(Met) biosynthesizing or methionine(Met) biosynthetic pathway.Can carry out the just adjusting of methionine(Met) biosynthetic pathway with any method, described method includes but not limited to transcribe and translates regulates and protein adjusting (by for example protein bound).The activity that increases comprises such activity, and this activity level is higher than the activity that shows before the microorganism operation or is higher than the activity that shows in the corresponding microorganism of not operating.Term " RXA00655 activity " comprises and causes the fine chemicals biosynthesizing, for example the biosynthetic any activity of methionine(Met).The active example of RXA00655 includes but not limited to the methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway are (as Escherichia Coli and Salmonella:Cellular and Molecular Biology, Neidhardt, Frederick C.Curtiss, Roy IIIIngraham, John L edits, second edition .1996, described in the ASM Press) negative adjusting, this negative adjusting causes that the methionine(Met) biosynthesizing reduces, the methionine(Met) biosynthetic pathway, the biosynthesizing of other compound of sulphur reduction approach or halfcystine biosynthetic pathway reduces.Can carry out the negative adjusting of methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway by any method, described method includes, but are not limited to transcribe and translates regulates and protein adjusting (by for example protein bound).The activity that reduces or prevent comprises such activity, and this activity level is lower than the activity that shows before the microorganism operation or is lower than the activity that shows in the corresponding microorganism of not operating.

The present invention relates to increase the method for producing by the sulfocompound of microorganism, this method is included under the condition of the production that increases sulfocompound and cultivates the biosynthetic positive regulator of overexpression methionine(Met), for example the microorganism of RXN02910.The present invention also relates to increase the method for producing by the sulfocompound of microorganism, this method is included in cultivates downward modulation under the condition of the production that increases sulfocompound and expresses the biosynthetic down regulator of methionine(Met), for example microorganism of RXA00655.

Term " sulfocompound " comprises any compound of sulfur-bearing or derivatives thereof.Sulfocompound comprises amino acid, and this amino acid includes, but are not limited to methionine(Met), halfcystine, S-adenosylmethionine and homocysteine.Here the RXA00655 nucleotide sequence is shown in SEQ ID NO:1, and here the RXA00655 peptide sequence shown in SEQ ID NO:2.Here the RXN02910 nucleotide sequence is shown in SEQ ID NO:5, and here the RXN02910 peptide sequence shown in SEQ IDNO:6.The homologous protein of RXA00655 such as SEQ ID NO:19 are shown in 21 and 23.The nucleotide sequence of the homologous protein of RXN02910 described here such as SEQ ID NO:18 are shown in 20 and 22.

SEQ ID NOs:16 and 17 represents the RXN02910 nucleic acid and the aminoacid sequence of sudden change respectively.RXN02910 molecule shown in the SEQ ID NO:16 is included in the single nucleotide alteration of nucleotide residue 556 positions in coding region from guanine (G) to VITAMIN B4 (A), this has caused the change that the aspartic acid (D) of amino-acid residue 186 positions of coded protein arrives l-asparagine (N), shown in SEQ IDNO:17.To with regard to the regulating effect of fine chemicals biosynthesizing (for example methionine(Met) biosynthesizing), this polymorphism can cause the modulation to this regulating effect, for example causes the methionine(Met) biosynthesizing of minimizing with regard to RXN02910.

Molecule of the present invention can be used for directly regulating from microorganism, fine chemicals such as corynebacterium glutamicum, for example methionine(Met) production (for example, the adjusting of the methionine(Met) biosynthesizing being regulated activity of proteins directly influences the output that this organism produces methionine(Met), productive rate and/or production efficiency), perhaps can produce and cause expectation compound output, the remote effect that productive rate and/or production efficiency increase (for example, with regard to regard to the proteinic adjusting of Nucleotide biosynthesizing, to influence the generation of organic acid in the bacterium or lipid acid to the modulation of this regulating effect, this possible reason is that the Nucleotide biosynthesizing of replying change is regulated and followed the adjusting of appearance to change in these chemical biosynthesizing or degradation pathway).

All respects of the present invention have been explained below in further detail.

Term ' fine chemicals ' by this area cognition, it comprises the molecule that is produced by organism that is used for multiple industry, wherein said industry is such as but not limited to pharmacy, agricultural and cosmetic industry.This compounds includes machine acid as tartrate, methylene-succinic acid and diaminopimelic acid, albumen originality (Proteinogenic) and non-proteinogen acidic amino acid, purine and pyrimidine bases, nucleosides and Nucleotide are (as Kuninaka in biotechnology (Biotechnology) the 6th volume of editing people such as Rehm, A. (1996) Nucleotide and related compound (Nucleotides and related compounds), the 561-612 page or leaf, described in VCH:Weinheim and the wherein contained reference), lipid, saturated and unsaturated fatty acids (as arachidonic acid), glycol (as propylene glycol and butyleneglycol), carbohydrate (as hyaluronic acid and trehalose), aromatic compound is (as aromatic amine, Vanillin and indigo), VITAMIN and cofactor (are seen as Ullmann industrial chemistry encyclopaedia (Ullmann ' s Encyclopedia of IndustrialChemistry), the A27 volume, " VITAMIN " (" Vitamins "), 443-613 page or leaf (1996) VCH:Weinheim and reference wherein; And Ong, A.S., Niki, E. and Packer, L. " the nutrition; lipid; healthy and disease " collection of thesis (" Nutrition; Lipids; Health; and Disease " Proceedings of the UNESCO/Confederation of Scientific and TechnologicalAssociations in Malaysia held of (1995) UNESCO/ Malaysia Science and Technology association and inferior state alliance of free radical research association, and the Society for Free Radical Research-Asia), 1-3 day in September, 1994 holds in Malaysian Penang, AOCS Press, (1995)), enzyme, polyketide people (1998) science (Science) 282:63-68 such as () Cane and at the chemical (Chemicals by Fermentation) of Gutcho (1983) by fermentation, Noyes DataCorporation, all other chemical described in ISBN:0818805086 and the reference wherein.The metabolism and the purposes of some chemical in these fine chemicals are further described below.

Element of the present invention and method

The present invention to small part based on to before the molecule (being called MR nucleic acid and protein molecule here) of unknown function, for example the function of RXA00655 nucleic acid and protein molecule and RXN02910 nucleic acid and protein molecule characterizes, one or more pathways metabolisms, for example methionine(Met) biosynthetic pathway in described molecular regulation or the modulation corynebacterium glutamicum.RXA00655 is the down regulator of methionine(Met) biosynthetic pathway, sulphur reduction approach and halfcystine biosynthetic pathway, and RXN02910 is the positive regulator of methionine(Met) biosynthetic pathway.Therefore, the present invention is characterised in that the production fine chemicals, other compound of methionine(Met), halfcystine and/or methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway and regulate fine chemicals for example, for example other compound of methionine(Met), halfcystine and/or methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway method of producing.These methods are included in the production fine chemicals, for example under the condition of other compound of methionine(Met), halfcystine and/or methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway, cultivate overexpression RXN02910 gene product or have the active microorganism of RXN02910 of increase.These class methods also comprise cultivating to have the RXA00655 gene product expression of inhibition or the active microorganism of RXA00655 of inhibition, to produce fine chemicals, other compound of methionine(Met), halfcystine and/or methionine(Met) biosynthetic pathway, sulphur reduction approach and/or halfcystine biosynthetic pathway for example.The present invention also provides the production fine chemicals, and the method for methionine(Met) for example, this method comprise cultivates overexpression RXN02910 gene product and show the RXA00655 gene product expression that suppresses simultaneously or the active microorganism of RXA00655 of inhibition.And, the present invention also provides the production fine chemicals, the method of methionine(Met) for example, this method comprise unites the microorganism of cultivating overexpression RXN02910 gene product and has the RXA00655 gene product expression of inhibition or the active microorganism of RXA00655 of inhibition.

In one embodiment, MR molecule of the present invention is being transcribed, the pathways metabolism in translation or the translation back horizontal adjustment corynebacterium glutamicum.In preferred embodiment, MR molecule of the present invention influences in this organism and expects fine chemicals, for example the production of methionine(Met) the adjusting activity of one or more corynebacterium glutamicum pathways metabolisms.In particularly preferred embodiments, adjust the activity of MR molecule of the present invention, to adjust the efficient or the output of the corynebacterium glutamicum pathways metabolism that MR protein of the present invention regulates, and this regulates directly or indirectly by corynebacterium glutamicum production expectation fine chemicals, for example output of methionine(Met), productive rate and/or production efficiency.

Term " MR protein " or " MR polypeptide " are included in the protein of transcribing, translate or translate pathways metabolism in the horizontal adjustment corynebacterium glutamicum of back.Term " the MR gene " or " the MR nucleotide sequence " comprising the proteic nucleotide sequence of coding MR, it is made up of coding region and corresponding 5 ' and 3 ' non-translated sequence district.Term " output " or " productivity " are this area cognition, are included in the concentration (for example, kg product/hour/liter) of the tunning (for example, the fine chemicals of expectation) that forms under preset time and the given fermentation volume.Term " production efficiency " comprise and desire to reach the required time of specific production level (for example, how long cell by reaching the specific output speed of fine chemicals).Term " productive rate " " product/carbon productive rate " or " production " be this area cognition, comprise the efficient that carbon source is converted into product (being methionine(Met)).It is typically expressed as kg product/kg carbon source.By increasing the productive rate or the production of compound, the amount of the recovery molecule of this compound or useful recovery molecule increases in the culture of given time durations specified rate.

Term " degraded " or " degradation pathway " and by this area cognition, be included in can be multistep rapid and be subjected to that cell decomposition compound (preferably organic compound) produces degraded product (generally speaking being less or more uncomplicated molecule) in the process of altitude mixture control.Term " metabolism " by this area cognition, comprise all biochemical reactions that carry out in the organism.The metabolism of specific compound (for example, the metabolism of amino acid such as glycine) then comprises all biosynthesizing, modification and the degradation pathway that relate to this compound in the cell.

Term " adjusting " is well known in the art, and comprises a kind of protein active control or regulate another kind of activity of proteins.Term " transcriptional regulatory " is well known in the art, and comprises that the DNA of prevention or activated code target protein is converted into the protein active of mRNA.Term " translation is regulated " is well known in the art, and comprises that the mRNA of prevention or activated code target protein is converted into the protein active of protein molecule.Term " translation then regulate " is well known in the art, and comprise by the covalent modification target protein (for example, by methylating, glucosylation, phosphorylation or by in conjunction with target protein), stop or improve the active protein active of target protein.

In another embodiment, MR molecule of the present invention can change the production of expecting molecule such as fine chemicals (as methionine(Met)) in microorganism such as the corynebacterium glutamicum.Use the genetic recombination technology, can operate of the present invention one or more and regulate protein, thereby adjust its function.For example, can improve the effect of biosynthetic enzyme, or destroy its other structure regulatory region to stop the feedback inhibition that this compound is produced.Similarly, can by replace, disappearance or add lacks or change degrading enzyme, thereby weaken it to the degrading activity of expectation compound and do not damage the viability of cell simultaneously.In each case, all can increase the overall yield or the throughput rate of one of these expectation fine chemicals.

This change also may improve fine chemicals in indirect mode in protein of the present invention and the nucleic acid molecule, for example production of methionine(Met).The regulation mechanism of pathways metabolism must be implicative of each other in the cell, and the activation of an approach can cause checking or activating of another approach in the mode of following.Therefore, by adjusting one or more activity of proteins of the present invention, can influence the production or the active efficient of another fine chemicals biosynthesizing or degradation pathway.For example, with regard to the proteinic gene of coding specific amino acids biosynthesizing,, can prevent other amino acid biosynthetic pathway simultaneously, because these approach are to be mutually related by reducing the ability that MR protein checks this genetic transcription.And, by modifying MR protein of the present invention, can be to a certain degree with growth and the division and their the extracellular environment uncoupling of cell; Just normally check when being suboptimum with regard to the biosynthetic MR protein of Nucleotide for growth and cell fission in extracellular conditions, thereby make it lack this now to check function by damaging this MR protein, when then even extracellular conditions is very poor, also can allow growth to take place.In the large scale fermentation growth, this is meaningful especially, because under fermentation conditions, temperature, nutrition supplement or ventilation usually are suboptimums in the culture, if but remove cell regulation system at these factors, then these conditions still can sustenticular cell growth and division.

Isolated nucleic acid sequences of the present invention is included in the genome of corynebacterium glutamicum strain system, and this strain system is deposited in American type culture collection (ATCC), and preserving number is ATCC 13032.At SEQ ID NOs:1, described Nucleotide and the aminoacid sequence of RXA00655 respectively in 18,20,22 and 2,19,21,23, and in SEQ ID NOs:5 and 6, described Nucleotide and the aminoacid sequence of RXN02910 respectively.SEQ ID NOs:16 and 17 represents the RXN02910 nucleic acid and the aminoacid sequence of sudden change respectively.RXN02910 molecule shown in the SEQ ID NO:16 is included in the single nucleotide alteration of nucleotide residue 556 positions in coding region from guanine (G) to VITAMIN B4 (A), this has caused the change of amino-acid residue 186 positions from aspartic acid (D) to l-asparagine (N) of coded protein, shown in SEQ ID NO:17.This polymorphism can be adjusted RXN02910 to methionine(Met) or the biosynthetic regulating effect of other fine chemicals, for example causes the methionine(Met) biosynthesizing of minimizing.

The present invention also relates to have in fact and SEQ ID NO:2, SEQ ID NO:6, SEQ IDNO:17, SEQ ID NO:19, the protein of the aminoacid sequence of SEQ ID NO:21 or SEQ ID NO:23 amino acid sequence homologous.As used herein, aminoacid sequence that it had and selected aminoacid sequence in fact homologous protein at least about 50% with coming from selected aminoacid sequence, for example complete selected aminoacid sequence.Aminoacid sequence that it had and selected aminoacid sequence homologous protein in fact also can be at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80%, 80-90%, or 90-95%, and most preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more the highland with coming from selected aminoacid sequence.

MR protein of the present invention or its biologically-active moiety or fragment can be transcribed ground, translation ground or translation and afterwards be regulated pathways metabolism in the corynebacterium glutamicum, and for example the methionine(Met) biosynthetic pathway perhaps has one or more activity described here.

All respects of the present invention have been described in the segmentation below in more detail.

A. transgene expression cassette, carrier and host cell

The invention provides the new transgenic expression cassette that comprises adjusting sequence and nucleic acid molecule, this nucleic acid molecule encoding metabolic pathway proteins matter (MR protein) or its fragment, wherein said adjusting sequence can mediate the expression of described nucleic acid molecule.Preferably, described adjusting sequence is and the allogenic promotor of described nucleic acid molecule.Preferably, regulating sequence is the promoter sequence that works in corynebacterium glutamicum.

MR protein can for example be carried out the enzymatic step of transcribing, translate or translate the back adjusting that participates in pathways metabolism in the corynebacterium glutamicum.The proteinic nucleic acid molecule of coding MR is called the MR nucleic acid molecule here.In preferred embodiment, MR protein participates in transcribing, translating of one or more pathways metabolisms or translates the back and regulate.

A) comprise NO:1 with SEQ ID, SEQ ID NO:5, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20 or SEQ ID NO:22 nucleotide sequence or its complementary sequence have the nucleic acid molecule of the nucleotide sequence of at least 60% identity;

B) comprise the segmental nucleic acid molecule of at least 30 Nucleotide of following nucleic acid, described nucleic acid comprises SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, nucleotide sequence or its complementary sequence of SEQ ID NO:20 or SEQ ID NO:22;

C) nucleic acid encoding molecule, described polypeptide comprises the NO:2 with SEQ ID, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21 or SEQ IDNO:23 aminoacid sequence have the aminoacid sequence at least about 60% identity; With

D) the segmental nucleic acid molecule of coded polypeptide, described polypeptide comprises SEQ ID NO:2, SEQ IDNO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21 or SEQID NO:23 aminoacid sequence, wherein this fragment comprises SEQ ID NO:2, SEQ IDNO:6, SEQ ID NO:17, SEQ ID NO:19, at least 10 continuous amino acid residues in SEQ ID NO:21 or the SEQID NO:23 aminoacid sequence.

In the preferred embodiment of the present invention, the nucleic acid molecule encoding polypeptide of coding MR protein (for example RXA00655 or RXA02910), this polypeptide comprises SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, the aminoacid sequence shown in SEQ ID NO:21 or the SEQ ID NO:23.Especially preferred described nucleic acid molecule comprises SEQ ID NO:1, SEQ IDNO:5, SEQ ID NO:16, SEQ ID NO:18, nucleotide sequence shown in SEQ ID NO:20 or the SEQ ID NO:22.Nucleic acid molecule also can coded polypeptide natural allelic variant, wherein this polypeptide comprises SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, the aminoacid sequence shown in SEQ ID NO:21 or the SEQ ID NO:23.

Terminology used here " nucleic acid molecule " is intended to comprise that dna molecular (for example, cDNA or genomic dna) and RNA molecule are (for example, mRNA) and utilize the DNA or the RNA analogue of nucleotide analog deposits yields.This term also comprises the non-translated sequence that is positioned at gene coding region 3 ' and 5 ' end: the sequence of downstream at least about 20 Nucleotide held at least about the sequence and the gene coding region 3 ' of 100 Nucleotide in 5 ' end upstream, coding region.Nucleic acid molecule can be strand or two strands, but preferably double-stranded DNA.

Terminology used here " transgene expression cassette " is intended to comprise all types of nucleic acid constructs that obtain by molecular biology method, wherein in described construct,

A) MR nucleic acid or its part (for example, comprise SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, the nucleic acid of SEQ ID NO:20 or SEQ IDNO:22 nucleotide sequence or its complementary sequence; The perhaps part of aforementioned nucleic acid), perhaps

B) with the adjusting sequence (for example, promoter sequence) that (a) makes up, perhaps

C) (a) and (b)

Be not arranged in their natural genotypic environment, perhaps they modified by molecular biology or gene engineering.This modification can comprise displacement, interpolation, disappearance, inversion or the insertion of one or more nucleotide residues." natural genotypic environment " is intended to describe the natural genetics of concrete gene.For example, the transgene expression construct can comprise the combination of MR nucleic acid and its natural promoter sequence, and prerequisite is that this combination is separated and/or is placed in the different genome environment from its natural gene group environment.Preferably, promoter sequence and described MR nucleic acid are allogenic.

" allogenic " is intended to describe the combination of MR nucleic acid and promoter sequence, do not regulate this same MR expression of nucleic acids under the natural situation of wherein said promoter sequence.

" genetically modified " relevant with carrier or host organisms is intended to describe carrier or the host cell that comprises above-mentioned transgene expression cassette.

" isolating " nucleic acid molecule is and other nucleic acid molecule isolated nucleic acid molecule that wherein said other nucleic acid molecule is present in the natural origin of this nucleic acid.

The sequence information that can utilize standard molecular biological technique and provide here, separate nucleic acid molecule of the present invention, for example has SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQID NO:18, nucleic acid molecule or its part of SEQ ID NO:20 or SEQ ID NO:22 nucleotide sequence.For example, utilize SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQID NO:18, all or part of one of SEQ ID NO:20 or SEQ ID NO:22 sequence as hybridization probe with standard hybridization technique (for example, Sambrook, J., Fritsh, E.F., and Maniatis, T.Molecular Cloning:A Laboratory Manual. second edition, Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY is described in 1989) can separate corynebacterium glutamicum MRDNA from the corynebacterium glutamicum library.And, comprise SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ IDNO:18, the nucleic acid molecule of all or part of one of SEQ ID NO:20 or SEQ ID NO:22 sequence, can utilize Oligonucleolide primers based on this sequences Design, separate by the polymerase chain reaction and (for example, can utilize based on this identical SEQ ID NO:1 SEQ ID NO:5, SEQID NO:16, SEQ ID NO:18, the Oligonucleolide primers of SEQ ID NO:20 or SEQ ID NO:22 sequences Design comprises SEQ ID NO:1 by the polymerase chain reaction separation, SEQ IDNO:5, SEQ ID NO:16, SEQ ID NO:18, the nucleic acid molecule of all or part of one of SEQ ID NO:20 or SEQ ID NO:22 sequence).For example, can be (for example from normal bacteria cellular segregation mRNA, by Chirgwin etc. the guanidine thiocyanate extracting method of (1979) Biochemistry 18:5294-5299), and can utilize reversed transcriptive enzyme (for example to prepare DNA, Moloney MLV reversed transcriptive enzyme, can be from Gibco/BRL, Bethesda, MD obtains; Perhaps AMV reversed transcriptive enzyme can be from Seikagaku America, Inc., and St.Petersburg, FL obtains).According to SEQ IDNO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, one of nucleotide sequence shown in SEQ ID NO:20 or the SEQ ID NO:22 can be designed for the synthetic oligonucleotide primer thing of polymerase chain reaction (PCR) amplification.According to the pcr amplification technology of standard, utilize cDNA or alternately genomic dna is as template and utilize suitable Oligonucleolide primers, nucleic acid of the present invention can increase.Can be in appropriate carrier with the nucleic acid clone that so increases, and characterize it by dna sequence analysis.And, by the standard synthetic technology, for example utilize automatic dna synthesizer also can prepare oligonucleotide corresponding to the MR nucleotide sequence.

In preferred embodiment, transgene expression cassette comprises SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, one of nucleotide sequence shown in SEQ ID NO:20 or the SEQ ID NO:22.SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, the sequence of SEQ ID NO:20 or SEQ ID NO:22 is corresponding to corynebacterium glutamicum MR DNA of the present invention.This DNA comprises the proteinic sequence of coding MR (promptly, at SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, " coding region " indicated in each sequence of SEQID NO:20 or SEQ ID NO:22) and also at SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18,5 ' non-translated sequence and the 3 ' non-translated sequence indicated among SEQ IDNO:20 or the SEQ ID NO:22.As alternative scheme, nucleic acid molecule can only comprise SEQ ID NO:1, SEQ ID NO:5, SEQID NO:16, SEQ ID NO:18, the coding region of arbitrary sequence among SEQ ID NO:20 or the SEQ ID NO:22.

Another preferred embodiment in, transgene expression cassette of the present invention comprises nucleic acid molecule, this nucleic acid molecule is SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ IDNO:18, the complementary sequence of one of nucleotide sequence or its part shown in SEQ ID NO:20 or the SEQ ID NO:22.With SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ IDNO:18, the complementary nucleic acid molecule of one of nucleotide sequence shown in SEQ ID NO:20 or the SEQ ID NO:22 is to be enough to the NO:1 with SEQ ID, SEQ ID NO:5, SEQ ID NO:16, SEQID NO:18, one of nucleotide sequence shown in SEQ ID NO:20 or the SEQ ID NO:22 complementary so that can with SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ IDNO:18, the hybridization of one of nucleotide sequence shown in SEQ ID NO:20 or the SEQ ID NO:22 also forms thus and stablizes double-helical nucleic acid molecule.

Another preferred embodiment in, transgene expression cassette of the present invention comprises nucleotide sequence, this nucleotide sequence and SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ IDNO:18, nucleotide sequence or its part have at least about 50%, 51% shown in SEQ ID NO:20 or the SEQ ID NO:22,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, even more preferably at least about 95%, 96%, 97%, 98%, 99% or higher homology.The present invention is also intended to comprise above-mentioned scope intermediary identity value range (for example, 70-90% identity or 80-95% identity).For example, be intended to comprise the identity value range that utilizes above-mentioned any value to combine as the upper limit and/or lower limit.Preferably implement in the originating party formula at another, transgene expression cassette of the present invention comprises nucleotide sequence, this nucleotide sequence can with SEQ ID NO:1, SEQ IDNO:5, SEQ ID NO:16, SEQ ID NO:18, one of nucleotide sequence shown in SEQ ID NO:20 or the SEQ ID NO:22 or the hybridization of its part, for example hybridize under stringent condition.

And, transgene expression cassette of the present invention can only comprise SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, the part of the coding region of one of sequence among SEQ ID NO:20 or the SEQ ID NO:22, for example, can be as the fragment of probe or primer, the fragment of the MR protein biologically-active moiety of perhaps encoding.

Can produce probe and primer from the definite nucleotide sequence of the MR gene clone that derives from corynebacterium glutamicum is feasible, being designed for the MR homologue (homologue) of identifying and/or cloning in other cell type and the organism, and come the MR homologue of other coryneform bacteria or relevant species.Probe/primer comprises the oligonucleotide of purifying basically usually.Oligonucleotide comprises under the stringent condition and SEQ ID NO:1 usually, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, the sense strand of one of sequence, SEQ ID NO:1 shown in SEQID NO:20 or the SEQ ID NO:22, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, shown in SEQ ID NO:20 or the SEQID NO:22 in the antisense strand of one of sequence or its natural mutation at least about 12, preferably about 25, the nucleotide sequence district of more preferably about 40,50 or 75 continuous nucleotides hybridization.Can in the PCR reaction, use based on SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, the primer of SEQ ID NO:20 or SEQ ID NO:22 nucleotide sequence is with clone MR homologue.The transcript or the genome sequence that can be used to detect the identical or homologous protein of coding based on the probe of MR nucleotide sequence.In preferred embodiment, probe further comprises the labelling groups that adheres on it, and for example this labelling groups can be radio isotope, fluorescent chemicals, enzyme or enzyme cofactor.This probe can be used as the part of diagnostic test kits, wherein this test kit is by the level such as MR coding nucleic acid in the detection cell sample, for example detect the MRmRNA level or whether detect that genome MR gene is suddenlyd change or lacks, be used to identify the proteinic cell of the wrong MR of expression.

In one embodiment, transgene expression cassette comprises the nucleic acid molecule of coded protein or its part, wherein this protein or its part comprise the NO:2 with SEQ ID, SEQ ID NO:6, SEQ IDNO:17, SEQ ID NO:19, the aminoacid sequence that SEQ ID NO:21 or SEQ ID NO:23 aminoacid sequence have enough homologys, this protein or its part are kept and are transcribed, translate or translate the ability of afterwards regulating pathways metabolism in the corynebacterium glutamicum like this.

Terminology used here " enough homologys " refers to have protein or its part of following aminoacid sequence, this aminoacid sequence comprises the identical of minimum quantity or is equal to SEQ ID NO:2, SEQID NO:6, SEQ ID NO:17, SEQ ID NO:19, the amino-acid residue of SEQ ID NO:21 or SEQ IDNO:23 aminoacid sequence (for example, with SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, amino-acid residue has the amino-acid residue of similar side chain in one of sequence of SEQ ID NO:21 or SEQ ID NO:23), thereby make this protein or its part to transcribe, pathways metabolism in the corynebacterium glutamicum, for example methionine(Met) biosynthesizing are regulated in translation or translation back.As described here, the protein member of this pathways metabolism can play the effect of regulating one or more fine chemicals biosynthesizing or degraded.This active example has also been described here.Therefore, " the proteinic function of MR " helps integrally-regulated one or more fine chemicals pathways metabolisms, perhaps helps one or more fine chemicals directly or indirectly, for example the output of methionine(Met), productive rate and/or production efficiency.

In another embodiment, protein and SEQ ID NO:2, SEQ ID NO:6, SEQID NO:17, SEQ ID NO:19, whole aminoacid sequences of SEQ ID NO:21 or SEQ ID NO:23 have at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90%, 90-95%, most preferably at least about 96%, 97%, 98%, 99% or higher homology.

As mentioned above, homology MR protein, preferably homology RXA00655 or RXN02910 protein can derive from other microorganism, preferably derive from prokaryotic micro-organisms.

Term " prokaryotic micro-organisms " is intended to comprise Gram-positive and gram negative bacterium.Preferably, this term comprises all genus and the kind of enterobacteriaceae (Enterobacteriaceae) or Nocardiaceae, enterobacteriaceae species Escherichia (Escherichia) for example, serratia (Serratia), proteus (Proteus), enterobacter (Enterobacter), Klebsiella (Klebsiella), salmonella (Salmonella), Shigella (Shigella), Edwardsiella (Edwardsielle), Citrobacter (Citrobacter), morganella morganii belongs to (Morganella), Providencia (Providencia) and Yersinia (Yersinia).And all Rhodopseudomonass (Pseudomonas) preferably, Burkholder Pseudomonas (Burkholderia), Nocardia (Nocardia), genus acetobacter (Acetobacter), streptomyces (Streptomyces), Gluconobacter (Gluconobacter), corynebacterium (Corynebacterium), brevibacterium sp (Brevibacterium), bacillus (Bacillus), fusobacterium (Clostridium), cyanobacteria (Cyanobacter), Staphylococcus (Staphylococcus), aerobacter (Aerobacter), Alcaligenes (Alcaligenes), Rhod (Rhodococcus) and Penicillium (Penicillium) species.Most preferably corynebacterium (Corynebacterium) and streptomyces (Streptomyces) species, resemble example coryneform bacteria species as shown in table 1, diphtheria corynebacterium (Corynebacterium diphtheriae), Corynebacterium efficiens and streptomyces coelicolor (Streptomyces coelicolor).

The proteinic example of homology MR can comprise:

A) preferably described, the RXA00655 protein that derives from diphtheria corynebacterium by the aminoacid sequence that comprises SEQ ID NO:19 sequence,

B) preferably described, the RXA00655 protein that derives from Corynebacterium efficiens YS-314 GenBank recording mechanism Nr.AP005223 by the aminoacid sequence that comprises SEQ ID NO:21 sequence,

C) preferably described, the RXA00655 protein that derives from streptomyces coelicolor Genbank recording mechanism NC_003888 by the aminoacid sequence that comprises SEQ ID NO:23 sequence.

Preferably, RXN02910 is intended to describe the polypeptide of nucleic acid molecule encoding, and this nucleic acid molecule comprises and is selected from following nucleic acid molecule:

A) comprise the nucleic acid molecule that has the nucleotide sequence of at least 60% identity with SEQ ID NO:5 or 16 nucleotide sequences;

B) comprise the segmental nucleic acid molecule of at least 30 Nucleotide of nucleic acid, wherein said nucleic acid comprises the nucleotide sequence of SEQ ID NO:5 or 16;

C) nucleic acid encoding molecule, this polypeptide comprise with SEQ ID NO:6 or 17 aminoacid sequences and have aminoacid sequence at least about 60% identity; With

D) coding comprises the segmental nucleic acid molecule of the polypeptide of SEQ ID NO:6 aminoacid sequence, and wherein this fragment comprises at least 10 successive amino-acid residues in SEQ ID NO:6 or 17 aminoacid sequences.

Preferably, RXA00655 is intended to describe the polypeptide of nucleic acid molecule encoding, and this nucleic acid molecule comprises and is selected from following nucleic acid molecule:

A) comprise the NO:1 with SEQ ID, 18,20 or 22 nucleotide sequences have the nucleic acid molecule of the nucleotide sequence of at least 60% identity;

B) comprise the segmental nucleic acid molecule of at least 30 Nucleotide of nucleic acid, described nucleic acid comprises SEQ ID NO: 1,18,20 or 22 nucleotide sequence;

C) nucleic acid encoding molecule, this polypeptide comprise the NO:2 with SEQ ID, and 19,21 or 23 aminoacid sequences have the aminoacid sequence at least about 60% identity;

D) coding comprises SEQ ID NO:2, the segmental nucleic acid molecule of the polypeptide of 19,21 or 23 aminoacid sequences, and wherein this fragment comprises SEQ ID NO:2, at least 10 successive amino-acid residues in 19,21 or 23 aminoacid sequences.

Preferably, the proteinic part of MR nucleic acid molecule encoding of the present invention is the biologically-active moiety of one of MR protein.Terminology used here " the proteinic biologically-active moiety of MR " is intended to comprise can transcribe, translate or translate afterwards to be regulated in the corynebacterium glutamicum pathways metabolism or has the proteinic part of active MR described here, for example territory/motif.Whether can transcribe, translate or translate and afterwards regulate pathways metabolism in the corynebacterium glutamicum in order to detect MR protein or its biologically-active moiety, can carry out the enzymic activity test.This test method is well known to those of ordinary skill in the art, describes in detail in the embodiment 8 of embodiment.

Can be by separating SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQID NO:19, the part of one of sequence among SEQ ID NO:21 or the SEQ ID NO:23, this encoding part (for example, passing through in-vitro recombination expression) of expression MR protein or peptide, with the activity of this encoding part of assessing MR protein or peptide, with other nucleic acid fragment of preparation coding MR protein biologically-active moiety.

The present invention further comprises following nucleic acid molecule, described nucleic acid molecule is owing to the degeneracy of genetic code is different from SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22, SEQ ID NO:18, SEQ ID NO:20, one of nucleotide sequence shown in the SEQ ID NO:22 (with its part), so this nucleic acid molecule and SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20 or SEQ ID NO:22, SEQ ID NO:18, nucleotide sequence coded identical MR protein shown in SEQ ID NO:20 or the SEQ IDNO:22.In another embodiment, isolated nucleic acid molecule of the present invention has the nucleotide sequence of coded protein, this protein has SEQID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21 or SEQ ID NO:23, SEQ ID NO:19, the aminoacid sequence shown in SEQ ID NO:21 or the SEQ ID NO:23.In further embodiment, nucleic acid molecule encoding total length corynebacterium glutamicum protein of the present invention, this protein is gone up substantially with coming from (by SEQ ID NO:1, SEQID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ IDNO:22, SEQ ID NO:18, the coding of open reading frame shown in SEQ ID NO:20 or the SEQ ID NO:22) SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21 or SEQ ID NO:23, SEQ ID NO:19, the aminoacid sequence of SEQ IDNO:21 or SEQ ID NO:23.

In one embodiment, the present invention includes identity percentage ratio with Nucleotide of the present invention or aminoacid sequence and be higher than Nucleotide and aminoacid sequence with the identity percentage ratio of prior art sequence (for example, Genbank sequence (or protein of this sequence encoding)).The identity percentage ratio score that the GAP of 3 each of hitting the highest by checking given sequence calculates, and by deduct the identity percentage ratio that the highest GAP calculates from 100%, those of ordinary skills can calculate the lower threshold at the identity percentage ratio of any given sequence of the present invention.Those of ordinary skills also will understand: the present invention also comprises nucleic acid and the aminoacid sequence (for example, at least 50% of identity percentage ratio greater than the lower threshold that calculates like this, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or higher identity).

Except SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22, SEQ ID NO:18 is shown in SEQ ID NO:20 or the SEQ ID NO:22 outside the corynebacterium glutamicum MR nucleotide sequence, those skilled in the art will appreciate that: cause the dna sequence polymorphism of MR Argine Monohydrochloride sequence variation in colony (for example, corynebacterium glutamicum colony), to exist.Because natural variation, this genetic polymorphism in the MR gene can exist between the individuality in colony.As used herein, term " gene " and " recombination " be meant and comprise coding MR albumen, the nucleic acid molecule of the proteic opening code-reading frame of corynebacterium glutamicum MR of preferably encoding.This type of natural variation generally can cause the 1-5% variation in the MR gene nucleotide series.Among the MR from natural variation and do not change active arbitrary and all these type of nucleotide diversities of MR protein function and due to amino acid polymorphism also within the scope of the present invention.

Can use corynebacterium glutamicum DNA or its part to separate under the hybridization conditions of strictness according to the hybridization technique of standard according to the homology of they and corynebacterium glutamicum MR nucleic acid disclosed herein corresponding to the nucleic acid molecule of the natural variant of corynebacterium glutamicum MR DNA of the present invention and non-glutamic acid coryneform bacteria homologue as hybridization probe.Therefore, in another embodiment, isolated nucleic acid molecule of the present invention is long to be at least 15 Nucleotide, and under stringent condition can with comprise SEQ IDNO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, SEQ ID NO:18, SEQ ID NO:20, or the making nucleic acid molecular hybridization of the nucleotide sequence of SEQ ID NO:22.In other embodiments, nucleic acid is long is at least 15,20,25,30,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220,225 or more a plurality of Nucleotide.As used herein, term " hybridize under stringent condition " be to be used for describing hybridization and wash conditions, with this understanding, but generally still phase mutual cross of at least 60% homologous nucleotide sequence each other.Preferably, stringent condition is such condition, have each other with this understanding at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or the sequence of higher homology generally still keep the phase mutual cross.This stringent condition is that those of ordinary skills are known, can be at molecular biology fresh approach (Current Protocols in Molecular Biology), John Wiley ﹠amp; Sons, N.Y. (1989) finds among the 6.3.1-6.3.6.One of stringent hybridization condition preferred, limiting examples is: in 6X sodium chloride/sodium citrate (SSC) in about 45 ℃ of hybridization, then at 0.2 X SSC, among the 0.1%SDS in 50-65 ℃ of washing one or many.Preferably, at stringent condition and SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, or SEQ ID NO:22, SEQ ID NO:18, SEQ ID NO:20, or the isolated nucleic acid molecule of the present invention of the sequence hybridization of SEQ IDNO:22 is corresponding to naturally occurring nucleic acid molecule.As used herein, " naturally occurring " nucleic acid molecule is meant RNA or the dna molecular (for example, its coding native protein) with the nucleotide sequence that exists at occurring in nature.In one embodiment, the natural corynebacterium glutamicum MR albumen of nucleic acid encoding.

The natural variant of the MR sequence that in colony, may exist, those of ordinary skills also will understand can be by sudden change to SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, or SEQ ID NO:22, SEQ ID NO:18, SEQ IDNO:20, or import variation in the nucleotide sequence of SEQ ID NO:22, cause the proteic aminoacid sequence of coded MR to change thus, but the proteic function of MR is constant.For example, can be at SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ IDNO:22, SEQ ID NO:18, SEQ ID NO:20, or in the sequence of SEQ ID NO:22 " nonessential " the amino-acid residue place introduces the Nucleotide cause amino acid to be replaced and replaces." nonessential " amino-acid residue is such residue, it can be at wild-type sequence (the SEQ IDNO:2 of one of MR albumen of the present invention, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23) be changed in, but its change does not cause the proteic activity change of described MR; And " essential " amino-acid residue is that the MR protein-active is required.Yet other amino-acid residue (for example, conservative or only be semiconservative those amino-acid residues in having MR active structures territory) can be nonessential to activity, and therefore may be suitable for accepting to change and do not change the MR activity simultaneously.

Therefore, another aspect of the present invention relates to such nucleic acid molecule, and the MR albumen of its coding includes the active nonessential amino-acid residue change to MR.Proteic aminoacid sequence of this type of MR and SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQID NO:21 or SEQ ID NO:23, SEQ ID NO:19, the sequence difference that contains among SEQ ID NO:21 or the SEQ IDNO:23, but keep at least a MR activity as herein described.In one embodiment, this isolated nucleic acid molecule comprises the nucleotide sequence of coded protein, wherein this protein comprises at least about 50% with coming from SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, the aminoacid sequence of SEQ ID NO:21 or SEQ ID NO:23 aminoacid sequence, and can transcribe, translate or translate and afterwards regulate pathways metabolism in the corynebacterium glutamicum, perhaps have one or more activity described here.Preferably, the protein of nucleic acid molecule encoding comes from SEQ ID NO:2, SEQ ID NO:6 together at least about 50-60% thus, SEQ ID NO:17, SEQ ID NO:19, one of sequence among SEQ ID NO:21 or the SEQ ID NO:23, more preferably come from SEQ ID NO:2 together at least about 60-70%, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, one of sequence among SEQ ID NO:21 or the SEQ ID NO:23, even more preferably at least about 70-80%, 80-90% is with coming from SEQ ID NO:2, SEQ ID NO:6, SEQID NO:17, SEQ ID NO:19, one of sequence among SEQ ID NO:21 or the SEQ ID NO:23, and most preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with coming from SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ IDNO:19, one of sequence among SEQ ID NO:21 or the SEQ ID NO:23.

In order (for example to determine two aminoacid sequences, SEQ ID NO:2, SEQ ID NO:6, SEQID NO:17, SEQ ID NO:19, one of sequence and its mutant form among SEQ ID NO:21 or the SEQ ID NO:23) or the percentage homology of two nucleic acid, with sequence alignment (for example, can in the sequence of protein or nucleic acid, import vacancy (gap), align so that carry out optimal sequence with another protein or nucleic acid) to reach best purpose relatively.Compare amino-acid residue or Nucleotide then at corresponding amino acid position or nucleotide position place.When a sequence (for example, SEQ ID NO:2, SEQ IDNO:6, SEQ ID NO:17, SEQ ID NO:19, one of sequence among SEQ ID NO:21 or the SEQ ID NO:23) certain position in and another sequence are (for example, SEQ ID NO:2, SEQ IDNO:6, SEQ ID NO:17, SEQ ID NO:19, the mutant form of the aminoacid sequence of SEQ ID NO:21 or SEQ ID NO:23) in corresponding position when being occupied by identical amino-acid residue or Nucleotide, these molecules in this position be homologous (that is amino acid or nucleic acid used herein, " homology " be equal to amino acid or nucleic acid " identity ").Percentage homology between two sequences is the function (that is % homology=identical positional number/total positional number x100) of the total same position number of two sequences.

Coding and SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or the proteic isolated nucleic acid molecule of SEQ ID NO:23 protein sequence homologous MR can be by replacing one or more Nucleotide, add or disappearance importing SEQ ID NO:1, SEQID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or the nucleotide sequence of SEQ IDNO:22, thus thus one or more amino acid are replaced, added or disappearance imports in the coded protein and produces.Can pass through standard technique, will suddenly change as site-directed mutagenesis and PCR-mediated mutagenesis imports SEQ ID NO:1, SEQ ID NO:5, and SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, or in one of the sequence of SEQ ID NO:22.Preferably, at a place or the non-essential amino acid residue place of many places predictions carry out conserved amino acid and replace." the conserved amino acid replacement " for amino-acid residue is replaced with the amino-acid residue with similar side chain.Amino-acid residue class with similar side chain defined in this area.These classes comprise have basic side chain amino acid (for example, Methionin, arginine, Histidine), amino acid with acid side-chain (for example, aspartic acid, L-glutamic acid), amino acid with uncharged polar side chain (for example, glycine, l-asparagine, glutamine, Serine, Threonine, tyrosine, halfcystine), amino acid with non-polar sidechain (for example, L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane), amino acid with β-branched building block (for example, Threonine, Xie Ansuan, Isoleucine) and have amino acid (for example, the tyrosine of aromatic side chain, phenylalanine, tryptophane, Histidine).Therefore, the non-essential amino acid residue of predicting in MR albumen is preferably used from another amino-acid residue of the same side chain class and is replaced.Perhaps, in another embodiment, can import sudden change at random along all or part of MR encoding sequence, and, keep the active mutant of MR to identify to the MR activity described in the gained screening mutant literary composition by for example saturation mutagenesis.To SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or after one of a SEQ ID NO:22 sequence carry out mutagenesis, can recombinant expressed proteins encoded, and activity of proteins is determined in use-case test (referring to the embodiment 6 of embodiment part) as described herein.

The present invention also provides the chimeric or fusion rotein of MR.As used herein, MR " chimeric protein " or " fusion rotein " comprise the MR polypeptide that is operably connected with non-MR polypeptide." MR polypeptide " refers to have the polypeptide with the corresponding aminoacid sequence of MR albumen." non-MR polypeptide " refers to have the polypeptide of following aminoacid sequence, and this sequence comes from the proteic protein of MR corresponding to being different in essence, and for example, described protein can be different with MR protein and derive from the protein of identical or different organism.In fusion rotein, term " is operably connected " and means MR polypeptide and non-MR polypeptide and merge to meet the form of reading frame mutually.Non-MR polypeptide can merge with the N-end or the C-end of MR polypeptide.For example, in one embodiment, fusion rotein is the GST-MR fusion rotein, and wherein the MR sequence merges the C-end to the GST sequence.This type of fusion rotein can be beneficial to the proteic purifying of reorganization MR.In another embodiment, fusion rotein is for containing the MR albumen of allos signal sequence at its N-end.In some host cell (for example, mammalian host cell), can increase proteic expression of MR and/or secretion by using the allos signal sequence.

Preferably, the chimeric or fusion rotein of MR of the present invention is by the recombinant DNA technology preparation of standard.For example, the dna fragmentation of different peptide sequences of encoding can link together to meet the mode of reading frame according to routine techniques, described technology is for for example, uses blunt ends or sticking terminal (being used for connecting), Restriction Enzyme digestion produces suitable end, fill and lead up cohesive end suitably the time, alkaline phosphatase treatment connects to avoid unwanted joint and enzymatic.In another embodiment, fusion gene can be synthetic by routine techniques, comprises automatic dna synthesizer.Alternatively, can use the pcr amplification that anchor primer carries out gene fragment, cause between two successive gene fragments, producing complementary overhang, can anneal subsequently and increase again to produce chimeric gene sequence (referring to for example, up-to-date molecular biology method (Current Protocolsin Molecular Biology), people such as Ausubel edit, John Wiley ﹠amp; Sons:1992).In addition, the expression vector sold of many merchants has been encoded and has been merged partly (as gst polypeptide).Can with the nucleic acid clone of coding MR in this type of expression vector, be connected as a single entity to meet the form of reading frame with MR albumen so that merge part.

The proteic homologue of MR can produce by mutagenesis, for example proteinic difference sudden change of MR or brachymemma.As used herein, term " homologue " be meant the proteic variant form of MR, it can be as the agonist or the antagonist of MR protein-active.The proteic agonist of MR can be held and the essentially identical biological activity of MR albumen or its part biological activity.The proteic antagonist of MR is passable, for example by competitive downstream or upstream member in conjunction with the MR adjusting cascade that comprises MR albumen, suppresses proteic one or more activity of MR of natural form.Therefore, corynebacterium glutamicum MR albumen of the present invention and homologue thereof can be adjusted the activity of one or more pathways metabolisms that MR albumen is regulated in this microorganism.

In an alternative embodiment, the proteic homologue of MR can be by identifying that at MR agonist or the proteic mutant combinatorial library of antagonistic activity screening MR for example, described library can be the truncated mutant library.In one embodiment, by on nucleic acid level, carrying out combinatorial mutagenesis, produce diversified MR variant library by diversified gene library coding.For example, variation MR variant library can prepare in the following way: synthetic oligonucleotide mixture enzymatic is connected to gene order so that the potential MR sequence of one group of degeneracy can be expressed as independent polypeptide, or alternatively, be expressed as wherein contain this group MR sequence one group than larger fusion protein (for example, being used for phage display).There is several different methods to can be used for producing potential MR homologue library from the oligonucleotide sequence of degeneracy.Can on automatic dna synthesizer, carry out the chemosynthesis of degeneracy gene order, then synthetic gene is connected into suitable expression vector.The application of one group of degeneracy gene makes can provide all sequences of one group of required potential MR sequence of coding in a mixture.Be used for that the method for synthetic degenerate oligonucleotide is known in the art (referring to for example, Narang, S.A (1983) Tetrahedron 39:3; People such as Itakura (1984) Annu.Rev.Biochem.53:323; People such as Itakura (1984) science (Science) 198:1056; People such as Ike (1983) nucleic acids research (Nucleic Acid Res) 11:477).

In addition, the segmental library of MR albumen coded sequence can be used for producing diversified MR segment group, reaches with screening and picks out the proteic homologue of MR subsequently.In one embodiment, can be by handling the double-stranded PCR fragment of MR encoding sequence (under the condition that described processing is carried out with nuclease, approximately once cutting only takes place in each molecule), with the double-stranded DNA sex change, make the DNA renaturation form double-stranded DNA (can comprise in this double-stranded DNA from different cleaved products have justice/antisense to), with the processing of S1 nuclease from the duplex of new formation, to remove the strand part, then the fragment library that produces is connected into expression vector, produces the segmental library of encoding sequence thus.Can form expression library with this method, N-end, C-end and the interior segments of the different sizes of described library coding MR albumen.

Be used to screen the gene product of the combinatorial library that produces by point mutation or brachymemma and be used to screen that the cDNA library is known in the art with some technology of the gene product that obtains to have selected character.These technology can be suitable for the gene library of rapid screening by the combinatorial mutagenesis generation of MR homologue through modification.The high throughput analysis that is suitable for of widespread use generally comprises with the technology of screening large-scale gene library gene library is cloned in the reproducible expression vector the most, transform suitable cell and express combination gene with obtaining vector library, under this expression condition, will be beneficial to the code carrier of the gene product that separation detection arrives required active test.The overall mutagenesis of recurrence (Recursive ensemblemutagenesis, REM) for increasing a kind of new technology of functional mutants frequency in the library, can with the shaker test combined utilization with identification of M R homologue (Arkin and Yourvan (1992) PNAS89:7811-7815; People such as Delgrave (1993) protein engineering (Protein Engineering) 6 (3): 327-331).

In another embodiment, can utilize based on the test of cell and analyze diversified MR library, method therefor is well known.

Except the proteic nucleic acid molecule of above-mentioned MR of encoding, another aspect of the present invention relates to the isolated nucleic acid molecule with these molecule antisenses." antisense " nucleic acid comprises such nucleotide sequence, and " justice is arranged " nucleic acid that it is complementary to coded protein for example is complementary to the coding strand of double chain DNA molecule or is complementary to the mRNA sequence.Therefore, antisense nucleic acid can and have the phosphorothioate odn bonding by hydrogen bond.Antisense nucleic acid can be complementary to the MR coding strand of total length or only be complementary to its part.In one embodiment, the antisense nucleic acid molecule antisense is in " coding region " of the coding strand of the proteic nucleotide sequence of coding MR.Term " coding region " is meant the nucleotides sequence column region that comprises the codon that can translate into amino-acid residue.In another embodiment, the antisense nucleic acid molecule antisense is in " non-coding region " of the coding strand of the nucleotide sequence of coding MR.Term " non-coding region " is meant and is not translated into amino acid whose 5 ' and the 3 ' sequence (that is, being also referred to as 5 ' and 3 ' non-translational region) that is positioned at the coding region flank.

Based on the coding strand sequence of coding disclosed herein MR molecule (for example, SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, sequence shown in SEQ ID NO:20 or the SEQID NO:22), can be according to Watson and Crick base pairing principle design antisense nucleic acid of the present invention.Antisense nucleic acid molecule can be complementary to the full length coding region of MR mRNA, but more preferably only antisense in the oligonucleotide of part MR mRNA coding region or non-coding region.For example, antisense oligonucleotide can be complementary to MR mRNA translation initiation site zone on every side.Antisense oligonucleotide can for example be about 5,10,15,20,25,30,35,40,45 or 50 Nucleotide.Antisense nucleic acid of the present invention can use synthetic the company with enzyme of method applied chemistry well known in the art to react structure.For example, the Nucleotide of available naturally occurring Nucleotide of antisense nucleic acid (as antisense oligonucleotide) or various modifications chemistry is synthetic, and the Nucleotide of wherein said modification is designed to increase the biologically stable of molecule or is used to the physical stability that increases antisense and the duplex that forms between the phosphorothioate odn is arranged.Alternatively and preferably, antisense nucleic acid can be used expression vector and carry out the biology preparation, in the described carrier antisense to subclone one section nucleic acid (that is, transcribing the RNA that inserts nucleic acid since then will be antisense orientation for the purpose target nucleic acid, and this will further describe in the subdivision below).

Another aspect of the present invention relates to the carrier that comprises at least one transgene expression cassette of the present invention, and expression vector preferably, this transgene expression cassette comprise the nucleic acid of coding MR protein (or its part).

As used herein, term " carrier " be meant such nucleic acid molecule, connected another nucleic acid of its Transshipment Permitted.One type carrier is " plasmid ", be meant the circular double stranded DNA ring that wherein can connect extra dna fragmentation.The carrier of another kind of type is a virus vector, wherein extra dna fragmentation can be connected in the viral genome.Some carrier can be in the host cell that they imported self-replicating (bacteria carrier that for example, has the bacterium replication orgin).Other carrier is integrated in the genome of host cell after importing host cell, and therefore along with host genome is duplicated together.And some carrier can instruct the expression of gene that is connected with their operability.Examples of such carriers is called herein " expression vector ".Generally, the expression vector that is used for recombinant DNA technology is generally the plasmid form.In this manual, " plasmid " and " carrier " be used interchangeably, because plasmid is the carrier format of frequent use.But the present invention is intended to comprise the expression vector of other form, and as phage vector, they play the effect that is equal to.

Transgene expression vector of the present invention comprises transgene expression cassette of the present invention with the form that is suitable for expression MR nucleic acid in host cell.Transgene expression cassette comprises one or more adjusting sequences (promotor), and the selection of regulating sequence is based on that the host cell that is used to express carries out, and they operationally are connected with nucleotide sequence to be expressed.In transgene expression cassette; " be operably connected " be meant that purpose nucleotide sequence and adjusting sequence connect in the mode that allows this nucleotide sequence to express (for example; in in-vitro transcription/translation system, or after carrier imports host cell expressing) in host cell.

Term " adjusting sequence " is intended to comprise promotor, enhanser and other expression controlling elements (for example, polyadenylation signal).For example, at Goeddel; Gene Expression Technology:Methods in En-zymology 185, Academic Press, San Diego, CA (1990) and at Vasicova P.Patek M.Nesvera J.Sahm H.Eikmanns B.Journal ofBacteriology (1999) 181 (19): 6188-91, at (1996) Microbiology142:1297-309 such as Patek M. with at Mateos etc. among (1994) Journal of Bacteriology 176:7362-71 this adjusting sequence has been described.Regulate sequence and be included in the adjusting sequence of guiding nucleus nucleotide sequence constitutive expression in many type host cells and adjusting sequence that only the guiding nucleus nucleotide sequence is expressed in some host cells.The preferred sequence of regulating is a promotor for example, such as cos-, and tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacI ^q-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, arny, SP02, λ-P _R-or λ-P _L, preferably in bacterium, use them.Also can use artificial promotor.The design that those skilled in the art will appreciate that expression vector can be depended on such as factors such as the selection of waiting to want transformed host cells, desirable protein matter expression levels.Expression vector of the present invention can be incorporated in the host cell,, comprise fusion rotein or peptide (for example, MR protein, the proteinic mutant forms of MR, fusion rotein etc.) with protein or the peptide that produces nucleic acid encoding described here.

Can design transgene expression cassette of the present invention or expression vector in prokaryotic organism or eukaryotic cells, to express MR protein.For example, can be at bacterial cell such as corynebacterium glutamicum, insect cell (utilizing rhabdovirus expression vector), yeast and other fungal cell, algae and metaphyte cell are perhaps expressed the MR gene in the mammalian cell.At Goeddel, GeneExpression Technology:Methods in Enzymology 185, Academic Press, SanDiego has further discussed the host cell that is fit among the CA (1990).As alternative scheme, for example, utilize the T7 promotor to regulate sequence and T7 polysaccharase, can in-vitro transcription and translation transgene expression cassette or expression vector.

Marking protein the most usually carries out with the carrier that comprises composing type or inducible promoter in prokaryotic organism, and described promotor guiding is merged or non-fused protein is expressed.

Fusion vector is to encoded protein matter wherein, and the N-terminal to recombinant protein adds a plurality of amino acid usually.Common fusion expression vector can merge glutathione-S-transferase (GST), maltose E conjugated protein or a-protein and target recombinant protein.

The example of suitable induction type intestinal bacteria (E.coli) expression vector comprises pTrc (Amann etc., (1988) Gene 69:301-315) pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHSI, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, λ gt11, pBdCl and pET 11d (Studier etc., Gene ExpressionTechnology:Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89; With editors such as Pouwels, (1985) Cloning Vectors.Elsevier:New York ISBN 0 444 904018).

At Eikmanns B.J., etc. other example of induction type intestinal bacteria corynebacterium glutamicum shuttle expression carrier can be found among (1991) Gene 102:93-8.The suitable cloning vector that uses in the corynebacterium glutamicum is Sinskey etc. for example, U.S. Patent number 4,649, disclosed cloning vector in 119, and disclose and (for example be used for corynebacterium glutamicum and relevant brevibacterium sp species, brevibacterium lactofermentum (B.lactofermentum)) Genetic Manipulative Technology (Yoshihama etc., J.Bacteriol.162:591-597 (1985); Katsumata etc., J.Bacteriol.159:306-311 (1984); With Santamaria etc., J.Gen.Microbiol.130:2237-2246 (1984)).

Expression of target gene from the pTrc carrier depends on host RNA polysaccharase transcribing from hybrid trp-lac promoter, fusion.Bacterium for other kind transforms, and can select the carrier that suits.For example, known plasmid pIJ101, pIJ364, pIJ702 and pIJ361 can be used for transforming streptomycete, and plasmid pUB110, pC194 or pBD214 are suitable for the conversion of genus bacillus species.Be used for transfer information and comprise pHM1519 to several plasmids of coryneform bacteria, pBL1, pSA77 or pAJ667 (Pouwels etc., editor. (1985) Cloning Vectors.Elsevier:New YorkIBSN 0 444 904018).The strategy that maximize recombinant protein matter is expressed is marking protein (Gottesman in the impaired host bacteria of the ability of enzymolysis cutting recombinant protein, S., GeneExpression Technology:Methods in Enzymology 185, Academic Press, SanDiego, California (1990) 119-128).Another strategy is to change the nucleotide sequence that will be inserted into the nucleic acid in the expression vector, making each amino acid whose each codon of coding all is the selected bacterium that is used to express, the codon that uses such as preference in the corynebacterium glutamicum (Wada etc. (1992) Nucleic Acids Res.20:2111-2118).Can carry out this change of nucleotide sequence of the present invention by the standard DNA synthetic technology.

Another aspect of the present invention relates to the genetically modified host cell of wherein having introduced transgene expression cassette of the present invention or expression vector.Here, can use term " host cell " and " genetically modified host cell " mutually with exchanging.Should be understood that this term not only refers to specific cell, also refer to the offspring or the potential offspring of this cell.Because sudden change or environmental influence some changes may occur in the generation subsequently, therefore in fact this offspring and parental cell may be inequality, but still are included in the scope of the invention.

Host cell can be any prokaryotic organism or eukaryotic cells.For example, can be at bacterial cell, such as corynebacterium glutamicum, insect cell is expressed MR protein in yeast or the mammalian cell.Other host cell that is fit to is that those of ordinary skills are known.The microorganism relevant with corynebacterium glutamicum has been shown in the table 1, and it can be used as the host cell of nucleic acid of the present invention and protein molecule easily.

Transform or rotaring dyeing technology by routine, carrier DNA can be incorporated in prokaryotic organism or the eukaryotic cells.Terminology used here " conversion " and " transfection " mean various well known in the art being used for and (for example introduce exogenous nucleic acid to host cell, linear DNA or RNA are (for example, be the carrier or the gene construct of line styleization, and carrier free)) or the nucleic acid of carrier format (for example, plasmid, phage, phagemid, phasmid, transposon or other DNA) technology, described technology comprises calcium phosphate or calcium chloride co-precipitation, the transfection of deae dextran mediation, fat transfection or electroporation.At Sambrook, Deng. (Molecular Cloning:A Laboratory Manual. second edition ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY, 1989) and can find in other lab guide to transform or the proper method of transfection host cell.

For microbial transformation, known expression vector and the transformation technology that depends on use has only the sub-fraction cell can mix the localized foreign DNA of additive type, perhaps by the process that relates to reorganization foreign DNA is incorporated in their genome.In order to identify and screen these transformant, the gene and the goal gene of the selected marker of will encoding usually (for example, to antibiotic resistance) are incorporated in the host cell together.Preferred selected marker comprises gives medicine, such as the selected marker of the resistance of kantlex, tsiklomitsin, bleomycin, paraxin, lincomycin.The nucleic acid of coding selected marker be directed into and is positioned in the host cell on the carrier identical with the proteinic carrier of coding MR, perhaps can be introduced on independent carrier.Can for example identify (for example, the cell that has mixed the selected marker will be survived, and other cell is with death) by the nucleic acid cell transformed of introducing by drug screening.

Can be at Tauch A.Puhler A.Kalinowski J.Thierbach G.Plasmid.44 (3): 285-91,2000, Kim HJ.Kim Y.Lee MS.Lee HS.Molecules ﹠amp; Cells.12 (1): 112-6,2001 Guerrero C.Mateos LM.Malumbres M.Martin JF.AppliedMicrobiology ﹠amp; Biotechnology.36 (6): 759-62,1992, Eikmanns BJ.Kleinertz E.Liebl W.Sahm H.Gene.102 (1): 93-8,1991, Yoshihama M.Higashiro K.Rao EA.Akedo M.Shanabruch WG.Follettie MT.WalkerGC.Sinskey AJ.Journal of Bacteriology.162 (2): 591-7,1985.Tauch A.Zheng Z.Puhler A.Kalinowski J.Plasmid.40 (2): 126-39,1998.CadenasRF.Martin JF.Gil JA.Gene.98 (1): 117-21 finds the example of the antibiotics resistance gene that can use in 1991 in corynebacterium glutamicum.

In another embodiment, can produce the transformed host biology (preferably microorganism) that contains selecting system, this system allows to regulate the expression of gene of introducing.For example, can allow only under the situation that IPTG exists, just to express the MR gene in comprising the MR gene under the lac operon control on the carrier.This regulation system is well known in the art, and at for example Eikmanns B.J. etc. among (1991) Gene 102:93-8 this regulation system has been described.

Can utilize the host cell of the present invention of cultivation, produce (that is, expressing) MR protein such as prokaryotic organism or Eukaryotic host cell.Therefore, the present invention further provides and utilize host cell of the present invention to produce the MR method of protein.In one embodiment, this method is included in cultivates host cell of the present invention (in cell in the suitable culture base, proteinic transgene expression cassette of coding MR or expression vector have been introduced, perhaps introduced the proteinic gene of MR of encoding wild type or change in the cellular genome), till producing MR protein.In another embodiment, this method further comprises from substratum or host cell separation MR protein.

The method of B. preventing the MR protein expression

There are a large amount of methods known to those skilled in the art to instruct the expression or the activity of how in organism, to induce the specific gene defective or how to prevent or to reduce described gene or corresponding gene product.In the preferred embodiment of the present invention, described method is applied to the biosynthetic down regulator of methionine(Met) (for example, RXA00655), has increased the biosynthesizing of methionine(Met) thus.

Described method can comprise one or more methods that are selected from following method.

A) rejecting of the gene of the described MR protein of coding (for example, negative regulator matter);

B) (for example, the negative regulator matter mutagenesis of) gene wherein can be induced described sudden change to the described MR protein of coding in the coding of described gene, non-coding or regulatory region;

C) expression of sense-rna, at least a portion complementation of the RNA of wherein said sense-rna and the described MR protein of coding (for example, negative regulator matter);

D) expression of dna binding protein dna, the genetic expression of its blocking-up or the minimizing described MR protein of coding (for example, negative regulator matter);

E) expression of the protein bound factor, the genetic expression of its blocking-up or the minimizing described MR protein of coding (for example, negative regulator matter);

F) expression of the dominant variant of described MR protein (for example, negative regulator matter);

With

G) stabilization removal of the mRNA of the described MR protein of coding (for example, negative regulator matter).

In preferred embodiment, (for example, by homologous recombination known in the art or other genetic method) destroys endogenous MR gene in the host cell, and its protein is no longer expressed.In another embodiment, use one or more point mutation, disappearance or inversion have changed the endogenous or MR gene introduced in the host cell.Described MR gene still may encoding function MR protein, but the non-functional MR protein or have the active MR protein of modifications (for example, increase or reduction) of also may encoding.In another embodiment, (for example, by disappearance, brachymemma, inversion or point mutation) changed one or more regulatory regions of MR gene in the microorganism (for example, promotor, repressor or inductor), to regulate the MR expression of gene.Those of ordinary skill in the art will understand and utilize method of the present invention easily to produce to contain the host cell of more than one described MR genes and protein modification, and comprise this host cell among the present invention.

In order to produce the homologous recombination microorganism, preparation contains the carrier of at least a portion MR gene, has wherein introduced disappearance, interpolation or displacement in this MR Gene Partial, so that Change Example is as, functional destruction MR gene.Preferably, this MR gene is a corynebacterium glutamicum MR gene, but it also can be to derive from Related Bacteria, perhaps even derive from the homologue in Mammals, yeast or insect source.In preferred embodiment, homologous recombination like this in case take place, with regard to the endogenous MR gene of functional destruction (that is encoding function protein no longer, in design vector; Also be called " rejecting " carrier).As alternative scheme, can design vector, so that in case homologous recombination takes place, just suddenly change or change endogenous MR gene (for example, can change the upstream regulation district) to change endogenous MR protein expression.In homologous recombination vector, the MR Gene Partial of change has other nucleic acid of MR gene at its 5 ' and 3 ' flank, to allow between the endogenous MR gene of endogenous MR gene that carrier has and microorganism homologous recombination taking place.This extra flank MR nucleic acid has for carrying out successfully enough length for the homologous recombination with native gene.Normally, (at 5 ' and 3 ' end) flanking DNA should have 100 base pairs to the length between several thousand base pairs, and be included in the carrier (referring to, Thomas for example, K.R., and Capecchi, M.R. (1987) Cell 51:503 is about the description of homologous recombination vector).In addition, the method that can utilize other screening mode to make up the karyomit(e) reorganization also is known, wherein recover selected marker by the method for positive-selecting, Schafer A.Tauch A.Jager W.Kalinowski J.Thierbach G.Puhler A.Gene.145 (1): 69-73,1994.(for example, passing through electroporation) be incorporated into carrier in the microorganism and utilize MR gene that technology screening known in the art wherein introduces with the cell of endogenous MR dna homolog reorganization.

By expressing the MR protein expression that orientation resists the sense-rna of the mRNA of coding MR to obtain to reduce.Described sense-rna has been described above.The method of utilizing the inverted defined gene regulatory gene to express be well known to those skilled in the art (referring to, Weintraub for example, H. etc., AntisenseRNA as a molecular tool for genetic analysis, Reviews Trends in Genetics, 1 (1) volume 1986).Usually use or produce antisense nucleic acid molecule of the present invention in position to cell, make their hybridization or, for example to transcribe and/or to translate, the expression of arrestin matter by suppressing in conjunction with coding proteinic cell mRNA of MR and/or genomic dna.Hybridization can be by conventional Nucleotide complementarity to form stable duplex, perhaps for example under the situation in conjunction with the antisense nucleic acid molecule of dna double spiral, hybridization is that the specificity in the duplex major groove interacts.Can modify antisense molecule, for example by with antisense nucleic acid molecule with is connected the acceptor or the antigen of expressing on the cell surface that the combination of its specificity is selected in conjunction with cell surface receptor or antigenic peptide or antibody.Also can utilize carrier described here, send antisense nucleic acid molecule to cell.In order to obtain antisense molecule concentration in enough cells, preferred wherein antisense nucleic acid molecule places the vector construction body under strong prokaryotic organism, virus or the promoter in eukaryote control.

In another embodiment, antisense nucleic acid molecule of the present invention is α-end group isomery nucleic acid molecule (α-anomeric nucleic acid molecule).α-end group isomery nucleic acid molecule and complementary RNA form special double-stranded heterozygote, and β-unit is opposite in this heterozygote with usually, that this two strands is moved towards is parallel to each other (Gaultier etc. (1987) Nucleic Acids.Res.15:6625-6641).Antisense nucleic acid molecule also can comprise 2 '-O-methyl ribonucleotides (Inoue etc. (1987) Nucleic Acids Res.15:6131-6148) or chimeric RNA-DNA analogue (Inoue etc. (1987) FEBS Lett.215:327-330).

In another embodiment, antisense nucleic acid of the present invention is a ribozyme.Ribozyme is the catalytic RNA molecule with ribonuclease activity, and it can cut the single-chain nucleic acid that has complementary district with it, such as mRNA.Therefore, ribozyme (for example, hammerhead ribozyme (described in Haselhoff and Gerlach (1988) Nature334:585-591)) can be used for catalyze cleavage MR mRNA transcript, to suppress the translation of MR mRNA.The nucleic acid that can design coding MR according to the nucleotide sequence (that is, SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20 or SEQ ID NO:22) of MR DNA disclosed herein has specific ribozyme.For example, can make up thermophilas (Tetrahymena) L-19 IVS RNA derivative, wherein nucleotide sequence complementation to be cut among the mRNA of the nucleotide sequence of avtive spot and coding MR.Referring to, U.S. Patent number .4 such as Cech for example, 987,071 and Cech etc. U.S. Patent number .5,116,742.As alternative scheme, the catalytic RNA that can utilize MR mRNA to have the specific ribonucleic acid enzymic activity from the screening of RNA library of molecules.Referring to, Bartel for example, D. and Szostak, J.W. (1993) Science 261:1411-1418.

As alternative scheme, can utilize with the regulatory region of MR nucleotide sequence (for example, MR promotor and/or enhanser) the complementary nucleotide sequence, by making this nucleotide sequence target target form stoping the triple helix structure of MR genetic transcription in the target cell, thereby suppress MR genetic expression.Usually referring to Helene, C. (1991) Anticancer Drug Des.6 (6): 569-84; Helene, C. etc. (1992) Ann.N.Y.Acad.Sci.660:27-36; And Maher, L.J. (1992) Bioassays 14 (12): 807-15.

In another embodiment, other or alternative method well known by persons skilled in the art can be used for regulating, for example prevent or (for example increase gene, the gene of coding methionine(Met) biosynthesizing down regulator) expression, perhaps being used to regulate for example increases or prevents or inhibitory phase is answered the activity of gene product.

Operable a kind of method is that the dominant variant of expressing gene product (for example, the gene of coding methionine(Met) biosynthesizing down regulator) is to prevent for example activity of methionine(Met) biosynthesizing down regulator.

Preferably, and methionine(Met) biosynthesizing down regulator (for example, as SEQ ID NO:1, the RXA00655 of 18,20 or 22 examples) be transcriptional.Known transcriptional exists as dimer, and it is in conjunction with the relative part that may contain the dna structure of tumor-necrosis factor glycoproteins with so-called double symmetry.Can in the aminoacid sequence of transcriptional, determine dna binding activity.In Schumacher M.A. and Brennan R.G. (2002) Molecular Microbiology.45:885-93, can find the example of this transcriptional dimer bonded architecture basics.For example, by the heterodimer protein that the not isoallele of expressing same protein in a cell is made up of the proteinic equipotential form of not suddenling change and suddenly change of adjusting with generation, can give this organism dominant phenotype.At Journal of Molecular Biology (2002) 322 (2): 311-24, with at Journal ofBiological Chemistry (1994) .269 (11): can be found use in the method that makes up mutant dominant aporepressor in the organism among the 8246-54, wherein this organism is also expressed the not allelotrope of sudden change of homologous genes.For example, DNA binding domains that can the described methionine(Met) biosynthesizing of inactivation down regulator.In another embodiment, mRNA is stable can be used as the method for life that increases or reduce given mRNA molecule with destabilization.At Smolke C.D. and Keasling J.D. (2002) Biotechnology ﹠amp; Bioengineering.78 (4): 412-24, at Smolke C.D. etc. (2001) Metabolic Engineering 3 (4): 313-21 and at Carrier T.A.and KeaslingJ.D. (1999) Biotechnology Progress 15 (1): can find to influence given mRNA method of life among the 58-64.The another kind of method that can be used for the regulatory gene expression relates to the expression of dna binding protein dna, and this dna binding protein dna increase or blocking-up or minimizing are from gene, and proteinic expression of gene is regulated in the methionine(Met) biosynthesizing of for example encoding.

By utilizing the specific DNA conjugated protein, for example gene can be blocked or reduce to zinc finger protein matter class transcription factor, the expression of gene of the methionine(Met) biosynthesizing negative regulator matter of for example encoding.The described factor can be oriented to the regulatory region of gene for example to be prevented.The application of the described factor allows to prevent expression, and does not change corresponding gene order.The artificial DNA binding factor that makes up energy binding specificity target sequence is a method known to those skilled in the art.By utilizing above-mentioned artificial DNA binding factor, they and transcriptional activation domain are merged, produce the manual transcription initiation factor thus, thereby can increase genetic expression, the genetic expression of the methionine(Met) biosynthesizing positive regulator of for example encoding.(Dreier B etc. (2001) J Biol Chem 276 (31): 29466-78; Dreier B etc. (2000) JMol Biol 303 (4): 489-502; Beerli RR etc. (2000) Proc Natl Acad Sci USA 97 (4): 1495-1500; Beerli RR etc. (2000) J Biol Chem 275 (42): 32617-32627; Segal DJ and Barbas CF 3rd. (2000) Curr Opin Chem Biol 4 (1): 34-39; KangJS and Kim JS (2000) J Biol Chem 275 (12): 8742-8748; Beerli RR etc. (1998) Proc Natl Acad Sci USA 95 (25): 14628-14633; Kim JS etc. (1997) Proc NatlAcad Sci USA 94 (8): 3616-3620; Klug A (1999) J Mol Biol 293 (2): 215-218; Tsai SY etc. (1998) Adv Drug Deliv Rev 30 (1-3): 23-31; Mapp AK etc. (2000) Proc Natl Acad Sci USA 97 (8): 3930-3935; Sharrocks AD etc. (1997) Int JBiochem Cell Biol 29 (12): 1371-1387; Zhang L etc. (2000) J Biol Chem 275 (43): 33850-33860).By utilizing, for example the lower molecular weight synthetic compound of the customization of polymeric amide type also can be realized (the Dervan PB and B ü rli RW (1999) the CurrentOpinion in Chemical Biology 3:688-693 of preventing of genetic expression; Gottesfeld JM etc. (2000) GeneExpr 9 (1-2): 77-91).For any specific DNA target sequence, can on reasonable basis, adopt these compounds to express, if perhaps this compound and transcriptional activation domain fusion use then allows initial gene to express to allow suppressor gene.Make up can the binding specificity target sequence the artificial DNA binding factor be method known to those skilled in the art (Bremer RE etc. (2001) Bioorg MedChem.9 (8): 2093-103; Ansari A.Z etc. (2001) Chem Biol.8 (6): 583-92; Gottesfeld J.M. etc. (2001) J Mol Biol.309 (3): 615-29; Wurtz N.R. etc. (2001) Org Lett 3 (8): 1201-3; Wang C.C. etc. (2001) Bioorg Med Chem 9 (3): 653-7; Urbach A.R. and Dervan P.B. (2001) Proc Natl Acad Sci USA 98 (8): 4343-8; Chiang S.Y. etc. (2000) J Biol Chem.275 (32): 24246-54).In another embodiment, can utilize the expression of the protein bound factor, proteinic expression of gene or activity are regulated in this protein bound factor activator or blocking-up or the biosynthesizing of minimizing coding methionine(Met).

Be suitable in conjunction with methionine(Met) biosynthesizing instrumentality, and therefore prevent the active protein bound factor can based on RNA such as, aptamer (Famulok M and Mayer G (1999) CurrTop Microbiol Immunol 243:123-36), antibody, antibody fragment or single-chain antibody.The structure of these protein bound factors and the method for utilization are well known by persons skilled in the art.

C. purposes of the present invention and method

Nucleic acid molecule described in the literary composition, protein, protein homologs, fusion rotein, primer, transgene expression cassette, expression vector and host cell can be used in one or more following methods: the cell yield of regulating desired compounds such as fine chemicals such as methionine(Met); Identify corynebacterium glutamicum and related organisms; Draw the Genome Atlas of the organism relevant with corynebacterium glutamicum; Identify and localizing objects corynebacterium glutamicum sequence; Study on Evolution; Determine to be in the MR albumen the required zone of function; Regulate the MR protein-active; And the activity of regulating one or more pathways metabolisms.

MR nucleic acid molecule of the present invention has various uses.To the operation of MR nucleic acid molecule of the present invention, for example prevent or increase the activity of expression of nucleic acid or protein molecule, can cause the biosynthetic adjusting of methionine(Met).For example, aspect the production of other compound of methionine(Met) or methionine(Met) biosynthetic pathway, overexpression RXN02910 or have the production that corynebacterium glutamicum strain system that the RXN02910 of increase expresses has shown remarkable increase, wherein this RXN02910 is the biosynthetic positive regulator of methionine(Met).And, aspect the production of other compound of methionine(Met) or methionine(Met) biosynthetic pathway, the corynebacterium glutamicum strain system that the RXN00655 that RXN00655 defective or have is prevented expresses has also shown the production of remarkable increase, and wherein this RXN00655 is the biosynthetic down regulator of methionine(Met).

In addition, MR nucleic acid molecule of the present invention is operated to be caused producing the MR albumen different with wild-type MR protein function.These protein can improve on effect or activity, can exist with many usually amounts in cell, or can reduce on effect or activity.

The method that the present invention also provides screening to regulate the molecule of MR protein-active, described molecule can interact with MR albumen itself or interact with proteic substrate of MR or bound fraction, perhaps regulates transcribing or translating of MR nucleic acid molecule of the present invention.In these class methods, will express the proteic microorganism of the one or more MR of the present invention and contact, and assess of the influence of each test-compound proteic activity of MR or expression level with one or more test-compounds.

This active change can directly be regulated by corynebacterium glutamicum and produce one or more fine chemicals, for example the output of methionine(Met), productive rate and/or production efficiency.For example, the proteinic gene of biosynthesizing for coding expectation fine chemicals, by optimizing the MR protein that activates this genetic transcription or translation, methionine(Met) biosynthesizing positive regulator for example, the activity of RXN02910 for example is perhaps by infringement or abolish the MR protein that checks this gene transcription or translation, for example methionine(Met) biosynthesizing down regulator, the activity of RXA00655 for example also can increase the activity or the activity rate of this biosynthetic pathway.Similarly, make its composition ground translate related protein in the fine chemicals degradation pathway of back inactivation expectation by changing the MR protein active, perhaps check this gene transcription or translation by changing the MR protein active with making its composition, because the degraded that this fine chemicals compound reduces, thereby can increase the productive rate and/or the throughput rate of cells produce fine chemicals.

And, because the interdependence of different metabolic approach by regulating one or more MR activity of proteins, can stimulate or improve the production or the productivity of one or more fine chemicals of cell indirectly.For example, for increasing output, productive rate and/or production efficiency by the expression that activates one or more Methionin biosynthetic enzymes, can increase other compound simultaneously, such as other amino acid whose expression, wherein at cell the demand of Methionin is increased also naturally can be required with bigger demand by cell when big for these other compounds.And the metabolism adjusting that can change cell integral body makes cell can grow better or duplicate down in the envrionment conditions (wherein the supply of possibility nutrition and oxygen is bad, and high-caliber Toxic waste is arranged in the possible environment) of fermentation culture.For example, the MR protein of---cytolemma produce required molecule---synthetic (in order to block the cell growth the suboptimum growth conditions under and to divide) of checking molecule to replying high-level refuse in the substratum of extracellular, carry out mutagenesis and make it no longer can check this synthesizing, can even under the suboptimum growth conditions, increase the growth and the propagation of cell in the culture.Because have the cell of the generation expectation fine chemicals of relative greater amt in the culture, the growth of this increase or viability also should increase productive rate and/or the productivity that fermenting culture is produced this expectation fine chemicals.

The aforementioned mutagenesis MR protein that is used for is not intended to restrictive with the mutagenesis strategy that causes being produced the gain in yield of fine chemicals (for example methionine(Met)) by corynebacterium glutamicum; The change that can carry out these strategies is that those skilled in the art are conspicuous.Utilize these strategies, and in conjunction with mechanism disclosed herein, nucleic acid of the present invention can be used to produce MR nucleic acid and the corynebacterium glutamicum of protein molecule or the relevant bacterial strain system of expressing sudden change with protein molecule, to improve the productive rate and/or the production efficiency of expectation compound.This expectation compound can be any natural product of corynebacterium glutamicum, comprise the end product of biosynthetic pathway and the intermediate of natural pathways metabolism, and not natural appearance in the corynebacterium glutamicum metabolism, be the molecule that produces still by corynebacterium glutamicum strain of the present invention.

MR molecule of the present invention also can be used to identify corynebacterium glutamicum organism or its close relative.And they can be used for Identifying micro-organisms population mixture corynebacterium glutamicum or its close relative's existence.The invention provides the nucleotide sequence of a large amount of corynebacterium glutamicum genes; Utilize the probe of crossing over the zone of this organism uniqueness in the corynebacterium glutamicum gene,, can determine whether this organism exists by under stringent condition, surveying the genomic dna that in the single of microorganism or population mixture culture, extracts.

Nucleic acid of the present invention and protein molecule also can be as the marks in genome specificity zone.This is not only useful in genomic mapping, and also can be used for the proteinic functional study of corynebacterium glutamicum.For example,, can digest the corynebacterium glutamicum genome in order to identify specific corynebacterium glutamicum dna binding protein dna bonded genome district, and resulting fragment of incubation and dna binding protein dna.Use nucleic acid molecule of the present invention, the nucleic acid molecule that preferably has easy certification mark can additionally be surveyed the fragment of conjugated protein; The bound energy of this nucleic acid molecule and genomic fragment is positioned fragment in the Genome Atlas of corynebacterium glutamicum, and when carrying out repeatedly with different enzymes, helps determining fast the nucleotide sequence of protein bound.And, nucleic acid molecule of the present invention may have enough homologys with the sequence of relevant species, these nucleic acid molecule can be as Related Bacteria, such as the mark of Genome Atlas structure in the brevibacterium lactofermentum (Brevibacterium lactofermentum) like this.

By the further example of the following examples the present invention, these embodiment should not be interpreted as restrictive.Here with all reference of being quoted among the application, accompanying drawing, patent application, patent, disclosed patent application, table and sequence table are incorporated as reference hereby.

Embodiment

Embodiment 1: the preparation of the total genomic dna of corynebacterium glutamicum ATCC 13032

The culture of corynebacterium glutamicum (ATCC 13032) 30 ℃ in BHI substratum (Difco) violent jolting spend the night.Centrifugal cell harvesting is abandoned supernatant and cell is resuspended in 5ml damping fluid-I (volume of culture 5%-all demonstrations of volume originally calculates gained for pressing the 100ml culture volume).The composition of damping fluid-I: 140.34g/l sucrose, 2.46g/l MgSO ₄X7H ₂O, 10ml/l KH ₂PO ₄Solution (100g/l is adjusted to pH 6.7 with KOH), 50ml/l M12 enriched material (10g/l (NH ₄) ₂SO ₄, 1g/l NaCl, 2g/l MgSO ₄X7H ₂O, 0.2g/l CaCl ₂, 0.5g/l yeast extract (Difco), 10ml/l trace elements mixture (200mg/l FeSO ₄XH ₂O, 10mg/l ZnSO ₄X7 H ₂O, 3mg/lMnCl ₂X4 H ₂O, 30mg/lH ₃BO ₃, 20mg/lCoCl ₂X6 H ₂O, 1mg/l NiCl ₂X6H ₂O, 3mg/l Na ₂MoO ₄X2H ₂O, 500mg/l complexing agent (EDTA or citric acid), 100ml/l VITAMIN-mixture (0.2mg/l vitamin H, 0.2mg/l folic acid, the 20mg/l para-amino benzoic acid, 20mg/l riboflavin, 40mg/l calcium pantothenate, 140mg/l nicotinic acid, 40mg/l pyridoxin hydrochloride, 200mg/l inositol).In suspension, add N,O-Diacetylmuramidase to final concentration 2.5mg/ml.37 ℃ hatch about 4 hours after, cell wall degradation, the protoplastis of centrifugal results gained.Precipitation is washed once with 5ml damping fluid-I and is washed once with 5ml TE-damping fluid (pH 8 for 10mM Tris-HCl, 1mM EDTA).Precipitation is resuspended in 4ml TE-damping fluid, and adds 0.5ml SDS solution (10%) and 0.5ml NaCl solution (5M).Add Proteinase K to final concentration 200 μ g/ml, suspension was hatched about 18 hours at 37 ℃.Use standard method phenol, phenol-chloroform-primary isoamyl alcohol and chloroform-isoamyl alcohol extracting and purify DNA.Then, add the 3 M sodium-acetates and 2 volume of ethanol of 1/50 volume, hatch 30 minutes in-20 ℃, and on supercentrifuge, use SS34 rotor (Sorvall) in 12,000 rev/mins centrifugal 30 minutes, deposit D NA.This DNA is dissolved in the 1ml TE-damping fluid that contains 20 μ g/ml RNaseA, and with 1000ml TE-damping fluid in 4 ℃ of dialysis at least 3 hours.During this period, exchange buffering liquid is 3 times.In the dna solution that the 0.4ml of packing dialysed, add 0.4ml 2M LiCl and 0.8ml ethanol.In-20 ℃ hatch 30 minutes after, centrifugal (13,000 rev/mins, Biofuge Fresco, Heraeus, Hanau, Germany) collect DNA.This DNA precipitation is dissolved in the TE-damping fluid.DNA with this method preparation can be used for all purposes, comprises the southern trace or makes up genomic library.

Embodiment 2: the genomic library that makes up corynebacterium glutamicum ATCC13032 in intestinal bacteria

Use is as the DNA of preparation as described in the embodiment 1, according to known and the method set up (referring to for example, Sambrook, J. wait people (1989) " molecular cloning: laboratory manual ", Cold Spring HarborLaboratory Press, or Ausubel, people such as F.M. (1994) " molecular biology fresh approach ", John Wiley ﹠amp; Sons.) make up clay and plasmid library.

Can use any plasmid or clay.Operable especially is plasmid pBR322 (Sutcliffe, newspaper (Proc.Natl.Acad.Sci.USA) 75:3737-3741 of institute of J.G. (1979) NAS); PACYC177 (Change ﹠amp; Cohen (1978) bacteriology magazine (J.Bacteriol) 134:1141-1156), the plasmid of pBS series (pBSSK+, pBSSK-and other; Stratagene, LaJolla, USA) or clay such as SuperCos1 (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T.J., Rosenthal A. and Waterson, R.H. (1987) gene (Gene) 53:283-286.Specificity is used for the gene library of corynebacterium glutamicum and can uses plasmid pSL109 (Lee, H.-S. and A.J.Sinskey (1994) J.Microbiol.Biotechnol.4:256-263) to make up.

Embodiment 3:DNA sequencing and computer function analysis

Be used for determined dna sequence as embodiment 2 described genomic libraries according to standard method, especially use the ABI377 sequenator to measure (referring to for example by chain termination method, Fleischman, R.D. wait people (1995) " the full genome stochastic sequence of Haemophilus influenzae (Haemophilus Influenzae) Rd is measured and assembling ", science ( Science), 269:496-512).Use has the sequencing primer of following nucleotide sequence: 5 '-GGAAACAGTATGACCATG-3 ' (SEQ ID NO:18) or 5 '-GTAAAACGACGGCCAGT-3 ' (SEQ ID NO:19).

Embodiment 4: mutagenesis in vivo

The mutagenesis in vivo of corynebacterium glutamicum can be implemented by plasmid (or other carrier) DNA is gone down to posterity in the intestinal bacteria that can not keep its genetic information integrity or other microorganism (for example bacillus kind or yeast such as yeast saccharomyces cerevisiae).Typical mutator strain has sudden change (mutHLS for example, mutD, mutT etc. at the gene that is used for the DNA repair system; Reference is seen intestinal bacteria and Salmonellas (Escherichia coli and Salmonella), the Rupp in ASM:Washington one book, W.D. (1996) DNA repair mechanism, 2277-2294 page or leaf).This type of bacterial strain is known for those of ordinary skills.The purposes of this type of bacterial strain is at for example Greener, A. and Callahan, M. (1994) StrategiesSet forth among the 7:32-34.

Embodiment 5

The growth of corynebacterium glutamicum---substratum and culture condition

Coryneform bacteria is cultivated in synthetic or natural growth medium.Being used for the different growth mediums of coryneform many kinds knows for people and is easy to obtain (people (1989) Appl.Microbiol.Biotechnol. such as Lieb, 32:205-210; People (1998) Biotechnology Letters such as von der Osten, 11:11-16; DE patent 4,120,867; At prokaryotic organism (The Procaryotes), Volume II, Balows, people such as A. edit the Liebl (1992) " corynebacterium " (" The Genus Corynebacterium ") in .Springer-Verlag one book).These substratum are made up of one or more carbon sources, nitrogenous source, inorganic salt, VITAMIN and trace elements.Preferred carbon source is a carbohydrate, as monose, disaccharides or polysaccharide.For example, glucose, fructose, seminose, semi-lactosi, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or Mierocrystalline cellulose can be used as good carbon source.Also can provide sugar to substratum by complex chemical compound such as molasses or other byproduct that refines from sugar.The mixture that different carbon sources are provided also can be favourable.Other possible carbon source is alcohols and organic acid, as methyl alcohol, ethanol, acetic acid or lactic acid.Nitrogenous source is generally the organic or inorganic nitrogen compound, or for comprising the material of these compounds.Exemplary nitrogenous source comprises ammonia or amine salt such as NH ₄Cl or (NH ₄) ₂SO ₄, NH ₄OH, nitrate, urea, amino acid or compound nitrogen source such as corn steep liquor, soyflour, soy-protein, yeast extract, meat extract etc.

Inorganic salt compound in the substratum be can be included in and muriate, microcosmic salt or the vitriol of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron comprised.Can in substratum, add inner complex to keep the solution metal ion.The inner complex that is particularly useful comprises dihydric phenol, as catechol or Protocatechuic Acid salt, or organic acid such as citric acid.Substratum generally also comprises other somatomedin such as VITAMIN or growth stimulant, and the example comprises vitamin H, riboflavin, VitB1, folic acid, nicotinic acid, pantothenate and vitamin B6.Somatomedin and salt are usually from the complex medium composition, as yeast extract, molasses, corn steep liquor etc.The definite composition of substratum compound depends on direct experiment strongly and determines separately under each particular case.The information of relevant medium optimization is at textbook " Applied Microbiol.Physiology, can find among the A Practical Approach (P.M.Rhodes, P.F.Stanbury edits, IRLPress (1997) 53-73 page or leaf, ISBN 0 19 963,577 3).Also can select growth medium from suppliers, as standard 1 (Merck) or BHI (grain heart infusion, DIFCO) or other.

By heating (1.5 crust, 121 ℃ were heated 20 minutes) or filtration sterilization all substratum moietys are sterilized.Composition can be sterilized together, or separately sterilization of the words that need.All substratum compositions can exist when the growth beginning, or they can randomly add continuously or add in batches.

Adopted culture condition is advised in each experiment separately.Temperature should be between the scope of 15 ℃ and 45 ℃.Temperature can keep constant and maybe can change in experimentation.The pH of substratum should preferably near 7.0, can keep by add damping fluid in substratum between 5 to 8.5 scope.The exemplary damping fluid that is used for this purpose is a potassium phosphate buffer.Can be alternatively or use synthetic damping fluid such as MOPS, HEPES, ACES and other simultaneously.Also can be by adding NaOH or NH in process of growth ₄OH keeps constant and cultivates pH.If use complex medium composition such as yeast extract, can reduce needs, because many complex chemical compounds have high surge capability to extra damping fluid.If use the fermentor cultivation microorganism, also available gaseous ammonia control pH.

Incubation time is usually between several hours to several days.Select incubation time to allow the product of accumulation maximum in the meat soup.Can in titer plate, Glass tubing, glass flask or the glass of multiple container such as different size or metal fermentor tank, implement disclosed growth experiment.For a large amount of clone of screening, should or be with or without the culturing micro-organisms in the bottle of shaking of baffle plate at titer plate, Glass tubing.Preferably use 100ml to shake bottle, the required growth medium of 10% (volume) wherein is housed.This flask should be gone up at gyrate shaker (amplitude 25mm) and shake with 100-300 rev/min of speed.Can reduce vaporization losses by keeping wet environment; Alternatively, should carry out mathematical correction to vaporization losses.

If the clone of test genetic modification, the contrast clone of no any insertion of basic plasmid is cloned or contains in the contrast that also should test unmodified.Use in 30 ℃ of cell inoculation substratum of on agar plate, growing of hatching to OD ₆₀₀0.5-1.5, wherein said agar plate such as CM plate (10g/l glucose, 2.5g/lNaCl, 2g/l urea, 10g/l gathers peptone, the 5g/l yeast extract, the 5g/l meat extract, 22g/l NaCl, 2g/l urea, 10g/l gathers peptone, the 5g/l yeast extract, the 5g/l meat extract, 22g/l agar is adjusted to pH 6.8 with 2M NaOH).The inoculation of substratum can be by introducing the corynebacterium glutamicum cell that obtains from the CM plate the salt aqueous suspensions or finish by the pre-culture of the liquid that adds this bacterium.

Embodiment 6: the analyzed in vitro of mutein function

Can be by the activity of proteins of several methods mensuration of having set up, as DNA Tape movement test (being also referred to as gel retardation assay) in conjunction with DNA.But this proteinoid is measured (as at Kolmar, described in the reference that J.14:3895-3904 people such as H. (1995) EMBO reaches wherein to be quoted) to the influence operation report gene test of other developed by molecule.The reporter gene test macro is known by people, and is established being applied to protokaryon and eukaryotic cell, the enzyme of use such as beta-galactosidase enzymes, green fluorescent protein and several other enzyme.

Embodiment 7: analyze the influence that mutain confrontation expectation product produces

Genetic modification in the corynebacterium glutamicum can be assessed in the following way to the influence that expectation compound (as amino acid) produces: modified microorganism is grown down in appropriate condition (those conditions as mentioned), and analyze substratum and/or cellular constituent at the increase that expectation product (being amino acid) produces.This type of analytical technology is known for those of ordinary skills, comprise spectroscopy, tlc, multiple staining, zymetology and micro-biological process and analyze chromatography such as high performance liquid chromatography (referring to for example, Ullman industrial chemistry encyclopaedia (Ullman, Encyclopediaof Industrial Chemistry), the A2 volume, 89-90 and 443-613 page or leaf, VCH:Weinheim (1985); Fallon, " application of HPLC in biological chemistry " in people such as A. (1987) biological chemistry and the Molecular Biology Lab's technology (LaboratoryTechniques in Biochemistry and Molecular Biology), 17 volumes; People such as Rehm (1993) biotechnology (Biotechnology), 3 volumes, III chapter " product reclaims and purifying ", 469-714 page or leaf, VCH:Weinheim; Belter, people such as P.A. (1988) bioseparation: the downstream processing in the biotechnology, John Wiley and Sons; Kennedy, J.F. and Cabral, the recovery method John Wileyand Sons of J.M.S. (1992) biological substance; Shaeiwitz in " Ulmann industrial chemistry encyclopaedia " book, J.A. and Henry, J.D. (1988) biological chemistry is separated, B3 volume, 11 chapters, 1-27 page or leaf, VCH:Weinheim; And Dechow, separation in F.J. (1989) biotechnology and purification technique (Separation andpurification techniques in biotechnology), Noyes Publications.).

Except measuring the fermentation end product, also can analyze overall productive rate, output and or production efficiency that other composition of the pathways metabolism that is used to produce the expectation compound such as intermediate and by product are determined compound.Analytical procedure comprises nutrient substance level (for example, carbohydrate, carbohydrate, nitrogenous source, phosphoric acid salt and other ion), the mensuration biomass composition of measuring in the substratum and grows, analyzes the generation of common metabolic thing in the biosynthetic pathway and measure the gas that produces in the fermenting process.The standard method of these mensuration is at " using microbe physiology, practical approach " (Applied Microbial Physiology, A PracticalApproach), and P.M.Rhodes and P.F.Stanbury edit, IRL Press, 103-129; List in 131-163 and 165-192 page or leaf (ISBN:0199635773) and the reference quoted thereof.

Embodiment 8: purifying expectation product from the corynebacterium glutamicum culture

Can reclaim the expectation product from corynebacterium glutamicum cell or above-mentioned culture supernatant by multiple method well known in the art.If the expectation product is not from the cell secreting outside, but low-speed centrifugal harvested cell, usefulness standard technique such as mechanical force or ultrasonic degradation cell from culture.Centrifugal removal cell debris keeps the supernatant fraction that contains soluble protein and is used to be further purified the expectation compound.If product is from corynebacterium glutamicum emiocytosis, then low-speed centrifugal is removed cell from culture, and reservation supernatant fraction is used to be further purified.

To carry out chromatography with appropriate resin from the supernatant fraction of any purification process, and expect that wherein molecule will be retained on the chromatographic resin and the many impurity in the sample are not retained on the chromatographic resin, perhaps impurity is retained on the chromatographic resin and sample does not keep.This chromatographic step must the time can use identical or different chromatographic resin to repeat.Those of ordinary skills are selecting suitable chromatographic resin and they are used for being proficient in very much aspect the specific molecule to be purified most effectively.Can pass through to filter or the ultrafiltration and concentration purified product, and be stored under the temperature that makes the product stability maximum.

It is known in this field that a large amount of purification process is arranged, and above-mentioned purification process is nonrestrictive.This type of purification technique is for example at Bailey, J.E. and Ollis, D.F. biochemical engineering basis (BiochemicalEngineering Fundamentals), McGraw-Hill: describe in New York (1986).

The identity of separating compound and purity can be by the standard technique evaluations of this area.These technology comprise high performance liquid chromatography (HPLC), spectrography, staining, tlc, NIRS, zymetology test or micro-biological process.The summary of this type of analytical procedure is seen: people such as Patek (1994) Appl.Environ.Microbiol.60:133-140; People such as Malakhova (1996) biotechnology (Biotekhnologiya) 11:27-32; With people (1998) biological processing engineering (Bioprocess Engineer) 19:67-70 such as Schmidt; The Ulmann industrial chemistry encyclopaedia (Ulmann ' s Encyclopedia of Industrial Chemistry) (1996) A27 volume, VCH:Weinheim, 89-90 page or leaf, 521-540 page or leaf, 540-547 page or leaf, 559-566 page or leaf, 575-581 page or leaf and 581-587 page or leaf; Michal, G. (1999) bio-chemical pathway: biological chemistry and molecular biology atlas (BiochemicalPathways:An Atlas of Biochemistry and Molecular Biology), John Wileyand Sons; Fallon in " laboratory technique in biological chemistry and the molecular biology " 17 volumes, the application of people such as A. (1987) HPLC in biological chemistry.

Embodiment 9: analysis of gene sequences of the present invention

Comparative sequences and definite percentage homology are technology well known in the art between two sequences, can use mathematical algorithm to finish, as the algorithm of Karlin and newspaper (Proc.Natl.Acad.Sci.USA) 87:2264-68 of institute of Altschul (1990) NAS, Karlin and newspaper (Proc.Natl.Acad.Sci.USA) 90:5873-77 of institute of Altschul (1993) NAS are seen in the improvement of this algorithm.This algorithm has been impregnated among the NBLAST and XBLAST program (2.0 version) of people such as Altschul (1990) molecular biology magazine (J.Mol.Biol.215:403-10).Available NBLAST program is carried out the retrieval of BLAST Nucleotide, and the score value of setting=100, word length=12 are to obtain and MR nucleic acid molecule homologous nucleotide sequence of the present invention.Available XBLAST program is carried out the retrieval of BLAST protein, and the score value of setting=50, word length=3 are to obtain and MR protein molecular homologous aminoacid sequence of the present invention.In order to obtain to be used for the breach comparison of comparison purpose, can use people (1997) nucleic acids research (Nucleic Acids Res.) 25 (17) such as Altschul: the Gapped BLAST described in the 3389-3402.When using BLAST and Gapped blast program, those of ordinary skill in the art will recognize that the concrete sequence analyzed optimizer (for example XBLAST and NBLAST) parameter how.

Another example that is used for the mathematical algorithm of sequence comparison is the algorithm of Meyers and Miller ((1988) Comput.Appl.Biosci.4:11-17).This algorithm is impregnated in ALIGN program (2.0 version), and wherein said ALIGN program is the part of GCG sequence alignment software package.When using ALIGN program comparing amino acid sequence, can use PAM120 weight residue table, be provided with the notch length point penalty be 12 and the breach point penalty be 4.Other sequential analysis algorithm is known in this field, comprises ADVANCE and ADAM that Torelli and Robotti (1994) Comput.Appl.Biosci.10:3-5 describes; And the FASTA of Pearson and Lipman (1988) P.N.A.S.85:2444-8 description.

Use the GCG software package (can Http:// www.gcg.comAcquisition) the GAP program in also can obtain two percentage homologys between aminoacid sequence, wherein uses Blosum 62 matrixes or PAM250 matrix, and the breach weight is 12,10,8,6 or 4, and the length weight is 2,3 or 4.Use the GAP program in the GCG software package can obtain two percentage homologys between nucleotide sequence, wherein use canonical parameter, as the breach weight be 50 and the length weight be 3.

Embodiment 10: make up and the operation dna microarray

Sequence of the present invention can be used in addition the structure of dna microarray and application (design of DNA array, methodology and purposes are well known, as Schena, people such as M. (1995) science (Science) 270:467-470; Wodicka, people such as L. (1997) Nature Biotechnol (NatureBiotechnology) 15:1359-1367; DeSaizieu, people such as A. (1998) Nature Biotechnol (Nature Biotechnology) 16:45-48; And DeRisi, describe among people such as J.L. (1997) science (Science) 278:680-686).

Dna microarray is solid or the softish upholder of being made up of soluble cotton, nylon, glass, siloxanes or other material.Nucleic acid molecule can be in an orderly way attached to its surface.Through behind the suitable mark, other nucleic acid or nucleic acid mixture can with the fixed making nucleic acid molecular hybridization, and marker can be used for monitoring and being determined at the strength of signal separately of regulation location hybrid molecule.This method allows simultaneously the quantitatively relative quantity or the absolute magnitude of all nucleic acid or selected nucleic acid in applied sample of nucleic acid or mixture.Therefore dna microarray allow a plurality of nucleic acid of parallel analysis (6800 of as many as or more a plurality of nucleic acid) expression (referring to for example, Schena, M. (1996) BioEssays 18 (5): 427-431).

Sequence of the present invention can be used for the design oligonucleotides primer, and wherein said Oligonucleolide primers can be by the localized area of nucleic acid amplification reaction such as one or more corynebacterium glutamicum genes of PCR amplification.Can covalently boundly on above-mentioned supporting dielectric surface, (, describe among people such as M. (1995) science (Science) 270:467-470) to the selection of 5 ' or 3 ' Oligonucleolide primers and design or to the PCR product that the selection and the design of suitable joint allows gained also referring to for example Schena.

Nucleic acid microarray also can be as Wodicka, and described synthesizing by the original position oligonucleotide of people such as L. (1997) Nature Biotechnol (NatureBiotechnology) 15:1359-1367 makes up.By photolithography, with the regional exposure of the accurate qualification of matrix.The unsettled protecting group of activation light carries out Nucleotide then and add, and any modification does not take place in the lucifuge zone thus.Ensuing protection allows at the synthetic different oligonucleotide in the position of regulation with the photoactivation circulation.The little localized area of gene of the present invention can be synthesized on microarray synthetic by the solid phase oligonucleotide.

Be present in the nucleic acid molecule of the present invention microarray hybridization therewith in sample or the mixture of ribonucleotides.These nucleic acid molecule can be according to the standard method mark.In brief, nucleic acid molecule (for example mRNA molecule or dna molecular) as reverse transcription or DNA between synthesis phase by mixing isotropic substance or fluorescently-labeled Nucleotide mark.The nucleic acid of mark and the hybridization of microarray are existing to be described (for example, Schena, people such as M. (1995) see above; Wodicka, people such as L. (1997) see above; With people (1998) such as DeSaizieu A., see above).The mark that the detection of hybrid molecule and quantitative basis are specifically mixed carries out.Radio-labeling can be as Schena, the described detection of people such as M. ((1995) see above), fluorescent mark can be for example method by people such as Shalon (1996) genome research (Genome Research) 6:639-645 detect.

As mentioned above, sequence of the present invention being applied to dna microarray makes can different corynebacterium glutamicum bacterial strain or other corynebacterium strain of comparative analysis.For example, the nucleic acid array method helps transcribing between spectrum research bacterial strain variation and identifying specific and/or aimed strain characteristic such as pathogenic, productivity and stress tolerance important function of gene based on individuality.And, can use the relatively express spectra of gene of the present invention during fermentation reaction of nucleic acid array technology.

Embodiment 11: cell protein group's (protein group) dynamic analysis

Gene of the present invention, composition and method can be used for studying protein group's interaction and kinetics, are called ' proteomics '.The gross protein group that the target protein group includes but not limited to corynebacterium glutamicum (for example, with the protein group of other organism relatively), those are at (for example, during the fermentation, under high temperature or the low temperature or at high or low pH the time) the active protein of tool under specific environment or the metabolism condition or those are at the active protein of the specific period of g and D tool.

Can be by multiple technology of knowing such as gel electrophoresis analysis protein group.Cell protein can for example obtain by cracking or extraction, and can use multiple electrophoretic technique to be separated from each other.SDS-PAGE (SDS-PAGE) isolated protein is to a great extent based on their molecular weight.Isoelectrofocusing polyacrylamide gel electrophoresis (IEF-PAGE) is based on isoelectric fractionation protein (iso-electric point not only reflects proteinic aminoacid sequence, also reflects proteinic posttranslational modification).Another preferred protein analysis method is continuously combination IEF-PAGE and SDS-PAGE, is called the 2-D-gel electrophoresis (for example, at people such as Hermann (1998) electrophoresis (Electrophoresis) 19:3217-3221; People such as Fountoulakis (1998) electrophoresis (Electrophoresis) 19:1193-1202; People such as Langen (1997) electrophoresis (Electrophoresis) 18:1184-1192; Description among people such as Antelmann (1997) electrophoresis (Electrophoresis) 18:1451-1463).Other isolation technique also can be used for protein separation, as capillary gel electrophoresis; This type of technology is known in the art.

Can use standard technique to show by the isolating protein of these methods, as showing by dyeing or mark.Suitable staining agent is known in this field, comprises that Xylene Brilliant Cyanine G, silver dye or fluorescence dye such as Sypro Ruby (Molcular Probos).(for example, in the substratum of corynebacterium glutamicum, comprise radiolabeled amino acid or other protein precursor ³⁵The S-methionine(Met), ³⁵The S-halfcystine, ¹⁴The amino acid of C-mark, ¹⁵N-amino acid, ¹⁵O ₃Or ¹⁵NH ₄ ⁺Or ¹³The amino acid of C-mark) will allow the protein of these cells before they separate, to be labeled.Similarly, can adopt fluorescent mark.Can extract, separate and separate the protein of these marks according to aforesaid technology.

Can further analyze by the protein that these technology show by the amount of measuring used dyestuff or mark.Can use as optical means and quantitatively determine given proteinic amount, and can compare with other the proteinic amount in same gel or other gel.On the gel proteinic comparison can be for example by optics comparison, spectroscopy, image scanning and analyze gel or by using photographic film and screen to carry out.This type of technology is known in the art.

For determining arbitrary given proteinic identity, can adopt direct sequence to measure or other standard technique.For example, can use N-and/or C-terminal amino acid sequencing (such as Edman degraded), also can use mass spectrum (particularly MALDI or ESI technology (referring to for example, people such as Langen (1997) electrophoresis (Electrophoresis) 18:1184-1192)).Protein sequence provided herein can be used for by these technical evaluation corynebacterium glutamicum protein.

The information that obtains by these methods can be used for comparison from protein existence, activity or modification pattern between the different samples of various biotic conditions (for example, especially, different organisms, fermentation time point, culture medium condition or different microhabitat etc.).Can be used for multiple use from independent these experiments or with data that other technical combinations obtains, as comparing the production efficiency of the behavior (under for example, the metabolism situation) multiple organism under the given situation, the productivity that increases the bacterial strain that produces fine chemicals or raising fine chemicals.

Embodiment 12

The corynebacterium glutamicum strain system of preparation methionine(Met) biosynthesizing down regulator (RXA00655) defective

Carry out the preparation of the corynebacterium glutamicum strain system of methionine(Met) biosynthesizing down regulator (RXA00655) defective by utilizing based on self clone technology of homologous recombination.Described know-why as shown in fig. 1.

Utilize standard method (Sambrook etc. (1989), Molecular Cloning.A LaboratoryManual, Cold Spring Harbor; Innis etc. (1990) PCR Protocols.A Guide toMethods and Applications, Academic Press), be connected among the carrier pCLiK5MCS_integrative_sacB (SEQ ID NO:4) by pcr amplified fragment RXA00655 (SEQ IDNO:1) 5 '-and 3 '-district, make up the plasmid that name is called pS_ Δ 655 (SEQ ID NO:3), this plasmid is used to prepare the corynebacterium glutamicum strain system of methionine(Met) biosynthesizing down regulator (RXA00655) defective.5 ' segmental 5 ' the terminal flank of pcr amplification is the XhoI site that primer is introduced, and 3 ' terminal flank is endogenous NheI site.3 ' segmental 5 ' the terminal flank of pcr amplification is by the NheI site of primer introducing and TAA terminator codon subsequently, and its 3 ' terminal flank is the MluI site that primer is introduced.

As described (Liebl etc. (1989) FEMS Microbiol Let 53:299-303), be among the ATCC13032 to the corynebacterium glutamicum strain with pS_ Δ 655 (SEQ ID NO:3) electroporation.Cross the transformant that intermolecular homologous recombination is integrated with plasmid at the CM agar plate top sieve gating of adding the 50mg/l kantlex.

CM agar

10.0g/L D-glucose

2.5g/L?NaCl

2.0g/L urea

10.0g/L bacterium is used peptone (Difco)

5.0g/L yeast extract (Difco)

5.0g/L extractum carnis (Difco)

22.0g/L agar (Difco)

Autoclaving (20 minutes, 121 ℃).

The clone who non-selectively (does not have kantlex) and spend the night the incubation kalamycin resistance in the CM substratum is to obtain the excision of plasmid with the RXA00655 chromosome copies.Culture is coated with flat board containing on the CM agar of 10% sucrose.Have only those clones that excised pS_ Δ 655 plasmids of integrating just can be grown on the CM agar that contains sucrose, because the sacB gene is to the deleterious levansucrase of corynebacterium glutamicum with sucrose inversion in the pS_ Δ 655.When excision, perhaps the disappearance construct of RXA00655 or the chromosome copies of RXA00655 are removed with plasmid.

In order to identify the clone who has got rid of the RXA00655 chromosome copies, the preparation might clone chromosomal DNA (Tauch etc. (1995) Plasmid 33:168-179 or Eikmanns etc. (1994) Microbiology 140:1817-1828), and according to Sambrook etc. (1989), Molecular Cloning.A Laboratory Manual, Cold Spring Harbor contrasts with the Southern engram analysis.The strain system of rejecting is called ATCC13032_ Δ _ rxa00655.

Embodiment 15

Prepare methionine(Met) with ATCC13032_ Δ _ rxa00655

At 30 ℃, incubation ATCC13032 and ATCC13032_ Δ rxa00655 bacterial strain on the CM agar plate.Scrape cell from flat board, and be suspended in the salt solution.Cultivate for main, with 10ml medium ii and the autoclaved CaCO of 0.5g in the cell suspending liquid inoculation 100ml erlenmeyer flask ₃(Riedel de Haen), reaching final OD600nm is 1.5.At 30 ℃, cultivated 72 hours.

Medium ii:

40g/1 sucrose

60g/l molasses (based on 100% sugared cubage)

25g/l(NH ₄) ₂SO ₄

0.4g/l?MgSO ₄＊7H ₂O

0.6g/lKH ₂PO ₄

0.3mg/l VitB1 * HCl

The 1mg/l vitamin H (derives from the mother liquor of 1mg/ml sterile filtration, uses NH ₄OH adjusts to 8.0 with pH)

2mg/l?FeSO ₄

2mg/l?MnSO ₄

Use NH ₄OH adjusts to 7.8 with pH.After, autoclaving substratum (121 ℃, 20 minutes).

Add the other vitamin B12 that derives from mother liquor (200 μ g/ml, sterile filtration), making final concentration is 200 μ g/l.

According to the method that comes from Agilent, utilizing Agilent 1100 serial LC is the amount that HPLC detects the methionine(Met) that forms.Go up the amino acid that separates with Phthalyldicarboxaldehyde (ortho-pthalaldehyde) column front derivation at Hypersil AA-post (Agilent).Strain is that ATCC13032_ Δ _ rxa00655 has produced significantly more methionine(Met) than ATCC13032.

Embodiment 16

The corynebacterium glutamicum strain system of preparation overexpression methionine(Met) biosynthesizing positive regulator (RXN02910)

Utilize standard method (Sambrook etc. (1989), Molecular Cloning.ALaboratory Manual, Cold Spring Harbor; Innis etc. (1990) PCR Protocols.A Guide to Methods and Applications, Academic Press), be connected to structure overexpression methionine(Met) biosynthesizing positive regulator (RXN02910 among the carrier pG (SEQ IDNO:12) by pcr amplified fragment (SEQ ID NO:9) with RXN02910; SEQ IDNO:5 or 6), name is called the plasmid of pGrxn02910 (SEQ ID NO:13).Oligonucleolide primers below using is to being used for amplification:

Adopted primer (SEQ ID NO:7) is arranged

5′-GAGAGACTCGAGTGGTTTAGGGGATGAGAAACCG-3′

Antisense primer (SEQ ID NO:8)

5′-CTCTCACTAGTCTACCGCGCCAACAACAGCG-3′

5 ' terminal flank of pcr amplified fragment is the XhoI site that primer is introduced, and 3 ' terminal flank is the BcuI site that primer is introduced.Other 5 ' the district that the fragment of amplification contains the open reading frame (ORF) of RXN02910 and comprises the corresponding gene of promotor.

As described (Liebl, etc. (1989) FEMS Microbiology Letters 53:299-303), be among the ATCC13032 with resulting plasmid pGrxn02910 (SEQ ID NO:13) electroporation to the corynebacterium glutamicum strain.Screen transformant (referring to above-mentioned) on the CM agar plate of 50mg/l kantlex adding.Resulting strain is called after ATCC13032/pGrxn02910.

Embodiment 17:

Prepare methionine(Met) with ATCC13032/pGrxn02910

At 30 ℃, incubation ATCC13032 and ATCC13032/pGrxn02910 bacterial strain on the CM agar plate.Scrape cell from flat board, and be suspended in the salt solution.Cultivate for main, with 10ml medium ii (referring to above-mentioned) and the autoclaved CaCO of 0.5g in the conical flask of cell suspending liquid inoculation 100ml ₃(Riedel de Haen), reaching final OD600nm is 1.5.At 30 ℃, cultivated 72 hours.Under the ATCC13032/pGrxn02910 situation, all flat boards and culture all contain 50 other μ g/l kantlex.According to the method that comes from Agilent, utilizing Agilent 1100 serial LC is the amount that HPLC detects the methionine(Met) that forms.Go up the amino acid that separates with the Phthalyldicarboxaldehyde column front derivation at HypersilAA-post (Agilent).Strain is that ATCC13032/pGrxn02910 has produced significantly more methionine(Met) than ATCC13032.

Embodiment 18

The corynebacterium glutamicum strain system of preparation methionine(Met) biosynthesizing positive regulator (RXN02910) defective

Carry out the preparation of the corynebacterium glutamicum strain system of methionine(Met) biosynthesizing positive regulator (RXN02910) defective by the insertion of kantlex selectivity carrier.

Utilize standard method (Sambrook etc. (1989), Molecular Cloning.A LaboratoryManual, Cold Spring Harbor; Innis etc. (1990) PCR Protocols.A Guide toMethods and Applications, Academic Press), be connected to the middle plasmid that is called pIntegrativ_ Δ 2910 (SEQ ID NO:15) that makes up of carrier pIntegrativ (SEQ ID NO:14) by the pcr amplified fragment with rxn02910, this plasmid is used to prepare the corynebacterium glutamicum strain system of methionine(Met) biosynthesizing positive regulator (rxn02910) defective.5 ' terminal flank of pcr amplified fragment (SEQ ID NO:11) is the XhoI site that primer is introduced, and 3 ' terminal flank is the EcoRV site that primer is introduced.

As described (Liebl etc. (1989) FEMS Microbiol Let 53:299-303), be among the ATCC13032 to the corynebacterium glutamicum strain with pIntegrativ_ Δ 2910 (SEQ ID NO:15) electroporation.Cross the transformant that intermolecular homologous recombination is integrated with plasmid at the CM agar plate top sieve gating of adding the 50mg/l kantlex.In order to identify the clone who has integrated plasmid and therefore destroyed the RXN02910 chromosome copies, preparation derive from the chromosomal DNA that may clone (Tauch etc. (1995) Plasmid 33:168-179 or Eikmanns etc. (1994) Microbiology 140:1817-1828), and according to Sambrook etc. (1989), Molecular Cloning.ALaboratory Manual, Cold Spring Harbor contrasts with the Southern engram analysis.

The strain system of rejecting is called ATCC13032_ Δ _ rxn02910.According to the method that comes from Agilent, utilizing Agilent 1100 serial LC is the amount that HPLC detects the methionine(Met) that forms.Go up the amino acid that separates with the Phthalyldicarboxaldehyde column front derivation at Hypersil AA-post (Agilent).

Strain is that ATCC13032_ Δ _ rxn02910 has produced methionine(Met) significantly still less than ATCC13032, therefore proves that rxn02910 is actually the biosynthetic positive regulator of methionine(Met).

Equivalent

Those of ordinary skills will recognize or only be to use conventional experiment can determine many equivalents of the specific embodiments of the present invention described in the literary composition.This type of equivalent is intended to be included in the following claim.

Table 1: can be used for coryneform bacteria and tyrothricin bacterial strain in the present invention's practice

Belong to

Kind

ATCC

?FERM

?NRRL

?CECT

?NCIMB

?CBS

?NCTC

?DSMZ

Tyrothricin

Brevibacterium ammoniagenes (Ammoniagenes)

21054

Tyrothricin

Brevibacterium ammoniagenes

19350

Tyrothricin

Brevibacterium ammoniagenes

19351

Tyrothricin

Brevibacterium ammoniagenes

19352

Tyrothricin

Brevibacterium ammoniagenes

19353

Tyrothricin

Brevibacterium ammoniagenes

19354

Tyrothricin

Brevibacterium ammoniagenes

19355

Tyrothricin

Brevibacterium ammoniagenes

19356

Tyrothricin

Brevibacterium ammoniagenes

21055

Tyrothricin

Brevibacterium ammoniagenes

21077

Tyrothricin

Brevibacterium ammoniagenes

21553

Tyrothricin

Brevibacterium ammoniagenes

21580

Tyrothricin

Brevibacterium ammoniagenes

39101

Tyrothricin

Butanicum

21196

Tyrothricin

Fork tyrothricin (Divaricatum)

21792

?P928

Tyrothricin

Brevibacterium flavum (Flavum)

21474

Tyrothricin

Brevibacterium flavum

21129

Tyrothricin

Brevibacterium flavum

21518

Tyrothricin

Brevibacterium flavum

B11474

Tyrothricin

Brevibacterium flavum

B11472

Tyrothricin

Brevibacterium flavum

21127

Tyrothricin

Brevibacterium flavum

21128

Tyrothricin

Brevibacterium flavum

21427

Tyrothricin

Brevibacterium flavum

21475

Tyrothricin

Brevibacterium flavum

21517

Tyrothricin

Brevibacterium flavum

21528

Tyrothricin

Brevibacterium flavum

21529

Tyrothricin

Brevibacterium flavum

B11477

Tyrothricin

Brevibacterium flavum

B11478

Tyrothricin

Brevibacterium flavum

21127

Tyrothricin

Brevibacterium flavum

B11474

Tyrothricin

Xi Shi tyrothricin (Healii)

15527

Tyrothricin

Ketoglutamicum

?21004

Tyrothricin

Ketoglutamicum

?21089

Tyrothricin

Ketosoreductum

?21914

Tyrothricin

Brevibacterium lactofermentum (Lactofermentum)

70

Tyrothricin

Brevibacterium lactofermentum

74

Tyrothricin

Brevibacterium lactofermentum

77

Tyrothricin

Brevibacterium lactofermentum

21798

Tyrothricin

Brevibacterium lactofermentum

21799

Tyrothricin

Brevibacterium lactofermentum

21800

Tyrothricin

Brevibacterium lactofermentum

21801

Tyrothricin

Brevibacterium lactofermentum

B11470

Tyrothricin

Brevibacterium lactofermentum

B11471

Tyrothricin

Brevibacterium lactofermentum

21086

Tyrothricin

Brevibacterium lactofermentum

21420

Tyrothricin

Brevibacterium lactofermentum

21086

Tyrothricin

Brevibacterium lactofermentum

31269

Tyrothricin

Linens

9174

Tyrothricin

Linens

19391

Tyrothricin

Linens

8377

Tyrothricin

Paraffinolyticum

11160

Tyrothricin

Kind

717.73

Tyrothricin

Kind

717.73

Tyrothricin

Kind

14604

Tyrothricin

Kind

21860

Tyrothricin

Kind

21864

Tyrothricin

Kind

21865

Tyrothricin

Kind

21866

Tyrothricin

Kind

19240

Coryneform bacteria

Have a liking for etheric acid coryneform bacteria (Acetoacidophilum)

21476

Coryneform bacteria

Have a liking for the etheric acid coryneform bacteria

13870

Coryneform bacteria

Acetoglutamicum

B11473

Coryneform bacteria

Acetoglutamicum

B11475

Coryneform bacteria

Acetoglutamicum

15806

Coryneform bacteria

Acetoglutamicum

21491

Coryneform bacteria

Acetoglutamicum

31270

Coryneform bacteria

Have a liking for acetyl coryneform bacteria (Acetophilum)

B3671

Coryneform bacteria

Corynebacterium ammoniagenes (Ammoniagenes)

6872

?2399

Coryneform bacteria

Corynebacterium ammoniagenes

15511

Coryneform bacteria

Fujiokense

21496

Coryneform bacteria

Corynebacterium glutamicum

14067

Coryneform bacteria

Corynebacterium glutamicum

39137

Coryneform bacteria

Corynebacterium glutamicum

21254

Coryneform bacteria

Corynebacterium glutamicum

21255

Coryneform bacteria

Corynebacterium glutamicum

31830

Coryneform bacteria

Corynebacterium glutamicum

13032

Coryneform bacteria

Corynebacterium glutamicum

14305

Coryneform bacteria

Corynebacterium glutamicum

15455

Coryneform bacteria

Corynebacterium glutamicum

13058

Coryneform bacteria

Corynebacterium glutamicum

13059

Coryneform bacteria

Corynebacterium glutamicum

13060

Coryneform bacteria

Corynebacterium glutamicum

21492

Coryneform bacteria

Corynebacterium glutamicum

21513

Coryneform bacteria

Corynebacterium glutamicum

21526

Coryneform bacteria

Corynebacterium glutamicum

21543

Coryneform bacteria

Corynebacterium glutamicum

13287

Coryneform bacteria

Corynebacterium glutamicum

21851

Coryneform bacteria

Corynebacterium glutamicum

21253

Coryneform bacteria

Corynebacterium glutamicum

21514

Coryneform bacteria

Corynebacterium glutamicum

21516

Coryneform bacteria

Corynebacterium glutamicum

21299

Coryneform bacteria

Corynebacterium glutamicum

21300

Coryneform bacteria

Corynebacterium glutamicum

39684

Coryneform bacteria

Corynebacterium glutamicum

21488

Coryneform bacteria

Corynebacterium glutamicum

21649

Coryneform bacteria

Corynebacterium glutamicum

21650

Coryneform bacteria

Corynebacterium glutamicum

19223

Coryneform bacteria

Corynebacterium glutamicum

13869

Coryneform bacteria

Corynebacterium glutamicum

21157

Coryneform bacteria

Corynebacterium glutamicum

21158

Coryneform bacteria

Corynebacterium glutamicum

21159

Coryneform bacteria

Corynebacterium glutamicum

21355

Coryneform bacteria

Corynebacterium glutamicum

31808

Coryneform bacteria

Corynebacterium glutamicum

21674

Coryneform bacteria

Corynebacterium glutamicum

21562

Coryneform bacteria

Corynebacterium glutamicum

21563

Coryneform bacteria

Corynebacterium glutamicum

21564

Coryneform bacteria

Corynebacterium glutamicum

21565

Coryneform bacteria

Corynebacterium glutamicum

21566

Coryneform bacteria

Corynebacterium glutamicum

21567

Coryneform bacteria

Corynebacterium glutamicum

21568

Coryneform bacteria

Corynebacterium glutamicum

21569

Coryneform bacteria

Corynebacterium glutamicum

21570

Coryneform bacteria

Corynebacterium glutamicum

21571

Coryneform bacteria

Corynebacterium glutamicum

21572

Coryneform bacteria

Corynebacterium glutamicum

21573

Coryneform bacteria

Corynebacterium glutamicum

21579

Coryneform bacteria

Corynebacterium glutamicum

19049

Coryneform bacteria

Corynebacterium glutamicum

19050

Coryneform bacteria

Corynebacterium glutamicum

19051

Coryneform bacteria

Corynebacterium glutamicum

19052

Coryneform bacteria

Corynebacterium glutamicum

19053

Coryneform bacteria

Corynebacterium glutamicum

19054

Coryneform bacteria

Corynebacterium glutamicum

19055

Coryneform bacteria

Corynebacterium glutamicum

19056

Coryneform bacteria

Corynebacterium glutamicum

19057

Coryneform bacteria

Corynebacterium glutamicum

19058

Coryneform bacteria

Corynebacterium glutamicum

19059

Coryneform bacteria

Corynebacterium glutamicum

19060

Coryneform bacteria

Corynebacterium glutamicum

19185

Coryneform bacteria

Corynebacterium glutamicum

13286

Coryneform bacteria

Corynebacterium glutamicum

21515

Coryneform bacteria

Corynebacterium glutamicum

21527

Coryneform bacteria

Corynebacterium glutamicum

21544

Coryneform bacteria

Corynebacterium glutamicum

21492

Coryneform bacteria

Corynebacterium glutamicum

B8183

Coryneform bacteria

Corynebacterium glutamicum

B8182

Coryneform bacteria

Corynebacterium glutamicum

B12416

Coryneform bacteria

Corynebacterium glutamicum

B12417

Coryneform bacteria

Corynebacterium glutamicum

B12418

Coryneform bacteria

Corynebacterium glutamicum

B11476

Coryneform bacteria

Corynebacterium glutamicum

21608

Coryneform bacteria

Corynebacterium lilium (lilium)

P973

Coryneform bacteria

nitrilophilus

?21419

?11594

Coryneform bacteria

Kind

P4445

Coryneform bacteria

Kind

P4446

Coryneform bacteria

Kind

31088

Coryneform bacteria

Kind

31089

Coryneform bacteria

Kind

31090

Coryneform bacteria

Kind

31090

Coryneform bacteria

Kind

31090

Coryneform bacteria

Kind

15954

?20145

Coryneform bacteria

Kind

21857

Coryneform bacteria

Kind

21862

Coryneform bacteria

Kind

21863

ATCC: American type culture collection (American Type Culture Collection), Rockville, MD, the U.S.

FERM: fermentation research institute (Fermentation Research Institute), Chiba, Japan

NRRL: agricultural research institute preservation center, northern research department (ARS Culture Collection, Northern Regional Research Laboratory), Peoria, IL, the U.S.

CECT: Spain typical case's culture collection center, Valencia, Spain

NCIMB: state-run industry and marine microorganism preservation company limited, Britain

CBS: fungi strain preservation center, NL

NCTC: state-run typical culture collection center (National Collection of TypeCultures), London, Britain

DSMZ: Germany microbial preservation center (Deutsche Sammlung vonMikroorganismen und Zellkulturen), Braunschweig, Germany

Reference is referring to Sugawara, H. etc. (1993) World directory of collectionsof cultures of microorganisms:Bacteria, fungi and yeasts (4 ' edn), Worldfederation for culture collections world data center on microorganisms, Saimata, Japen.

Claims

1. method that the sulfocompound production of corynebacterium glutamicum microorganism is regulated, this method is included under the condition of the production of regulating sulfocompound and cultivates the described microorganism that the metY expression of gene is regulated, wherein said method comprises the positive transcription regulatory protein RXN02910 of overexpression metY gene, wherein RXN02910 is by nucleic acid molecule encoding, and this nucleic acid molecule is made up of nucleic acid molecule SEQID NO:5 or 16.

2. the method for claim 1, wherein said sulfocompound is selected from methionine(Met), halfcystine, S-adenosylmethionine and homocysteine.

3. method as claimed in claim 1 or 2 wherein increases the production of methionine(Met).

4. method of producing methionine(Met), this method is included in the active corynebacterium glutamicum microorganism of RXN02910 of cultivating overexpression RXN02910 under the condition of producing methionine(Met) or having increase, wherein RXN02910 is by nucleic acid molecule encoding, and this nucleic acid molecule is made up of nucleic acid molecule SEQ IDNO:5 or 16.

5. method of producing methionine(Met), this method are included in and cultivate corynebacterium glutamicum microorganism, this microorganism under the condition of producing methionine(Met)

A) overexpression RXN02910 or have the RXN02910 activity of increase, and

B) have RXA00655 expression of preventing or the RXA00655 activity that reduces,

Wherein RXA00655 is by nucleic acid molecule encoding, and this nucleic acid molecule is made up of coded polypeptide SEQ IDNO:2,19,21 or 23 nucleic acid molecule,

Wherein RXN02910 is by nucleic acid molecule encoding, and this nucleic acid molecule is made up of nucleic acid molecule SEQ IDNO:5 or 16.

6. the described method of claim 5, wherein said RXA00655 is by nucleic acid molecule encoding, and this nucleic acid molecule is made up of nucleic acid molecule SEQ ID NO:1,18,20 or 22.

7. as each described method in the claim 1,2,4,5 or 6, it further comprises the recovery methionine(Met).

8. regulate the transgene expression cassette of the sulfocompound production of corynebacterium glutamicum microorganism, described transgene expression cassette comprise regulate sequence and coding as metY genetic transcription regulate the nucleic acid molecule of proteinic polypeptide SEQ ID NO:6 or SEQ ID NO:17

Wherein said adjusting sequence can mediate the expression of described nucleic acid molecule in the corynebacterium glutamicum microorganism.

9. transgene expression cassette as claimed in claim 8, wherein said nucleic acid molecule are nucleic acid molecule SEQ ID NO:5, SEQ ID NO:16 or its complementary molecule.

10. transgene expression cassette as claimed in claim 8, wherein said transgene expression cassette also comprise coding as metY genetic transcription regulate the nucleic acid molecule of proteinic polypeptide SEQ ID NO:2, SEQ ID NO:19, SEQ ID NO:21 or SEQ ID NO:23.

11. transgene expression cassette as claimed in claim 10, wherein said nucleic acid molecule are nucleic acid molecule SEQ ID NO:1, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22 or its complementary molecule.

12. as each described transgene expression cassette in the claim 8 to 11, wherein said adjusting sequence is and the allogenic promoter sequence of described nucleic acid molecule.

13. as each described transgene expression cassette in the claim 8 to 11, wherein with described promoter sequence antisense or have justice direction place described nucleic acid molecule.

14. regulate the carrier that the sulfocompound of corynebacterium glutamicum microorganism is produced, described carrier comprises among the claim 8-13 each transgene expression cassette.

15. carrier as claimed in claim 14, wherein said carrier is an expression vector.

16. regulate the genetically modified host cell that the sulfocompound of corynebacterium glutamicum microorganism is produced, each transgene expression cassette among the wherein said genetically modified host cell usefulness claim 8-13, or the carrier transfection in claim 14 or 15.