CN100554426C - Corynebacterium glutamicum with genetic modification produces L-Methionin - Google Patents
Corynebacterium glutamicum with genetic modification produces L-Methionin Download PDFInfo
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Abstract
The present invention relates to bar shaped bacteria and reach the method for preparing compound by these bacteriums of fermenting, described bacterium is except having coded protein or RNA synthetic open reading frame (ORF), gene or allelic at least one copy at natural site (locus), in each case, in each case second, randomly the 3rd or the 4th site also have this open reading frame (ORF) that is integrated into the karyomit(e) form, gene or allelic second, randomly the 3rd or the 4th copy.
Description
Prior art
Compound, especially L-amino acid, VITAMIN, nucleosides and Nucleotide and D-amino acid are used for people's medication, are used for pharmacy industry, are used for makeup, are used for grocery trade and are used for Animal nutrition.
These numerous compounds are fermentative preparation from bar shaped bacteria especially paddy ammonia coryneform bacterial strains.Because itself and importance thereof have continued to carry out the trial of improved production method.The improvement of production method can relate to the fermentation measure.As stirring and oxygen supply, or the sugared concentration between the component of nutritional medium such as yeast phase, or for example by the processing of ion exchange chromatography to the product form, or the intrinsic nature of production of microorganism itself.
For improveing the nature of production of these microorganisms, can use methods such as mutagenesis, selection and mutant selection, can obtain metabolic antagonist is had resistance or has the metabolite of regulating importance to be auxotrophic and to produce amino acid whose bacterial strain with this method.
For some years, the method for recombinant DNA technology also has been used for the improvement of coryneform bacterial strains, and it is by each amino acid bio synthetic gene of amplification, and research improves the effect of amino acid production.
A kind of method in common comprises by some biosynthesis gene in the episomal replication plasmid amplification specified microorganisms.The shortcoming of this method is during fermentation (to ferment in commercial run and carry out many generations usually), the spontaneous forfeiture of described plasmid (segregational instability).
Another kind method comprises plasmid that utilization is not duplicated some biosynthesis genes that double in specified microorganisms.In this method, the plasmid integration that will comprise clone's biosynthesis gene advances that (Reinscheid etc. use and environmental microbiology 60 (1) 126-132 (1994) in the karyomit(e) biosynthesis gene of this microorganism; Jetten etc., applied microbiology and biotechnics 43 (1): 76-82 (1995)).The shortcoming of this method is that the nucleotide sequence of plasmid and the nucleotide sequence of selecting necessary antibiotics resistance gene are retained in the microorganism, and this is a unfavorable factor to the processing and the utilization of biomass for example.In addition, the expert expect this bacterial strain since in corresponding to industrial fermentation in the commonly used generation by the desintegration (disintegration) of " Campbell's typical exchange (Campbell typecross over) " instability.
Goal of the invention
The inventor provides improvement to use the new measure of bar shaped bacteria fermentative production compound.
Summary of the invention
The invention provides the bar shaped bacteria of production compound, be characterised in that these bacteriums except have coded protein or RNA synthetic open reading frame (ORF) at natural site (locus), outside gene or allelic at least one copy, at second, randomly the 3rd or the 4th site also have this open reading frame (ORF) that is integrated into the karyomit(e) form, gene or allelic second, randomly the 3rd or the 4th copy, this specific second, randomly the 3rd or the 4th site do not have can/make can be in microorganism the nucleotide sequence of episomal replication or swivel base (is capable of/enables episomal replication or transposition), reach the nucleotide sequence of not authorizing antibiotics resistance, this second, randomly the 3rd or the 4th site and bacterial growth and desirable compound are produced essential open reading frame (ORF), gene or allelotrope are uncorrelated.
The present invention also provides the method for preparing one or more compound, wherein carries out following steps:
A) the such bar shaped bacteria of fermentation,
A1) these bacteriums are except existing coded protein or RNA synthetic open reading frame (ORF) at natural site (locus), outside gene or allelic at least one copy, at second, randomly the 3rd or the 4th site have this open reading frame (ORF) that is integrated into the karyomit(e) form, gene or allelic second, randomly the 3rd or the 4th copy, this specific second, randomly the 3rd or the 4th site do not have can/make can be in microorganism the nucleotide sequence of episomal replication or swivel base, reach the nucleotide sequence of not authorizing antibiotics resistance, this second, randomly the 3rd or the 4th site and bacterial growth and desirable compound are produced essential open reading frame (ORF), gene or allelotrope are uncorrelated
A2) the interior activity of the born of the same parents of respective egg white matter improves in the described bacterium, and especially the nucleotide sequence of encoding such proteins is crossed and expressed,
B) compound in concentrated fermenting broth and/or the bacterial cell,
C) separate described compound, randomly
D) with＞(greater than) component and/or the biomass of the fermenting broth of 0-100wt%.
The present invention also provides the method for preparing one or more compound, may further comprise the steps:
A) make described open reading frame (ORF), under the condition that gene or allelotrope are expressed, fermentation of coryneform bacteria, particularly excellent bacillus Pseudomonas, these bacteriums are except existing coded protein or RNA synthetic open reading frame (ORF) at natural site (locus), outside gene or allelic at least one copy, in each case, second in each case, randomly the 3rd or the 4th site also have this open reading frame (ORF) of integration form, gene or allelic second, randomly the 3rd or the 4th copy, this specific second, randomly the 3rd or the 4th site do not have can/make can be in microorganism the nucleotide sequence of episomal replication or swivel base, reach the nucleotide sequence of not authorizing antibiotics resistance
B) compound in concentrated fermenting broth and/or the bacterial cell,
C) separate described compound, randomly
D) with＞(greater than) component and/or the biomass of the fermenting broth of 0-100wt%.
Detailed Description Of The Invention
Should understand described compound and be meant amino acid especially, VITAMIN, nucleosides and Nucleotide.Prior art is known and can obtain the biosynthetic pathway of these compounds.
Amino acid preferably is meant L-amino acid, and especially albumen originality L-amino acid is selected from the L-aspartic acid, altheine, the L-Threonine, L-Serine, L-L-glutamic acid, L-glutaminate, glycine, L-L-Ala, L-halfcystine, L-Xie Ansuan, the L-methionine(Met), L-Isoleucine, L-leucine, L-tyrosine, the L-phenylalanine, L-Histidine, L-Methionin, L-tryptophane, L-proline(Pro) and L-arginine, and their salt, particularly L-Methionin, L-methionine(Met) and L-Threonine.Preferred especially L-Methionin.
The amino acid that the proteinogen acidic amino acid is interpreted as existing in natural protein, natural protein are microorganism, plant, animal and human's protein.
VITAMIN is meant VITMAIN B1 (VitB1) especially, Wei ShengsuB2 (riboflavin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxol), vitamin B12 (cyanocobalamin), nicotinic acid/niacinamide, vitamin(e) M (folic acid) and vitamin-E (tocopherol) and their salt, preferred pantothenic acid.
Nucleosides and Nucleotide are meant S-adenosylmethionine especially, inosine-5 '-single phosphoric acid and guanosine-5 '-single phosphoric acid and their salt.
Bar shaped bacteria is those bacteriums of Corynebacterium particularly.Preferred Corynebacterium glutamicum in Corynebacterium produces ammonia rod bacillus (Corynebacterium ammoniagenes) and thermophilic product ammonia rod bacillus (Corynebacterium thermoaminogenes).The classified information of this group bacterial isolates sees Kampfer and Kroppenstedt (Canadian Journal of Microbiology 42,989-1005 (1996)) and US-A-5, and 250,434 is described.
The suitable bacterial strain of Corynebacterium glutamicum bacterial classification is those known wild type strains particularly:
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium lilium ATCC15990
Corynebacterium melassecola ATCC17965
Corynebacterium herculisATCC13868
Arthrobacter sp.ATCC243
Brevibacterium chang-fua ATCC14017
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869
Brevibacterium divaricatum ATCC14020
Brevibacterium taipei ATCC13744 and
Microbacterium ammoniaphilum ATCC21645
And the mutant or the bacterial strain of the production compound that therefrom produces, as known in the art those mutant or bacterial strain.
Produce the particularly known wild type strain of suitable bacterial strain of ammonia rod bacillus (C.ammoniagenes) bacterial classification:
Brevibacterium ammoniagenes ATCC6871
Brevibacterium ammoniagenes ATCC15137 and
Corynebacterium sp.ATCC21084
And the mutant or the bacterial strain of the production compound that therefrom produces, as known in the art those mutant or bacterial strain.
The particularly known wild type strain of suitable bacterial strain of thermophilic product ammonia rod bacillus (C.thermoaminogenes) bacterial classification:
Corynebacterium thermoaminogenes FERM BP-1539
Corynebacterium thermoaminogenes FERM BP-1540
Corynebacterium thermoaminogenes FERM BP-1541 and
Corynebacterium thermoaminogenes FERM BP-1542
And the mutant or the bacterial strain of the production compound that therefrom produces, as known in the art those mutant or bacterial strain.
Bacterial strain with ATCC label can derive from American type culture collection (Manassas, VA, the U.S.).Bacterial strain with FERM label can derive from state-run industrial technology synthetic study (the National Institute of Advanced Industrial Science andTechnology of institute, AIST Tsukuba Central 6,1-1-1 Higashi, Tsukuba Ibaraki, Japan).The thermophilic product ammonia coryneform bacterial strains of mentioning (FERM BP-1539, FERM BP-1540, FERM BP-1541 and FERM BP-1542) is at US-A 5,250, describes in 434.
Open reading frame (ORF) has been described the nucleotide sequence of one section coding or a kind of protein of codified or polypeptide or Yeast Nucleic Acid, in the prior art not to the description of its function.
After a kind of function was assigned to the nucleotide sequence sections of mentioning, it generally was called gene.
Allelotrope generally is meant the other form of given gene.This form is distinguished by the difference in the nucleotide sequence.
In the present invention, preferably using endogenous is species characteristic open reading frame, gene or allelotrope.These are interpreted as being present in open reading frame, gene or allelotrope or its nucleotide sequence in species group style such as the Corynebacterium glutamicum.
In the literary composition of the present invention " open reading frame (ORF), gene or the allelic copy that exist at natural site (locus) " be meant ORF or gene or allelotrope with respect to adjacent ORF or gene or allelic position and situation as in being present in corresponding wild-type or corresponding parental generation organism or initial organism.
Therefore, the lysC of feedback resistance E.C. 2.7.2.4. of lysC gene or coding Corynebacterium glutamicum for example
FBRAllelic natural site is lysC site or lysC locus or lysC gene locus, and the one side is gene or the open reading frame orfX and the leuA of direct neighbor, and opposite side is the asd gene.
Feedback resistance E.C. 2.7.2.4. is meant, compare with wild form, the inhibition of the mixture of the mixture of Methionin and Threonine or AEC (amino-ethyl halfcystine) and Threonine or Methionin self or AEC self is had E.C. 2.7.2.4. than Wheat Protein.The bacterial strain typical case who produces L-Methionin is contained the E.C. 2.7.2.4. of this feedback resistance or desensitization.
The chromosomal nucleotide sequence of known Corynebacterium glutamicum, and be found in the nucleotide sequence database accession number AX114121 of patent application EP-A-1108790 and European Molecular Bioglogy Laboratory (EMBL, Heidelberg, Germany and Cambridge, Britain).The nucleotide sequence of orfX, the accession number of leuA gene and asd gene are AX120364 (orfX), AX123517 (leuA) and AX123519 (asd).
In addition, also can use for example state-run biotechnology of other database database (NCBI of information center, Bethesda, MD, the U.S.) or Switzerland information biology institute database (Swissprot, Geneva, Switzerland) or protein information resource database (PIR, Washington, DC, the U.S.).
" second in each case, randomly the 3rd or the 4th site " is meant a site different with natural site.Be also referred to as " target site " or " target sequence " hereinafter.It also can be called " integration site " or " conversion site ".This second, randomly the 3rd or the 4th site perhaps are present in the nucleotide sequence in these corresponding sites, preferably in karyomit(e), and are nonessential to the generation of growth and desirable compound generally.
For producing bar shaped bacteria of the present invention, separate the desirable ORF that randomly comprises expression and/or conditioning signal, gene or allelic nucleotide sequence, the nucleotide sequence of target site is provided at its end, then preferably by means of in bar shaped bacteria, not duplicating or the only limited carrier that duplicates shifts these sequences in the into desirable bar shaped bacteria, separate desirable ORF, gene or allelotrope mix those bacteriums of target site, this target site do not have can/make can be in microorganism the nucleotide sequence of episomal replication, do not have energy swivel base/feasible nucleotide sequence that swivel base takes place, and do not authorize the nucleotide sequence of antibiotics resistance.
The present invention also provides a kind of method of producing bar shaped bacteria, and described bacterium produces one or more compound, and described method comprises:
A) the desirable ORF of separating at least one, gene or allelic nucleotide sequence, it randomly comprises expresses and/or conditioning signal,
B) the target site nucleotide sequence is offered described ORF, gene or allelic 5 ' and 3 ' end,
C) desirable ORF, gene or the allelic nucleotide sequence that preferably will have the target site nucleotide sequence mixes in the carrier, described carrier in bar shaped bacteria, do not duplicate or only limited extent duplicate,
D) with b) or nucleotides sequence column jump c) advance in the bar shaped bacteria,
E) separate such bar shaped bacteria, a) nucleotide sequence is incorporated in target site in the described bar shaped bacteria, this target site do not have can/make can be in microorganism the nucleotide sequence of episomal replication, do not have energy swivel base/feasible nucleotide sequence that swivel base takes place, and do not authorize the nucleotide sequence of antibiotics resistance.
Preferably, do not have the sequence of used carrier or the remnants of species foreign DNA, for example limit cleavage site at described target site.Randomly, keep maximum 24 of this DNA in ORF, gene or the allelotrope upstream or the downstream of mixing at this target site, preferred maximum 12, preferred especially maximum 6 Nucleotide.
By measure of the present invention, the productive rate of the fermentation process of bar shaped bacteria or preparation compound is improved 0.5-1.0% or 1.0-1.5% or 1.5-2.0% at least at least at least being selected from aspect one or more of next group: the concentration (compound of formation, based on unit volume), output (the compound of formation, based on the carbon source that consumes), product forms speed (compound of formation is based on the time).
Guidance about the genetic engineering method of routine for example separates chromosomal DNA, plasmid DNA, and performance constraint enzymes etc. see (molecular cloning experiment instruction (1989), press of cold spring harbor laboratory) such as Sambrook.See (applied microbiology and biotechnologys 29 such as Thierbach about the guidance that in bar shaped bacteria, transforms and engage, 356-362 (1988)), (bacteriology magazine 172 such as Schafer, 1663-1666 (1990) and gene 145,69-73 (1994)), and Schwarzer and Puhler (biology/technology 9,84-87 (1991)).
Only the carrier that duplicates of limited extent is meant plasmid vector, and it duplicates or do not duplicate down according to host or carrier culture condition.Therefore, only be lower than 31 ℃ just the temperature sensitivity plasmid of the bar shaped bacteria of reproducibles (US-A-6,303,383) are described by Nakamura etc.
The present invention also provides the bar shaped bacteria that produces L-Methionin, particularly excellent bacillus Pseudomonas, it is characterized in that these bacteriums are used for the open reading frame (ORF) that Methionin is produced except existing at natural site (locus), outside gene or allelic at least one copy, in each case, second in each case, randomly the 3rd or the 4th site have the corresponding open reading frame (ORF) of integration form, gene or allelic second, randomly the 3rd or the 4th copy, this specific second, randomly the 3rd or the 4th site do not have can/make can be in microorganism the nucleotide sequence of episomal replication, do not have energy swivel base/feasible nucleotide sequence that swivel base takes place, and do not authorize the nucleotide sequence of antibiotics resistance.
The present invention also provides the method for producing L-Methionin, may further comprise the steps:
A) under the condition that described open reading frame (ORF), gene or allelotrope are expressed, fermentation of coryneform bacteria is Corynebacterium glutamicum especially, and these characteristic of bacteria are except in natural site
(locus) exists and is used for the open reading frame (ORF) that Methionin is produced, outside gene or allelic at least one copy, in each case, second in each case, randomly the 3rd or the 4th site also have the corresponding open reading frame (ORF) of integration form, gene or allelic second, randomly the 3rd or the 4th copy, this specific second, randomly the 3rd or the 4th site do not have can/make can be in microorganism the nucleotide sequence of episomal replication, there is not energy swivel base/feasible nucleotide sequence that swivel base takes place, reach the nucleotide sequence of not authorizing antibiotics resistance
B) the L-Methionin in the concentrated fermenting broth,
C) from fermenting broth, separate L-Methionin, randomly
D) with＞(greater than) component and/or the biomass of the fermenting broth of 0-100%.
" be used for open reading frame (ORF), gene or allelic copy that Methionin is produced " and be meant all preferred endogenic open reading frame, gene or allelotrope, its enhancing/mistake expression can have the effect that improvement Methionin is produced.Strengthen the intracellular concentration or active raising that are meant special genes product, protein or enzyme.
These are particularly including following open reading frame, gene or allelotrope: accBC, accDA, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dapA, dapB, dapC, dapD, dapE, dapF, ddh, dps, eno, gap, gap2, gdh, gnd, lysC, lysC
FBR, lysE, msiK, opcA, oxyR, ppc, ppc
FBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwa1, zwf and zwf A213T.These are summarized and explanation in table 1.
These are particularly including the lysC of encoder feedback resistance E.C. 2.7.2.4.
FBRAllelotrope.Various lysC
FBRAllelotrope is summarized and explanation in table 2.
Preferred following lysC
FBRAllelotrope: lysC A279T (with proteic the 279th L-Ala of E.C. 2.7.2.4. of encoding shown in the Threonine displacement SEQ IDNO:2), lysC A279V (with proteic the 279th L-Ala of E.C. 2.7.2.4. of encoding shown in the Xie Ansuan displacement SEQ ID NO:2), lysC S301F (with proteic the 301st Serine of E.C. 2.7.2.4. of encoding shown in the phenylalanine displacement SEQ ID NO:2), lysC T308I (with proteic the 308th Threonine of E.C. 2.7.2.4. of encoding shown in the Isoleucine displacement SEQ ID NO:2), lysCS301Y (with proteic the 301st Serine of E.C. 2.7.2.4. of encoding shown in the tyrosine displacement SEQ ID NO:2), lysC G345D (with proteic the 345th glycine of E.C. 2.7.2.4. of encoding shown in the aspartic acid displacement SEQ ID NO:2), lysC R320G (with proteic the 320th arginine of E.C. 2.7.2.4. of encoding shown in the glycine displacement SEQ ID NO:2), lysC T311I (with proteic the 311st Threonine of E.C. 2.7.2.4. of encoding shown in the Isoleucine displacement SEQ ID NO:2), lysC S381F (with proteic the 381st Serine of E.C. 2.7.2.4. of encoding shown in the phenylalanine displacement SEQ ID NO:2).
Particularly preferably be lysC
FBRAllelotrope lysC T311I (with proteic the 311st Threonine of E.C. 2.7.2.4. of encoding shown in the Isoleucine displacement SEQID NO:2), its nucleotides sequence is shown in SEQ ID NO:3; The proteic aminoacid sequence of E.C. 2.7.2.4. of coding is shown in SEQ ID NO:4.
The described open reading frame that Methionin produces, gene or allelic the second of being used for, randomly the 3rd or the 4th copy can be integrated into second, randomly the 3rd or the 4th site in each case.Can use following open reading frame, gene or nucleotide sequence: aecD, ccpA1, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, menE, mqo, pck, pgi, poxB and zwa2, particularly gene aecD, gluA, gluB, gluC, gluD and pck.These are summarized and explanation in table 3.
Described site not only comprises the coding region of described open reading frame or gene certainly, also comprise be positioned at the upstream with express and regulate relevant zone or nucleotide sequence, ribosome bind site for example, promotor, regulate proteic binding site, regulate the binding site and the attenuator of Yeast Nucleic Acid.These zones generally are positioned at the 1-800 of upstream of coding region, 1-600, and 1-400,1-200 is in 1-100 or 1-50 the Nucleotide scope.Equally, also comprise the zone that is positioned at the downstream, transcription terminator for example, these zones generally are positioned at the 1-400 in coding downstream, 1-200,1-100 is in 1-50 or 1-25 the Nucleotide scope.
Can use intrachromosomal intergenic region in addition, promptly not have the nucleotide sequence of encoding function.At last, can also use prophage or the defective phage that comprises in the karyomit(e).
Prophage is meant a kind of phage, its genome particularly, and its genome with the host duplicates and does not take place the formation of infectious particles.Defective phage is meant a kind of prophage, its genome particularly, and it loses the ability that forms so-called infectious particles owing to various results of mutation.Defective phage is also referred to as recessive phage.Prophage and defective phage are present in its host's the karyomit(e) with integration form usually.In the prior art about its for example teaching material described (bacterium and the phage genetics of Edward A.Birge of being described in further detail, 3rded., Springer-Verlag, USA New York 1994), perhaps described (the Bakterienviren of teaching material of S.Klaus etc., Gustav Fischer Verlag, Jena, Germany 1992).
Table 1: be used for open reading frame, gene and allelotrope that Methionin is produced
| Title | Coded enzyme or proteinic description | Reference | Accession number |
| accBC | Acyl-CoA carboxylase EC 6.3.4.14 (acyl-CoA carboxylase) | Jager et al.Archives ofMicrobiology(1996)166：76-82EP1108790；WO0100805 | U35023 AX123524 AX066441 |
| accDA | Acyl-CoA carboxylase EC 6.4.1.2 (acyl group axle enzyme A carboxylase) | EP1055725EP1108790WO0100805 | AX121013 AX066443 |
| cstA | The hungry albumin A of carbon (the hungry albumin A of carbon) | EP1108790WO0100804 | AX120811 AX066109 |
| cysD | Sulfate adenylyl transferase subunit II EC 2.7.7.4 (sulfate adenylyl transferase chainlet) | EP1108790 | AX123177 |
| cysE | Serine acetyltransferase EC 2.3.1.30 (serine acetyltransferase) | EP1108790WO0100843 | AX122902 AX063961 |
| cysH | 3 '-adenosine phosphate sulfate reduction enzyme EC 1.8.99.4 (3 '-adenosine phosphate-5 '-phosphinylidyne sulfate reduction enzyme) | EP1108790WO0100842 | AX123178 AX066001 |
| cysK | Cysteine synthase EC 4.2.99.8 (cysteine synthase) | EP1108790WO0100843 | AX122901 AX063963 |
| cysN | Sulfate adenylyl transferase subunit I EC 2.7.7.4 (sulfate adenylyl transferase) | EP1108790 | AX123176 AX127152 |
| cysQ | Translocator CysQ (translocator cysQ) | EP1108790WO0100805 | AX127145 AX066423 |
| dapA | Dihydro-2, dipicolimic acid 2 synthase EC 4.2.1.52 (dihydro-2, dipicolimic acid 2 synthase) | Bonnassie et al.NucleicAcids Research 18：6421(1990)Pisabarro et al.，Journal ofBacteriology 175：2743-2749(1993)EP1108790WO0100805EP0435132EP1067192EP1067193 | X53993 Z21502 AX123560 AX063773 |
| dapB | Dihydro-2, dipicolimic acid 2 reductase enzyme EC 1.3.1.26 (dihydro-2, dipicolimic acid 2 reductase enzyme) | EP1108790WO0100843EP1067192 | AX127149 AX063753 AX137723 |
| EP1067193 Pisabarro et al.，Journal of Bacteriology175： 2743-2749(1993) JP1998215883 JP1997322774 JP1997070291 JP1995075578 | AX137602 X67737 Z21502 E16749 E14520 E12773 E08900 | ||
| dapC | N-succinyl-keto-amine pimelic acid transaminase EC 2.6.1.17 (N-succinyl-keto-amine pimelic acid transaminase) | EP1108790 WO0100843 EP1136559 | AX127146 AX064219 |
| dapD | Tetrahydrochysene-2, dipicolimic acid 2 succinylation enzyme EC 2.3.1.117 (tetrahydrochysene-2, dipicolimic acid 2 succinylation enzyme) | EP1108790 WO0100843 Wehrmann et al.Journal of Bacteriology 180：3159-3165(1998) | AX127146 AX063757 AJ004934 |
| dapE | N-succinyl-diamino acid pimelic acid takes off succinylation enzyme EC 3.5.1.18 (N-succinyl-diamino acid pimelic acid takes off the succinylation enzyme) | EP1108790 WO0100843 Wehrmann et al. Microbiology 140：3349-3356 (1994) | AX127146 AX063749 X81379 |
| dapF | EC 5.1.1.7 (diamino acid pimelic acid epimerase) | EP1108790 WO0100843 EP1085094 | AX127149 AX063719 AX137620 |
| ddh | Diamino acid pimelic acid desaturase EC 1.4.1.16 (diamino acid pimelic acid desaturase) | EP1108790 WO0100843 Ishino et al.，Nucleic Acids Research 15：3917-3917 (1987) JP1997322774 JP1993284970 Kim et al.，Journal of Microbiology and Biotechnology 5：250-256 (1995) | AX127152 AX063759 Y00151 E14511 E05776 D87976 |
| dps | DNA protected protein (protected protein in the hungry process) | EP1108790 | AX127153 |
| eno | Hydratase, phosphoenolpyruvate EC 4.2.1.11 (Hydratase, phosphoenolpyruvate) | EP1108790 WO0100844 EP1090998 Hermann et al.， Electrophoresis 19：3217-3221(1998) | AX127146 AX064945 AX136862 |
| gap | Glyceraldehyde-3-phosphate dehydrogenase EC 1.2.1.12 | EP1108790 WO0100844 | AX127148 AX064941 |
| (glyceraldehyde-3-phosphate dehydrogenase) | Eikmanns et al.，Journal of Bacteriology 174：6076-6086(1992) | X59403 | |
| gap2 | Glyceraldehyde-3-phosphate dehydrogenase EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase 2) | EP1108790 WO0100844 | AX127146 AX064939 |
| gdh | Glutamate dehydrogenase EC 1.4.1.4 (glutamate dehydrogenase) | EP1108790 WO0100844 Boermann et al.，Molecular Microbiology 6：317-326 (1992). Guyonvarch et al.，NCBI | AX127150 AX063811 X59404 X72855 |
| gnd | 6-Phosphogluconic dehydrogenase EC 1.1.1.44 (6-Phosphogluconic dehydrogenase) | EP1108790 WO0100844 | AX127147 AX121689 AX065125 |
| lysC | E.C. 2.7.2.4. EC 2.7.2.4 (E.C. 2.7.2.4.) | EP1108790 WO0100844 Kalinowski et al.，Molecular Microbiology 5：1197-204 (1991) | AX120365 AX063743 X57226 |
| lysC FBR | E.C. 2.7.2.4. feedback resistance (fbr) EC 2.7.2.4 (E.C. 2.7.2.4. fbr) | See Table 2 | |
| lysE | Methionin output albumen (Methionin output albumen) | EP1108790 WO0100843 Vrljic et al.，Molecular Microbiology 22：815-826 (1996) | AX123539 AX123539 X96471 |
| msiK | Sugar input albumen (multiple sugar input albumen) | EP1108790 | AX120892 |
| opcA | Glucose-6-phosphate dehydrogenase (G6PD) (glucose-6-phosphate dehydrogenase (G6PD) subunit) | WO0104325 | AX076272 |
| oxyR | Transcriptional (transcriptional) | EP1108790 | AX122198 AX127149 |
| ppc FBR | Phosphoenolpyruvate carboxylase feedback resistance EC 4.1.1.31 (Phosphoenolpyruvate carboxylase feedback resistance) | EP0723011 WO0100852 | |
| ppc | Phosphoenolpyruvate carboxylase EC 4.1.1.31 (Phosphoenolpyruvate carboxylase) | EP1108790 O′Reagan et al.，Gene 77 (2)：237-251(1989) | AX127148 AX123554 M25819 |
| pgk | Phosphoglyceric kinase EC 2.7.2.3 (phosphoglyceric kinase) | EP1108790 WO0100844 Eikmanns，Journal of Bacteriology 174： | AX121838 AX127148 AX064943 X59403 |
| 6076-6086(1992) | |||
| pknA | Protein kinase A (protein kinase A) | EP1108790 | AX120131 AX120085 |
| pknB | Protein kinase B (protein kinase B) | EP1108790 | AX120130 AX120085 |
| pknD | Protein kinase D (protein kinase D) | EP1108790 | AX127150 AX122469 AX122468 |
| pknG | Protein kinase G (protein kinase G) | EP1108790 | AX127152 AX123109 |
| ppsA | Phosphoenolpyruvate synthase EC 2.7.9.2 (phosphoenolpyruvate synthase) | EP1108790 | AX127144 AX120700 AX122469 |
| ptsH | Phosphotransferase system albumen H EC 2.7.1.69 (phosphotransferase system composition H) | EP1108790 WO0100844 | AX122210 AX127149 AX069154 |
| ptsI | Phosphotransferase system enzyme I EC 2.7.3.9 | EP1108790 | AX122206 AX127149 |
| ptsM | Glucose specificity phosphotransferase system enzyme II EC 2.7.1.69 (glucose phosphotransferase system enzyme II) | Lee et al.，FEMS Microbiology Letters 119(1-2)：137-145(1994) | L18874 |
| pyc | Pyruvate carboxylase EC 6.4.1.1 (pyruvate carboxylase) | WO9918228 Peters-Wendisch et al.， Microbiology 144：915-927 (1998) | A97276 Y09548 |
| pyc P458S | Pyruvate carboxylase EC 6.4.1.1 (pyruvate carboxylase) amino acid exchange P458S | EP1108790 | |
| sigC | Sigma factor C EC 2.7.7.6 (the outer substituting sigma factor of the function C of kytoplasm) | EP1108790 | AX120368 AX120085 |
| sigD | RNA polymerase Sigma factor D EC 2.7.7.6 (the RNA polymerase sigma factor) | EP1108790 | AX120753 AX127144 |
| sigE | Sigma factor E EC 2.7.7.6 (the outer substituting sigma factor of the function E of kytoplasm) | EP1108790 | AX127146 AX121325 |
| sigH | Sigma factor H EC 2.7.7.6 (sigma factor S igH) | EP1108790 | AX127145 AX120939 |
| sigM | Sigma factor M EC 2.7.7.6 (sigma factor S igM) | EP1108790 | AX123500 AX127145 |
| tal | Transaldolase | WO0104325 | AX076272 |
| EC 2.2.1.2 (transaldolase) | |||
| thyA | Thymidylate synthase EC 2.1.1.45 (thymidylate synthase) | EP1108790 | AX121026 AX127145 |
| tkt | Transketolase EC 2.2.1.1 (transketolase) | Ikeda et al.，NCBI | AB023377 |
| tpi | Triose-phosphate isomerase EC 5.3.1.1 (triose-phosphate isomerase) | Eikmanns，Journal of Bacteriology 174： 6076-6086 (1992) | X59403 |
| zwa1 | Growth factor-21 (growth factor-21) | EP1111062 | AX133781 |
| zwf | G-6-P-1-desaturase EC 1.1.1.49 (G-6-P-1-desaturase) | EP1108790 WO0104325 | AX127148 AX121827 AX076272 |
| zwf A213T | G-6-P-1-desaturase EC 1.1.1.49 (G-6-P-1-desaturase) amino acid exchange A213T | EP1108790 |
The lysC of table 2 encoder feedback resistance E.C. 2.7.2.4.
FBRAllelotrope
| The allelotrope title | Further information | Reference | Accession number |
| lysC FBR-E05108 | JP 1993184366-A (sequence 1) | E05108 | |
| lysC FBR-E06825 | lysC A279T | JP 1994062866-A (sequence 1) | E06825 |
| lysC FBR-E06826 | lysC A279T | JP 1994062866-A (sequence 2) | E06826 |
| lysC FBR-E06827 | JP 1994062866-A (sequence 3) | E06827 | |
| lysC FBR-E08177 | JP 1994261766-A (sequence 1) | E08177 | |
| lysC FBR-E08178 | lysC A279T | JP 1994261766-A (sequence 2) | E08178 |
| lysC FBR-E08179 | lysC A279V | JP 1994261766-A (sequence 3) | E08179 |
| lysC FBR-E08180 | lysC S301F | JP 1994261766-A (sequence 4) | E08180 |
| lysC FBR-E08181 | lysC T308I | JP 1994261766-A (sequence 5) | E08181 |
| lysC FBR-E08182 | JP 1994261766-A (sequence 6) | E08182 | |
| lysC FBR-E12770 | JP 1997070291-A (sequence 13) | E12770 | |
| lysC FBR-E14514 | JP 1997322774-A (sequence 9) | E14514 | |
| lysC FBR-E16352 | JP 1998165180-A (sequence 3) | E16352 | |
| lysC FBR-E16745 | JP 1998215883-A (sequence 3) | E16745 | |
| lysC FBR-E16746 | JP 1998215883-A (sequence 4) | E16746 | |
| lysC FBR-I74588 | US 5688671-A (sequence 1) | 174588 | |
| lysC FBR-I74589 | lysC A279T | US 5688671-A (sequence 2) | I74589 |
| lysC FBR-I74590 | US 5688671-A (sequence 7) | I74590 | |
| lysC FBR-I74591 | lysC A279T | US 5688671-A (sequence 8) | I74591 |
| lysC FBR-I74592 | US 5688671-A (sequence 9) | I74592 | |
| lysC FBR-I74593 | lysC A279T | US 5688671-A (sequence 10) | I74593 |
| lysC FBR-I74594 | US 5688671-A (sequence 11) | I74594 | |
| lysC FBR-I74595 | lysC A279T | US 5688671-A (sequence 12) | I74595 |
| lysC FBR-I74596 | US 5688671-A (sequence 13) | I74596 | |
| lysC FBR-I74597 | lysC A279T | US 5688671-A (sequence 14) | I74597 |
| lysC FBR-X57226 | lysC S301Y | EP0387527 Kalinowski et al.， Molecular and General Genetics 224：317-324(1990) | X57226 |
| lysC FBR-L16848 | lysC G345D | Follettie and Sinskey NCBI Nucleotide Database(1990) | L16848 |
| lysC FBR-L27125 | lysC R320G lysC G345D | Jetten et al.，Applied Microbiology Biotechnology 43：76-82(1995) | L27125 |
| lysC FBR | lysC T311I | WO0063388 (sequence 17) | |
| lysC FBR | lysC S301F | US3732144 | |
| lysC FBR | lysC S381F | ||
| lysC FBR | JP6261766 (sequence 1) | ||
| lysC FBR | lysC A279T | JP6261766 (sequence 2) | |
| lysC FBR | lysC A279V | JP6261766 (sequence 3) | |
| lysC FBR | lysC S301F | JP6261766 (sequence 4) | |
| lysC FBR | lysC T308I | JP6261766 (sequence 5) |
Table 3 is used to integrate open reading frame, gene and the allelic target site that Methionin is produced
| The gene title | Coded enzyme or proteinic description | Reference | Accession number |
| aecD | β C-S lyase EC 2.6.1.1 (β C-S lyase) | Rossol et al..Journal of Bacteriology 174(9)： 2968-77(1992) | M89931 |
| ccpA1 | Catabolite control albumen (catabolite control albumin A 1) | WO0100844 EP1108790 | AX065267 AX127147 |
| ccpA2 | Catabolite control albumen (catabolite control albumin A 2) | WO0100844 EP1108790 | AX065267 AX121594 |
| citA | Sensor kinase c itA (sensor kinase c itA) | EP1108790 | AX120161 |
| citB | Transcriptional CitB (transcriptional CitB) | EP1108790 | AX120163 |
| citE | Citrate lyase EC 4.1.3.6 (citrate lyase) | WO0100844 EP1108790 | AX065421 AX127146 |
| fda | Fructose-bis phosphate aldolase EC 4.1.2.13 (fructose-1,6-diphosphate zymohexase) | von der Osten et al.，Molecular Microbiology 3(11)：1625-37 (1989) | X17313 |
| gluA | Glutamate transport ATP conjugated protein (glutamate transport ATP is conjugated protein) | Kronemeyer et al.，Journal of Bacteriology 177(5)：1152-8 (1995) | X81191 |
| gluB | L-glutamic acid conjugated protein (L-glutamic acid is conjugated protein) | Kronemeyer et al.，Journal of Bacteriology 177(5)：1152-8 (1995) | X81191 |
| gluC | Glutamate transport permease (glutamate transport system permease) | Kronemeyer et al.，Journal of Bacteriology 177(5)：1152-8 (1995) | X81191 |
| gluD | Glutamate transport permease (glutamate transport system permease) | Kronemeyer et al.，Journal of Bacteriology 177(5)：1152-8 (1995) | X81191 |
| luxR | Transcriptional LuxR (transcriptional LuxR) | WO0100842 EP1108790 | AX065953 AX123320 |
| luxS | Histidine kinase LuxS (histidine kinase LuxS) | EP1108790 | AX123323 AX127145 |
| lysR1 | Transcriptional LysR1 (transcriptional LysR1) | EP1108790 | AX064673 AX127144 |
| lysR2 | Transcriptional activator LysR2 (transcriptional LysR2) | EP1108790 | AX123312 |
| lysR3 | Transcriptional LysR3 (transcriptional LysR3) | WO0100842 EP1108790 | AX065957 AX127150 |
| menE | O-succinyl-phenylformic acid coenzyme A lyase EC 6.2.1.26 (O-succinyl-phenylformic acid coenzyme A lyase) | WO0100843 EP1108790 | AX064599 AX064193 AX127144 |
| mqo | Oxysuccinic acid-quinone oxidoreductase (oxysuccinic acid-quinone oxidoreductase) | Molenaar et al.，Eur.Journal of |
AJ224946 |
| pck | Phosphoenolpyruvate carboxykinase (phosphoenolpyruvate carboxykinase) | WO0100844 | AJ269506 AX065053 |
| pgi | G-6-P isomerase EC 5.3.1.9 (G-6-P isomerase) | EP1087015 EP1108790 | AX136015 AX127146 |
| poxB | Pyruvic oxidase EC 1.2.3.3 (pyruvic oxidase) | WO0100844 EP1096013 | AX064959 AX137665 |
| zwa2 | Cell growth factor 2 (growth factor-2) | EP1106693 EP1108790 | AX113822 AX127146 |
The present invention also provides a kind of method of producing bar shaped bacteria, and described bacterium produces L-Methionin, and described method comprises:
A) separate at least one desirable ORF, gene or the allelic nucleotide sequence that is used for Methionin production, it randomly comprises expresses and/or conditioning signal,
B) nucleotide sequence with target site offers described ORF, gene or allelic 5 ' and the 3 ' end that Methionin is produced that be used for,
C) desirable ORF, gene or the allelic nucleotide sequence that preferably will have the target site nucleotide sequence mixes in the carrier, described carrier in bar shaped bacteria, do not duplicate or only limited extent duplicate,
D) with b) or nucleotides sequence column jump c) advance in the bar shaped bacteria,
E) separate such bar shaped bacteria, in the described bar shaped bacteria a) nucleotide sequence mix target site, this target site do not have can/make can be in microorganism the nucleotide sequence of episomal replication, do not have energy swivel base/feasible nucleotide sequence that swivel base takes place, and do not authorize the nucleotide sequence of antibiotics resistance.
The present invention also provides the bar shaped bacteria that produces L-methionine(Met) and/or L-Threonine, particularly excellent bacillus Pseudomonas, it is characterized in that except be used for the open reading frame (ORF) that methionine(Met) production or Threonine are produced in natural site (locus) existence, outside gene or allelic at least one copy, in each case, at second, randomly the 3rd or the 4th site have the corresponding open reading frame (ORF) of integration form, gene or allelic second, randomly the 3rd or the 4th copy, this specific second, randomly the 3rd or the 4th site do not have can/make can be in microorganism the nucleotide sequence of episomal replication, do not have energy swivel base/feasible nucleotide sequence that swivel base takes place, and do not authorize the nucleotide sequence of antibiotics resistance.
The present invention also provides the method for a kind of L-of production methionine(Met) and/or L-Threonine, and it may further comprise the steps:
A) make described open reading frame (ORF), the condition bottom fermentation bar shaped bacteria that gene or allelotrope are expressed, Corynebacterium glutamicum particularly, these bacteriums are characterised in that the open reading frame (ORF) that is used for methionine(Met) production or Threonine production except existing at natural site (locus), outside gene or allelic at least one copy, in each case at second, randomly the 3rd or the 4th site have the open reading frame (ORF) of integration form, gene or allelic second, randomly the 3rd or the 4th copy, this specific second, randomly the 3rd or the 4th site do not have can/make can be in microorganism the nucleotide sequence of episomal replication, there is not energy swivel base/feasible nucleotide sequence that swivel base takes place, reach the nucleotide sequence of not authorizing antibiotics resistance
B) L-methionine(Met) and/or the L-Threonine in the concentrated fermenting broth,
C) from fermenting broth, separate L-methionine(Met) and/or L-Threonine, randomly
D) randomly with＞(greater than) component and/or the biomass of the fermenting broth of 0-100%.
" be used for open reading frame (ORF), gene or allelic copy that methionine(Met) is produced " and be meant all preferably endogenous open reading frame, gene or allelotrope, its enhancing/mistake expression can have the effect that the improvement methionine(Met) is produced.
These comprise following open reading frame, gene or allelotrope: accBC, accDA, aecD, cstA, cysD, cysE, cysH, cysR, cysN, cysQ, dps, eno, fda, gap, gap2, gdh, gnd, glyA, hom, hom
FBR, lysC, lysC
FBR, metA, metB, metE, metH, metY, msiK, opcA, oxyR, ppc, ppc
FBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwa1, zwf and zwf A213T.These are summarized and explanation in table 4.These are particularly including the lysC of encoder feedback resistance E.C. 2.7.2.4.
FBRAllelotrope (seeing Table 2), and the hom of encoder feedback resistance homoserine dehydrogenase
FBRAllelotrope.
Describedly be used for open reading frame (ORF) that methionine(Met) produces, gene or allelic second, randomly the 3rd or the 4th copy can be incorporated into second, randomly the 3rd or the 4th site in each case.Can use following open reading frame, gene or nucleotide sequence: brnE, brnF, brnQ, ccpA1, ccpA2, citA, citB, citE, ddh, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, menE, metD, metK, pck, pgi, poxB and zwa2.These are summarized and explanation in table 5.。
Described site not only comprises the coding region of described open reading frame or gene certainly, also comprise be positioned at the upstream with express and regulate relevant zone or nucleotide sequence, ribosome bind site for example, promotor, regulate proteic binding site, regulate the binding site and the attenuator of Yeast Nucleic Acid.These zones generally are positioned at the 1-800 of upstream of coding region, 1-600, and 1-400,1-200 is in 1-100 or 1-50 the Nucleotide scope.Equally, also comprise the zone that is positioned at the downstream, transcription terminator for example, these zones generally are positioned at the 1-400 in downstream, coding region, 1-200,1-100 is in 1-50 or 1-25 the Nucleotide scope.
Can use intrachromosomal intergenic region in addition, promptly not have the nucleotide sequence of encoding function.At last, can also use prophage or the defective phage that comprises in the karyomit(e).
Table 4: be used for open reading frame, gene and allelotrope that methionine(Met) is produced
| Title | Coded enzyme or proteinic description | Reference | Accession number |
| accBC | Acyl-CoA carboxylase EC 6.3.4.14 (acyl-CoA carboxylase) | Jager et al.Archives of Microbiology (1996)166：76-82 EP1108790； WO0100805 | U35023 AX123524 AX066441 |
| accDA | Acyl-CoA carboxylase EC 6.4.1.2 (acyl-CoA carboxylase) | EP1055725 EP1108790 WO0100805 | AX121013 AX066443 |
| aecD | Cystathionine-β-lyase EC 4.4.1.8 (cystathionine-β-lyase) | Rossol et al.，Journal of Bacteriology 174：2968-2977(1992) | M89931 |
| cstA | The hungry albumin A of carbon (the hungry albumin A of carbon) | EP1108790 WO0100804 | AX120811 AX066109 |
| cysD | Sulfate adenylyl transferase subunit II EC 2.7.7.4 (sulfate adenylyl transferase chainlet) | EP1108790 | AX123177 |
| cysE | Serine acetyltransferase EC 2.3.1.30 (serine acetyltransferase) | EP1108790 WO0100843 | AX122902 AX063961 |
| cysH | 3 '-adenosine phosphate sulfate reduction enzyme EC 1.8.99.4 (3 '-adenosine phosphate-5 '-phosphinylidyne sulfate reduction enzyme) | EP1108790 WO0100842 | AX123178 AX066001 |
| cysK | Cysteine synthase EC 4.2.99.8 (cysteine synthase) | EP1108790 WO0100843 | AX122901 AX063963 |
| cysN | Sulfate adenylyl transferase subunit I EC 2.7.7.4 (sulfate adenylyl transferase) | EP1108790 | AX123176 AX127152 |
| cysQ | Translocator CysQ (translocator cysQ) | EP1108790 WO0100805 | AX127145 AX066423 |
| dps | DNA protected protein (protected protein in the hungry process) | EP1108790 | AX127153 |
| eno | Hydratase, phosphoenolpyruvate EC 4.2.1.11 (Hydratase, phosphoenolpyruvate) | EP1108790 WO0100844 EP1090998 Hermann et al.， Electrophoresis 19：3217-3221(1998) | AX127146 AX064945 AX136862 |
| fda | Fructose-bis phosphate aldolase EC 4.1.2.13 (fructose-1,6-diphosphate zymohexase) | von der Osten et al.， Molecular Microbiology 3 (11)：1625-37(1989) | X17313 |
| gap | Glyceraldehyde-3-phosphate dehydrogenase | EP1108790 | AX127148 |
| EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase) | WO0100844 Eikmanns et al.，Journal of Bacteriology 174：6076-6086(1992) | AX064941 X59403 | |
| gap2 | Glyceraldehyde-3-phosphate dehydrogenase EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase 2) | EP1108790 WO0100844 | AX127146 AX064939 |
| gdh | Glutamate dehydrogenase EC 1.4.1.4 (glutamate dehydrogenase) | EP1108790 WO0100844 Boermann et al.，Molecular Microbiology 6：317-326 (1992). Guyonvarch et al.，NCBI | AX127150 AX063811 X59404 X72855 |
| glyA | Glycine/serine hydroxymethylase EC 2.1.2.1 (glycine/serine hydroxymethylase) | EP1108790 | AX127146 AX121194 |
| gnd | 6-Phosphogluconic dehydrogenase EC 1.1.1.44 (6-Phosphogluconic dehydrogenase) | EP1108790 WO0100844 | AX127147 AX121689 AX065125 |
| hom | Homoserine dehydrogenase EC 1.1.1.3 (homoserine dehydrogenase) | Peoples et al.，Molecular Microbiology 2：63-72(1988) | Y00546 |
| hom FBR | Homoserine dehydrogenase feedback resistance EC 1.1.1.3 (homoserine dehydrogenase fbr) | Reinscheid et al.，Journal of Bacteriology 173：3228-30 (1991) | |
| lysC | E.C. 2.7.2.4. EC 2.7.2.4 (E.C. 2.7.2.4.) | EP1108790 WO0100844 Kalinowski et al.，Molecular Microbiology 5：1197-204 (1991) | AX120365 AX063743 X57226 |
| lysC FBR | E.C. 2.7.2.4. feedback resistance (fbr) EC 2.7.2.4 (E.C. 2.7.2.4. fbr) | See Table 2 | |
| metA | Homoserine acetyltransferase EC 2.3.1.31 (homoserine acetyltransferase) | Park et al.，Molecular Cells 8：286-94(1988) | AF052652 |
| metB | Cystathionine-γ-lyase EC 4.4.1.1 (cystathionine-γ-synthase) | Hwang et al.，Molecular Cells 9：300-308(1999) | AF126953 |
| metE | Homoserine methyltransgerase EC 2.1.1.14 (homoserine methyltransgerase) | EP1108790 | AX127146 AX121345 |
| metH | Homoserine methyltransgerase (vitamin B12 dependency) EC 2.1.1.14 (homoserine methyltransgerase) | EP1108790 | AX127148 AX121747 |
| metY | Acetylhomoserine sulfhydrolase (acetylhomoserine sulfhydrolase) | EP1108790 | AX120810 AX127145 |
| msiK | Sugar input albumen (multiple sugar input albumen) | EP1108790 | AX120892 |
| opcA | Glucose-6-phosphate dehydrogenase (G6PD) (glucose-6-phosphate dehydrogenase (G6PD) subunit) | WO0104325 | AX076272 |
| oxyR | Transcriptional (transcriptional) | EP1108790 | AX122198 AX127149 |
| ppc FBR | Phosphoenolpyruvate carboxylase feedback resistance EC 4.1.1.31 (Phosphoenolpyruvate carboxylase feedback resistance) | EP0723011 WO0100852 | |
| ppc | Phosphoenolpyruvate carboxylase EC 4.1.1.31 (Phosphoenolpyruvate carboxylase) | EP1108790 O′Reagan et al.，Gene 77 (2)：237-251(1989) | AX127148 AX123554 M25819 |
| pgk | Phosphoglyceric kinase EC 2.7.2.3 (phosphoglyceric kinase) | EP1108790 WO0100844 Eikmanns，Journal of Bacteriology 174： 6076-6086(1992) | AX121838 AX127148 AX064943 X59403 |
| pknA | Protein kinase A (protein kinase A) | EP1108790 | AX120131 AX120085 |
| pknB | Protein kinase B (protein kinase B) | EP1108790 | AX120130 AX120085 |
| pknD | Protein kinase D (protein kinase D) | EP1108790 | AX127150 AX122469 AX122468 |
| pknG | Protein kinase G (protein kinase G) | EP1108790 | AX127152 AX123109 |
| ppsA | Phosphoenolpyruvate synthase EC 2.7.9.2 (phosphoenolpyruvate synthase) | EP1108790 | AX127144 AX120700 AX122469 |
| ptsH | Phosphotransferase system albumen H EC 2.7.1.69 (phosphotransferase system composition H) | EP1108790 WO0100844 | AX122210 AX127149 AX069154 |
| ptsI | Phosphotransferase system enzyme I EC 2.7.3.9 | EP1108790 | AX122206 AX127149 |
| ptsM | Glucose specificity phosphotransferase system enzyme II EC 2.7.1.69 (glucose phosphotransferase system enzyme II) | Lee et al.，FEMS Microbiology Letters 119(1-2)：137-145(1994) | L18874 |
| pyc | Pyruvate carboxylase EC 6.4.1.1 (pyruvate carboxylase) | WO9918228 Peters-Wendisch et al.， Microbiology 144：915-927 (1998) | A97276 Y09548 |
| pyc P458S | Pyruvate carboxylase EC 6.4.1.1 (pyruvate carboxylase) amino acid exchange P458S | EP1108790 | |
| sigC | Sigma factor C EC 2.7.7.6 (the outer substituting sigma factor of the function C of kytoplasm) | EP1108790 | AX120368 AX120085 |
| sigD | RNA polymerase Sigma factor D EC 2.7.7.6 (the RNA polymerase sigma factor) | EP1108790 | AX120753 AX127144 |
| sigE | Sigma factor E EC 2.7.7.6 (the outer substituting sigma factor of the function E of kytoplasm) | EP1108790 | AX127146 AX121325 |
| sigH | Sigma factor H EC 2.7.7.6 (sigma factor S igH) | EP1108790 | AX127145 AX120939 |
| sigM | Sigma factor M EC 2.7.7.6 (sigma factor S igM) | EP1108790 | AX123500 AX127145 |
| tal | Transaldolase EC 2.2.1.2 (transaldolase) | WO0104325 | AX076272 |
| thyA | Thymidylate synthase EC 2.1.1.45 (thymidylate synthase) | EP1108790 | AX121026 AX127145 |
| tkt | Transketolase EC 2.2.1.1 (transketolase) | Ikeda et al.，NCBI | AB023377 |
| tpi | Triose-phosphate isomerase EC 5.3.1.1 (triose-phosphate isomerase) | Eikmanns，Journal of Bacteriology 174： 6076-6086 (1992) | X59403 |
| zwa1 | Growth factor-21 (growth factor-21) | EP1111062 | AX133781 |
| zwf | G-6-P-1-desaturase EC 1.1.1.49 (G-6-P-1-desaturase) | EP1108790 WO0104325 | AX127148 AX121827 AX076272 |
| zwf A213T | G-6-P-1-desaturase EC 1.1.1.49 (G-6-P-1-desaturase) amino acid exchange A213T | EP1108790 |
Table 5 is used to integrate open reading frame, gene and the allelic target site that methionine(Met) is produced
| The gene title | Coded enzyme or proteinic description | Reference | Accession number |
| brnE | Branched-chain amino acid translocator (branched-chain amino acid translocator) | EP1096010 | AX137709 AX137714 |
| brnF | Branched-chain amino acid translocator (branched-chain amino acid translocator) | EP1096010 | AX137709 AX137714 |
| brnQ | Branched-chain amino acid carrier proteins (branched-chain amino acid movement system carrier proteins) | Tauch et al.，Archives of Microbiology 169(4)：303-12(1998) WO0100805 EP1108790 | M89931 AX066841 AX127150 |
| ccpA1 | Catabolite control albumen (catabolite control albumin A 1) | WO0100844 EP1108790 | AX065267 AX127147 |
| ccpA2 | Catabolite control albumen (catabolite control albumin A 2) | WO0100844 EP1108790 | AX065267 AX121594 |
| citA | Sensor kinase c itA (sensor kinase c itA) | EP1108790 | AX120161 |
| citB | Transcriptional CitB (transcriptional CitB) | EP1108790 | AX120163 |
| citE | Citrate lyase EC 4.1.3.6 (citrate lyase) | WO0100844 EP1108790 | AX065421 AX127146 |
| ddh | Diamino acid pimelic acid desaturase EC 1.4.1.16 (diamino acid pimelic acid desaturase) | Ishino et al.，Nucleic Acids Research 15：3917-3917(1987) EP1108790 | S07384 AX127152 |
| gluA | Glutamate transport ATP conjugated protein (glutamate transport ATP is conjugated protein) | Kronemeyer et al.，Journal of Bacteriology 177(5)：1152-8 (1995) | X81191 |
| gluB | L-glutamic acid conjugated protein (L-glutamic acid is conjugated protein) | Kronemeyer et al.，Journal of Bacteriology 177(5)：1152-8 (1995) | X81191 |
| gluC | Glutamate transport permease (glutamate transport system permease) | Kronemeyer et al.，Journal of Bacteriology 177(5)：1152-8 (1995) | X81191 |
| gluD | Glutamate transport permease (glutamate transport system permease) | Kronemeyer et al.，Journal of Bacteriology 177(5)：1152-8 (1995) | X81191 |
| luxR | Transcriptional LuxR (transcriptional LuxR) | WO0100842 EP1108790 | AX065953 AX123320 |
| luxS | Histidine kinase LuxS (histidine kinase LuxS) | EP1108790 | AX123323 AX127145 |
| lysR1 | Transcriptional LysR1 (transcriptional LysR1) | EP1108790 | AX064673 AX127144 |
| lysR2 | Transcriptional activator LysR2 | EP1108790 | AX123312 |
| (transcriptional LysR2) | |||
| lysR3 | Transcriptional LysR3 (transcriptional LysR3) | WO0100842 EP1108790 | AX065957 AX127150 |
| menE | O-succinyl-phenylformic acid coenzyme A lyase EC 6.2.1.26 (O-succinyl-phenylformic acid coenzyme A lyase) | WO0100843 EP1108790 | AX064599 AX064193 AX127144 |
| metD | Transcriptional MetD (transcriptional MetD) | EP1108790 | AX123327 AX127153 |
| metK | Methionine(Met) acyl glycosides transferring enzyme EC 2.5.1.6 (S-adenosylmethionine synthetic enzyme) | WO0100843 EP1108790 | AX063959 AX127148 |
| pck | Phosphoenolpyruvate carboxykinase (phosphoenolpyruvate carboxykinase) | WO0100844 | AJ269506 AX065053 |
| pgi | G-6-P isomerase EC 5.3.1.9 (G-6-P isomerase) | EP1087015 EP1108790 | AX136015 AX127146 |
| poxB | Pyruvic oxidase EC 1.2.3.3 (pyruvic oxidase) | WO0100844 EP1096013 | AX064959 AX137665 |
| zwa2 | Cell growth factor 2 (growth factor-2) | EP1106693 EP1108790 | AX113822 AX127146 |
" be used for open reading frame (ORF), gene or allelic copy that Threonine is produced " and be meant that its all enhancing/mistake expression can have open reading frame, gene or the allelotrope of the effect of improvement Threonine production.
These comprise following open reading frame, gene or allelotrope: accBC, accDA, cstA, cysD, cysE, cysH, cysI, cysN, cysQ, dps, eno, fda, gap, gap2, gdh, gnd, hom, hom
FBR, lysC, lysC
FBR, msiK, opcA, oxyR, ppc, ppc
FBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, thrB, thrC, thrE, zwa1, zwf and zwfA213T.These are summarized and explanation in table 6.These are particularly including the lysC of encoder feedback resistance E.C. 2.7.2.4.
FBRThe hom of allelotrope (seeing Table 2) and encoder feedback resistance homoserine dehydrogenase
FBRAllelotrope.
Be used for the open reading frame, gene of Threonine production or allelic second, randomly the 3rd or the 4th copy can be integrated into second, randomly the 3rd or the 4th site in each case.Can use following open reading frame, gene or nucleotide sequence: ccpA1, ccpA2, citA, citB, citE for this reason, ddh, gluA, gluB, gluC, gluD, glyA, ilvA, ilvBN, ilvC, ilvD, luxR, luxS, lysR1, lysR2, lysR3, mdh, menE, metA, metD, pck, poxB, sigB and zwa2.These are summarized and explanation in table 7.
Described site not only comprises the coding region of described open reading frame or gene certainly, also comprise be positioned at the upstream with express and regulate relevant zone or nucleotide sequence, ribosome bind site for example, promotor, regulate proteic binding site, regulate the binding site and the attenuator of Yeast Nucleic Acid.These zones generally are positioned at the 1-800 of upstream of coding region, 1-600, and 1-400,1-200 is in 1-100 or 1-50 the Nucleotide scope.Equally, also comprise the zone that is positioned at the downstream, transcription terminator for example, these zones generally are positioned at the 1-400 in coding downstream, 1-200,1-100 is in 1-50 or 1-25 the Nucleotide scope.
Can use intrachromosomal intergenic region in addition, promptly not have the nucleotide sequence of encoding function.At last, can also use prophage or the defective phage that comprises in the karyomit(e).
Table 6 is used for open reading frame, gene and the allelotrope that Threonine is produced
| Title | Coded enzyme or proteinic description | Reference | Accession number |
| accBC | Acyl-CoA carboxylase EC 6.3.4.14 (acyl-CoA carboxylase) | Jager et al.Archives of Microbiology (1996)166：76-82 EP1108790； WO0100805 | U35023 AX123524 AX066441 |
| accDA | Acyl-CoA carboxylase EC 6.4.1.2 (acyl-CoA carboxylase) | EP1055725 EP1108790 WO0100805 | AX121013 AX066443 |
| cstA | The hungry albumin A of carbon (the hungry albumin A of carbon) | EP1108790 WO0100804 | AX120811 AX066109 |
| cysD | Sulfate adenylyl transferase subunit II EC 2.7.7.4 (sulfate adenylyl transferase chainlet) | EP1108790 | AX123177 |
| cysE | Serine acetyltransferase EC 2.3.1.30 (serine acetyltransferase) | EP1108790 WO0100843 | AX122902 AX063961 |
| cysH | 3 '-adenosine phosphate sulfate reduction enzyme EC 1.8.99.4 (3 '-adenosine phosphate-5 '-phosphinylidyne sulfate reduction enzyme) | EP1108790 WO0100842 | AX123178 AX066001 |
| cysK | Cysteine synthase EC 4.2.99.8 (cysteine synthase) | EP1108790 WO0100843 | AX122901 AX063963 |
| cysN | Sulfate adenylyl transferase subunit I EC 2.7.7.4 (sulfate adenylyl transferase) | EP1108790 | AX123176 AX127152 |
| cysQ | Translocator CysQ (translocator cysQ) | EP1108790 WO0100805 | AX127145 AX066423 |
| dps | DNA protected protein (protected protein in the hungry process) | EP1108790 | AX127153 |
| eno | Hydratase, phosphoenolpyruvate EC 4.2.1.11 (Hydratase, phosphoenolpyruvate) | EP1108790 WO0100844 EP1090998 Hermann et al.， Electrophoresis 19：3217-3221(1998) | AX127146 AX064945 AX136862 |
| fda | Fructose-bis phosphate aldolase EC 4.1.2.13 (fructose-1,6-diphosphate zymohexase) | von der Osten et al.， Molecular Microbiology 3 (11)：1625-37(1989) | X17313 |
| gap | Glyceraldehyde-3-phosphate dehydrogenase EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase) | EP1108790 WO0100844 Eikmanns etal.，Journal of Bacteriology 174：6076-6086(1992) | AX127148 AX064941 X59403 |
| gap2 | Glyceraldehyde-3-phosphate dehydrogenase EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase 2) | EP1108790 WO0100844 | AX127146 AX064939 |
| gdh | Glutamate dehydrogenase EC 1.4.1.4 (glutamate dehydrogenase) | EP1108790 WO0100844 Boermann et al.，Molecular Microbiology 6：317-326 (1992). Guyonvarch et al.，NCBI | AX127150 AX063811 X59404 X72855 |
| gnd | 6-Phosphogluconic dehydrogenase EC 1.1.1.44 (6-Phosphogluconic dehydrogenase) | EP1108790 WO0100844 | AX127147 AX121689 AX065125 |
| hom | Homoserine dehydrogenase EC 1.1.1.3 (homoserine dehydrogenase) | Peoples et al.，Molecular Microbiology 2：63-72(1988) | Y00546 |
| hom FBR | Homoserine dehydrogenase feedback resistance EC 1.1.1.3 (homoserine dehydrogenase fbr) | Reinscheid et al.，Journal of Bacteriology 173：3228-30 (1991) | |
| lysC | E.C. 2.7.2.4. EC 2.7.2.4 (E.C. 2.7.2.4.) | EP1108790 WO0100844 Kalinowski et al.，Molecular Microbiology 5：1197-204 (1991) | AX120365 AX063743 X57226 |
| lysC FBR | E.C. 2.7.2.4. feedback resistance (fbr) EC 2.7.2.4 (E.C. 2.7.2.4. fbr) | See Table 2 | |
| msiK | Sugar input albumen (multiple sugar input albumen) | EP1108790 | AX120892 |
| opcA | Glucose-6-phosphate dehydrogenase (G6PD) (glucose-6-phosphate dehydrogenase (G6PD) subunit) | WO0104325 | AX076272 |
| oxyR | Transcriptional (transcriptional) | EP1108790 | AX122198 AX127149 |
| ppc FBR | Phosphoenolpyruvate carboxylase feedback resistance EC 4.1.1.31 (Phosphoenolpyruvate carboxylase feedback resistance) | EP0723011 WO0100852 | |
| ppc | Phosphoenolpyruvate carboxylase EC 4.1.1.31 (Phosphoenolpyruvate carboxylase) | EP1108790 O′Reagan et al.，Gene 77 (2)：237-251(1989) | AX127148 AX123554 M25819 |
| pgk | Phosphoglyceric kinase EC 2.7.2.3 (phosphoglyceric kinase) | EP1108790 WO0100844 Eikmanns，Journal of Bacteriology 174： 6076-6086(1992) | AX121838 AX127148 AX064943 X59403 |
| pknA | Protein kinase A | EP1108790 | AX120131 |
| (protein kinase A) | AX120085 | ||
| pknB | Protein kinase B (protein kinase B) | EP1108790 | AX120130 AX120085 |
| pknD | Protein kinase D (protein kinase D) | EP1108790 | AX127150 AX122469 AX122468 |
| pknG | Protein kinase G (protein kinase G) | EP1108790 | AX127152 AX123109 |
| ppsA | Phosphoenolpyruvate synthase EC 2.7.9.2 (phosphoenolpyruvate synthase) | EP1108790 | AX127144 AX120700 AX122469 |
| ptsH | Phosphotransferase system albumen H EC 2.7.1.69 (phosphotransferase system composition H) | EP1108790 WO0100844 | AX122210 AX127149 AX069154 |
| ptsI | Phosphotransferase system enzyme I EC 2.7.3.9 | EP1108790 | AX122206 AX127149 |
| ptsM | Glucose specificity phosphotransferase system enzyme II EC 2.7.1.69 (glucose phosphotransferase system enzyme II) | Lee et al.，FEMS Microbiology Letters 119(1-2)：137-145(1994) | L18874 |
| pyc | Pyruvate carboxylase EC 6.4.1.1 (pyruvate carboxylase) | WO9918228 Peters-Wendisch et al.， Microbiology 144：915-927 (1998) | A97276 Y09548 |
| pyc P458S | Pyruvate carboxylase EC 6.4.1.1 (pyruvate carboxylase) amino acid exchange P458S | EP1108790 | |
| sigC | Sigma factor C EC 2.7.7.6 (the outer substituting sigma factor of the function C of kytoplasm) | EP1108790 | AX120368 AX120085 |
| sigD | RNA polymerase Sigma factor D EC 2.7.7.6 (the RNA polymerase sigma factor) | EP1108790 | AX120753 AX127144 |
| sigE | Sigma factor E EC 2.7.7.6 (the outer substituting sigma factor of the function E of kytoplasm) | EP1108790 | AX127146 AX121325 |
| sigH | Sigma factor H EC 2.7.7.6 (sigma factor S igH) | EP1108790 | AX127145 AX120939 |
| sigM | Sigma factor M EC 2.7.7.6 (sigma factor S igM) | EP1108790 | AX123500 AX127145 |
| tal | Transaldolase EC 2.2.1.2 (transaldolase) | WO0104325 | AX076272 |
| thrB | Homoserine kinase EC 2.7.1.39 (homoserine kinase) | Peoples et al.，Molecular Microbiology 2：63-72(1988) | Y00546 |
| thrC | Threonine synthase EC 4.2.99.2 (threonine synthase) | Han et al.，Molecular Microbiology 4：1693-1702 (1990) | X56037 |
| thrE | Threonine output albumen (Threonine output carrier) | EP1085091 | AX137526 |
| thyA | Thymidylate synthase EC 2.1.1.45 (thymidylate synthase) | EP1108790 | AX121026 AX127145 |
| tkt | Transketolase EC 2.2.1.1 (transketolase) | Ikeda et al.，NCBI | AB023377 |
| tpi | Triose-phosphate isomerase EC 5.3.1.1 (triose-phosphate isomerase) | Eikmanns，Journal of Bacteriology 174： 6076-6086 (1992) | X59403 |
| zwa1 | Growth factor-21 (growth factor-21) | EP1111062 | AX133781 |
| zwf | G-6-P-1-desaturase EC 1.1.1.49 (G-6-P-1-desaturase) | EP1108790 WO0104325 | AX127148 AX121827 AX076272 |
| zwf A213T | G-6-P-1-desaturase EC 1.1.1.49 (G-6-P-1-desaturase) amino acid exchange A213T | EP1108790 |
Table 7 is used to integrate open reading frame, gene and the allelic target site that Threonine is produced
| The gene title | Coded enzyme or proteinic description | Reference | Accession number |
| ccpA1 | Catabolite control albumen (catabolite control albumin A 1) | WO0100844 EP1108790 | AX065267 AX127147 |
| ccpA2 | Catabolite control albumen (catabolite control albumin A 2) | WO0100844 EP1108790 | AX065267 AX121594 |
| citA | Sensor kinase c itA (sensor kinase c itA) | EP1108790 | AX120161 |
| citB | Transcriptional CitB (transcriptional CitB) | EP1108790 | AX120163 |
| citE | Citrate lyase EC 4.1.3.6 (citrate lyase) | WO0100844 EP1108790 | AX065421 AX127146 |
| ddh | Diamino acid pimelic acid desaturase EC 1.4.1.16 (diamino acid pimelic acid desaturase) | Ishino et al.，Nucleic Acids Research 15：3917-3917(1987) EP1108790 | S07384 AX127152 |
| gluA | Glutamate transport ATP conjugated protein (glutamate transport ATP is conjugated protein) | Kronemeyer et al.，Journal of Bacteriology 177(5)：1152-8 (1995) | X81191 |
| gluB | L-glutamic acid conjugated protein (L-glutamic acid is conjugated protein) | Kronemeyer et al.，Journal of Bacteriology 177(5)：1152-8 (1995) | X81191 |
| gluC | Glutamate transport permease (glutamate transport system permease) | Kronemeyer et al.，Journal of Bacteriology 177(5)：1152-8 (1995) | X81191 |
| gluD | Glutamate transport permease (glutamate transport system permease) | Kronemeyer et al.，Journal of Bacteriology 177(5)：1152-8 (1995) | X81191 |
| glyA | Glycine hydroxymethyltransferase EC 2.1.2.1 (glycine hydroxymethyltransferase) | WO0100843 | AX063861 AF327063 |
| ilvA | Threonine dehydra(ta)se EC4.2.1.16 (threonine dehydra(ta)se) | Mockel et al.，Journal of Bacteriology 174(24)， 8065-8072(1992) EP1108790 | A47044 L01508 AX127150 |
| ilvBN | Acetolactate synthase EC 4.1.3.18 (acetolactate synthase) | Keilhauer et al.，Journal of Bacteriology 175(17)：5595-603 (1993) EP1108790 | L09232 AX127147 |
| ilvC | Reduction isomerase EC 1.1.1.86 (ketol-acid reductoisomerase) | Keilhauer et al.，Journal of Bacteriology 175(17)：5595-603 (1993) EP1108790 | C48648 AX127147 |
| ilvD | Dihydroxyacid dehydratase | EP1006189 | AX136925 |
| EC 4.2.1.9 (dihydroxyacid dehydratase) | |||
| luxR | Transcriptional LuxR (transcriptional LuxR) | WO0100842 EP1108790 | AX065953 AX123320 |
| luxS | Histidine kinase LuxS (histidine kinase LuxS) | EP1108790 | AX123323 AX127145 |
| lysR1 | Transcriptional LysR1 (transcriptional LysR1) | EP1108790 | AX064673 AX127144 |
| lysR2 | Transcriptional activator LysR2 (transcriptional LysR2) | EP1108790 | AX123312 |
| lysR3 | Transcriptional LysR3 (transcriptional LysR3) | WO0100842 EP1108790 | AX065957 AX127150 |
| mdh | Malate dehydrogenase (malic acid dehydrogenase) EC 1.1.1.37 (malate dehydrogenase (malic acid dehydrogenase)) | WO0100844 | AX064895 |
| menE | O-succinyl-phenylformic acid coenzyme A lyase EC 6.2.1.26 (O-succinyl-phenylformic acid coenzyme A lyase) | WO0100843 EP1108790 | AX064599 AX064193 AX127144 |
| metA | Homoserine-O-Transacetylase EC 2.3.1.31 (homoserine-O-Transacetylase) | Park et al.，Molecular Cells30； 8(3)：286-94(1998) WO0100843 EP1108790 | AX063895 AX127145 |
| metD | Transcriptional MetD (transcriptional MetD) | EP1108790 | AX123327 AX127153 |
| pck | Phosphoenolpyruvate carboxykinase (phosphoenolpyruvate carboxykinase) | WO0100844 | AJ269506 AX065053 |
| poxB | Pyruvic oxidase EC 1.2.3.3 (pyruvic oxidase) | WO0100844 EP1096013 | AX064959 AX137665 |
| sigB | The rna polymerase transcribe factor (the rna polymerase transcribe factor) | EP1108790 | AX127149 |
| zwa2 | Cell growth factor 2 (growth factor-2) | EP1106693 EP1108790 | AX113822 AX127146 |
The present invention also provides the method for producing bar shaped bacteria, and described bacterium produces L-methionine(Met) and/or L-Threonine, and described method comprises:
A) separate at least one desirable ORF, gene or the allelic nucleotide sequence that is used for methionine(Met) production or Threonine production, it randomly comprises expresses and/or conditioning signal,
B) nucleotide sequence with target site offers ORF, gene or allelic 5 ' and the 3 ' end that is used for Methionin production,
C) desirable ORF, gene or the allelic nucleotide sequence that preferably will have the target site nucleotide sequence mixes in the carrier, described carrier in bar shaped bacteria, do not duplicate or only limited extent duplicate,
D) with b) or nucleotides sequence column jump c) advance in the bar shaped bacteria,
E) separate such bar shaped bacteria, described middle a) nucleotide sequence mixes target site, this target site do not have can/make can be in microorganism the nucleotide sequence of episomal replication, not having can swivel base/make the nucleotide sequence that swivel base takes place, and does not authorize the nucleotide sequence of antibiotics resistance.
The present invention also provides the bar shaped bacteria that produces the L-Xie Ansuan, particularly excellent bacillus Pseudomonas, it is characterized in that being used for the open reading frame (ORF) that Xie Ansuan is produced except existing at natural site (locus), outside gene or allelic at least one copy, in each case at second, randomly the 3rd or the 4th site also have the corresponding open reading frame (ORF) of integration form, gene or allelic second, randomly the 3rd or the 4th copy, this specific second, randomly the 3rd or the 4th site do not have can/make can be in microorganism the nucleotide sequence of episomal replication, do not have energy swivel base/feasible nucleotide sequence that swivel base takes place, and do not authorize the nucleotide sequence of antibiotics resistance.
The present invention also provides a kind of method of the L-of preparation Xie Ansuan, and it may further comprise the steps:
A) make described open reading frame (ORF), the condition bottom fermentation bar shaped bacteria that gene or allelotrope are expressed, Corynebacterium glutamicum particularly, these bacteriums are characterised in that except existing at natural site (locus) and are used for the open reading frame (ORF) that Xie Ansuan is produced, outside gene or allelic at least one copy, in each case at second, randomly the 3rd or the 4th site also have the corresponding open reading frame (ORF) of integration form, gene or allelic second, randomly the 3rd or the 4th copy, this specific second, randomly the 3rd or the 4th site do not have can/make can be in microorganism the nucleotide sequence of episomal replication, there is not energy swivel base/feasible nucleotide sequence that swivel base takes place, reach the nucleotide sequence of not authorizing antibiotics resistance
B) the L-Xie Ansuan in the concentrated fermenting broth,
C) from fermenting broth, separate the L-Xie Ansuan, randomly
D) with＞(greater than) component and/or the biomass of the fermenting broth of 0-100%.
" be used for open reading frame (ORF), gene or allelic copy that Xie Ansuan is produced " and be meant that its all enhancing/mistake expression can have open reading frame, gene or the allelotrope of the effect of improvement Xie Ansuan production.
These comprise following open reading frame, gene or allelotrope: brnE, brnF, brnEF, cstA, cysD, dps, eno, fda, gap, gap2, gdh, ilvB, ilvN, ilvBN, ilvC, ilvD, ilvE, msiK, pgk, ptsH, ptsI, ptsM, sigC, sigD, sigE, sigH, sigM, tpi, zwa1.These are summarized and explanation in table 8.These are particularly including the ilvBN allelotrope of the acetolactate synthase of coding Xie Ansuan resistance.
Be used for open reading frame (ORF) that Xie Ansuan produces, gene or allelic second, randomly the 3rd or the 4th copy can be integrated into second, randomly the 3rd or the 4th site in each case.Can use following open reading frame, gene or nucleotide sequence: aecD, ccpA1, ccpA2, citA, citB, citE, ddh, gluA, gluB, gluC, gluD, glyA, ilvA, luxR, lysR1, lysR2, lysR3, panB, panC, poxB and zwa2 for this reason.These are summarized and explanation in table 9.
Described site not only comprises the coding region of described open reading frame or gene certainly, also comprise be positioned at the upstream with express and regulate relevant zone or nucleotide sequence, ribosome bind site for example, promotor, regulate proteic binding site, regulate the binding site and the attenuator of Yeast Nucleic Acid.These zones generally are positioned at the 1-800 of upstream of coding region, 1-600, and 1-400,1-200 is in 1-100 or 1-50 the Nucleotide scope.Equally, also comprise the zone that is positioned at the downstream, transcription terminator for example, these zones generally are positioned at the 1-400 in downstream, coding region, 1-200,1-100 is in 1-50 or 1-25 the Nucleotide scope.
Can use intrachromosomal intergenic region in addition, promptly not have the nucleotide sequence of encoding function.At last, can also use prophage or the defective phage that comprises in the karyomit(e).
Table 8 is used for open reading frame, gene and the allelotrope that Xie Ansuan is produced
| Title | Coded enzyme or proteinic description | Reference | Accession number |
| brnEF | The output albumen of branched-chain amino acid (the output albumen of branched-chain amino acid) | EP1096010 Kennerknecht et al.，NCBI | AF454053 |
| cstA | The hungry albumin A of carbon (the hungry albumin A of carbon) | EP1108790 WO0100804 | AX120811 AX066109 |
| dps | DNA protected protein (protected protein in the hungry process) | EP1108790 | AX127153 |
| eno | Hydratase, phosphoenolpyruvate EC 4.2.1.11 (Hydratase, phosphoenolpyruvate) | EP1108790 WO0100844 EP 1090998 Hermann et al.， Electrophoresis 19：3217-3221(1998) | AX127146 AX064945 AX136862 |
| fda | Fructose-bis phosphate aldolase EC 4.1.2.13 (fructose-1,6-diphosphate zymohexase) | von der Osten et al.， Molecular Microbiology 3 (11)：1625-37(1989) | X17313 |
| gap | Glyceraldehyde-3-phosphate dehydrogenase EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase) | EP1108790 WO0100844 Eikmanns et al.，Journal of Bacteriology 174：6076-6086(1992) | AX127148 AX064941 X59403 |
| gap2 | Glyceraldehyde-3-phosphate dehydrogenase EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase 2) | EP1108790 WO0100844 | AX127146 AX064939 |
| gdh | Glutamate dehydrogenase EC 1.4.1.4 (glutamate dehydrogenase) | EP1108790 WO0100844 Boermann et al.，Molecular Microbiology 6：317-326 (1992). Guyonvarch et al.，NCBI | AX127150 AX063811 X59404 X72855 |
| ilvBN | Acetolactate synthase EC 4.1.3.18 (acetolactate synthase) | Keilhauer et al.，Journal of Bacteriology 175(17)：5595-603(1993) EP1108790 | L09232 AX127147 |
| ilvC | Reduction isomerase EC 1.1.1.86 (acetohydroxy acid reduction isomerase) | Keilhauer et al.，Journal of Bacteriology 175(17)：5595-603(1993) EP1108790 | C48648 AX127147 |
| ilvD | Dihydroxyacid dehydratase EC 4.2.1.9 (dihydroxyacid dehydratase) | EP1006189 | AX136925 |
| ilvE | Transaminase B EC2.6.1.42 (Transaminase B) | EP1108790 | AX136925 |
| msiK | Sugar input albumen (multiple sugar input albumen) | EP1108790 | AX120892 |
| pgk | Phosphoglyceric kinase EC 2.7.2.3 (phosphoglyceric kinase) | EP1108790 WO0100844 Eikmanns，Journal of Bacteriology 174： 6076-6086(1992) | AX121838 AX127148 AX064943 X59403 |
| ptsH | Phosphotransferase system albumen H EC 2.7.1.69 (phosphotransferase system composition H) | EP1108790 WO0100844 | AX122210 AX127149 AX069154 |
| ptsI | Phosphotransferase system enzyme I EC 2.7.3.9 | EP1108790 | AX122206 AX127149 |
| ptsM | Glucose specificity phosphotransferase system enzyme II EC 2.7.1.69 (glucose phosphotransferase system enzyme II) | Lee et al.，FEMS Microbiology Letters 119(1-2)：137-145(1994) | L18874 |
| sigC | Sigma factor C EC 2.7.7.6 (the outer substituting sigma factor of the function C of kytoplasm) | EP1108790 | AX120368 AX120085 |
| sigD | RNA polymerase Sigma factor D EC 2.7.7.6 (the RNA polymerase sigma factor) | EP1108790 | AX120753 AX127144 |
| sigE | Sigma factor E EC 2.7.7.6 (the outer substituting sigma factor of the function E of kytoplasm) | EP1108790 | AX127146 AX121325 |
| sigH | Sigma factor H EC 2.7.7.6 (sigma factor S igH) | EP1108790 | AX127145 AX120939 |
| sigM | Sigma factor M EC 2.7.7.6 (sigma factor S igM) | EP1108790 | AX123500 AX127145 |
| tpi | Triose-phosphate isomerase EC 5.3.1.1 (triose-phosphate isomerase) | Eikmanns，Journal of Bacteriology 174： 6076-6086 (1992) | X59403 |
| zwa1 | Growth factor-21 (growth factor-21) | EP1111062 | AX133781 |
Table 9 is used to integrate open reading frame, gene and the allelic target site that Xie Ansuan is produced
| The gene title | Coded enzyme or proteinic description | Reference | Accession number |
| aecD | β C-S lyase EC 2.6.1.1 (β C-S lyase) | Rossol et al.，Journal of Bacteriology 174(9)： 2968-77(1992) | M89931 |
| ccpA1 | Catabolite control albumen (catabolite control albumin A 1) | WO0100844 EP1108790 | AX065267 AX127147 |
| ccpA2 | Catabolite control albumen (catabolite control albumin A 2) | WO0100844 EP1108790 | AX065267 AX121594 |
| citA | Sensor kinase c itA (sensor kinase c itA) | EP1108790 | AX120161 |
| citB | Transcriptional CitB (transcriptional CitB) | EP1108790 | AX120163 |
| citE | Citrate lyase EC 4.1.3.6 (citrate lyase) | WO0100844 EP1108790 | AX065421 AX127146 |
| ddh | Diamino acid pimelic acid desaturase EC 1.4.1.16 (diamino acid pimelic acid desaturase) | Ishino et al.，Nucleic Acids Research 15：3917-3917(1987) EP1108790 | S07384 AX127152 |
| gluA | Glutamate transport ATP conjugated protein (glutamate transport ATP is conjugated protein) | Kronemeyer et al.，Journal of Bacteriology 177(5)：1152-8 (1995) | X81191 |
| gluB | L-glutamic acid conjugated protein (L-glutamic acid is conjugated protein) | Kronemeyer et al.，Journal of Bacteriology 177(5)：1152-8 (1995) | X81191 |
| gluC | Glutamate transport permease (glutamate transport system permease) | Kronemeyer et al.，Journal of Bacteriology 177(5)：1152-8 (1995) | X81191 |
| gluD | Glutamate transport permease (glutamate transport system permease) | Kronemeyer et al.，Journal of Bacteriology 177(5)：1152-8 (1995) | X81191 |
| glyA | Glycine hydroxymethyltransferase EC 2.1.2.1 (glycine hydroxymethyltransferase) | WO0100843 | AX063861 AF327063 |
| ilvA | Threonine dehydra(ta)se EC 4.2.1.16 (threonine dehydra(ta)se) | Mockel et al.，Journal of Bacteriology 174(24)， 8065-8072(1992) EP1108790 | A47044 L01508 AX127150 |
| luxR | Transcriptional LuxR (transcriptional LuxR) | WO0100842 EP1108790 | AX065953 AX123320 |
| lysR1 | Transcriptional LysR1 (transcriptional LysR1) | EP1108790 | AX064673 AX127144 |
| lysR2 | Transcriptional activator LysR2 (transcriptional LysR2) | EP1108790 | AX123312 |
| lysR3 | Transcriptional LysR3 (transcriptional LysR3) | WO0100842 EP1108790 | AX065957 AX127150 |
| panB | Ketopantoic acid hydroxymethyl transferases EC 2.1.2.11 (ketopantoic acid hydroxymethyl transferases) | US6177264 | X96580 |
| panC | Pantothenate synthetase EC 6.3.2.1 (pantothenate synthetase) | US6177264 | X96580 |
| poxB | Pyruvic oxidase EC 1.2.3.3 (pyruvic oxidase) | WO0100844 EP1096013 | AX064959 AX137665 |
| zwa2 | Cell growth factor 2 (growth factor-2) | EP1106693 EP1108790 | AX113822 AX127146 |
The invention provides a kind of method of producing bar shaped bacteria, described bacterium produces the L-Xie Ansuan, and described method comprises:
A) separate at least one desirable ORF, gene or the allelic nucleotide sequence that is used for Xie Ansuan production, it randomly comprises expresses and/or conditioning signal,
B) nucleotide sequence with target site offers ORF, gene or allelic 5 ' and the 3 ' end that is used for Methionin production,
C) desirable ORF, gene or the allelic nucleotide sequence that preferably will have the target site nucleotide sequence mixes in the carrier, described carrier in bar shaped bacteria, do not duplicate or only limited extent duplicate,
D) with b) or nucleotides sequence column jump c) advance in the bar shaped bacteria,
E) separate such bar shaped bacteria, in the described bar shaped bacteria a) nucleotide sequence mix target site, this target site do not have can/make can be in microorganism the nucleotide sequence of episomal replication, do not have energy swivel base/feasible nucleotide sequence that swivel base takes place, and do not authorize the nucleotide sequence of antibiotics resistance.
During implementing the present invention, can be with lysC
FBRAllelic second copy mixes in the gluB gene of Corynebacterium glutamicum, make simultaneously do not have in this gluB gene locus can/make can be in microorganism the nucleotide sequence of episomal replication, there is not energy swivel base/feasible nucleotide sequence that swivel base takes place, the nucleotide sequence of the antibiotics resistance of not authorizing.This is called the bacterial strain of DSM13994glu::lysC, carries lysC in its natural lysC site
FBRAllelotrope lysC T311I is that the gluB gene carries lysC in second site (target site)
FBRSecond copy of allelotrope lysCT311I.Can be used for realizing with lysC
FBRThe plasmid that allelotrope mixes the gluB gene is shown in Fig. 1.Described plasmid is called pK18mobsacBglu1_1.
During implementing the present invention, can also be with lysC
FBRAn allelic copy mixes in the target site of gluB gene of Corynebacterium glutamicum, make simultaneously this gluB gene locus do not have can/make can be in microorganism the nucleotide sequence of episomal replication, do not have energy swivel base/feasible nucleotide sequence that swivel base takes place, do not authorize the nucleotide sequence of antibiotics resistance.This is called the bacterial strain of DSM12866glu::lysC, carries the lysC gene of wild-type form in its natural lysC site, is that the gluB gene carries lysC in second site (target site)
FBRSecond copy of the lysC gene of allelotrope lysC T311I form.This bacterial strain has been deposited in Germany microorganism and cell culture preservation center, and preserving number is DSM15039.Can be used for realizing with lysC
FBRThe plasmid that allelotrope mixes the gluB gene is shown in Fig. 1.Described plasmid is called pK18mobsacBglu1_1.
During implementing the present invention, can also be with lysC
FBRAn allelic copy mixes in the target site of aecD gene of Corynebacterium glutamicum, make simultaneously this aceD gene locus do not have can/make can be in microorganism the nucleotide sequence of episomal replication, do not have energy swivel base/feasible nucleotide sequence that swivel base takes place, and do not authorize the nucleotide sequence of antibiotics resistance.This is called the bacterial strain of DSM12866aecD::lysC, carries the lysC gene of wild-type form in its natural lysC site, is that the aecD gene carries lysC in second site (target site)
FBRSecond copy of the lysC gene of allelotrope lysC T311I form.Can be used for realizing with lysC
FBRThe plasmid that allelotrope mixes the aecD gene is shown in Fig. 2.Described plasmid is called pK18mobsacBaecD1_1.
During implementing the present invention, can also be with lysC
FBRAn allelic copy mixes in the target site of pck gene of Corynebacterium glutamicum, make simultaneously this pck gene locus do not have can/make can be in microorganism the nucleotide sequence of episomal replication, do not have energy swivel base/feasible nucleotide sequence that swivel base takes place, and do not authorize the nucleotide sequence of antibiotics resistance.This is called the bacterial strain of DSM12866pck::lysC, carries the lysC gene of wild-type form in its natural lysC site, is that the pck gene carries lysC in second site (target site)
FBRSecond copy of the lysC gene of allelotrope lysCT311I form.Can be used for realizing that the plasmid that mixes the pck gene is shown in Fig. 3.Described plasmid is called pK18mobsacBpck1_1.
During implementing the present invention, a copy of ddh gene can also be mixed in the gluB gene of Corynebacterium glutamicum, make simultaneously this gluB gene locus do not have can/make can be in microorganism the nucleotide sequence of episomal replication, do not have energy swivel base/feasible nucleotide sequence that swivel base takes place, and do not authorize the nucleotide sequence of antibiotics resistance.This is called the bacterial strain of DSM12866glu::ddh, carries a copy of ddh gene in its natural ddh site, is second copy that the gluB gene carries the ddh gene in second site (target site).Can be used to realize that the plasmid that the ddh gene is mixed the gluB gene is shown in Fig. 4.Described plasmid is called pK18mobsacBgluB2_1.
During implementing the present invention, a copy of dapA gene can also be mixed in the aecD gene of Corynebacterium glutamicum, make simultaneously this aecD gene locus do not have can/make can be in microorganism the nucleotide sequence of episomal replication, do not have energy swivel base/feasible nucleotide sequence that swivel base takes place, and do not authorize the nucleotide sequence of antibiotics resistance.This is called the bacterial strain of DSM12866aecD::dapA, carries a copy of dapA gene in its natural dapA site, is second copy that the aecD gene carries the dapA gene in second site (target site).Can be used to realize that the plasmid that the dapA gene is mixed the aecD gene is shown in Fig. 5.Described plasmid is called pK18mobsacBaecD2_1.
During implementing the present invention, a copy of pyc gene can be mixed in the target site of pck gene of Corynebacterium glutamicum, make simultaneously this pck gene locus do not have can/make can be in microorganism the nucleotide sequence of episomal replication, do not have energy swivel base/feasible nucleotide sequence that swivel base takes place, and do not authorize the nucleotide sequence of antibiotics resistance.This is called the bacterial strain of DSM12866pck::pyc, carrying a copy of the pyc gene of wild-type form in its natural pyc site, is second copy that the pck gene carries the pyc gene of pyc allelotrope pyc P458S form in second site (target site).Can be used to realize that the plasmid that pyc allelotrope is mixed the pck gene is shown in Fig. 6.Described plasmid is called pK18mobsacBpck1_3.
Be to produce compound, the bar shaped bacteria that produces according to the present invention can cultured continuously or discontinuous cultivation, and described discontinuous cultivation is batch culture or fed batch cultivation or repeated fed-batch culture.Teaching material that Chmiel shows (Bioprozesstechnik1.Einfhrung in dieBioverfahrenstechnik (Gustav Fischer Verlag is seen in the general introduction of known cultural method, Stuttgart, 1991)) or teaching material that Storhas shows (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
Used substratum must meet the demand of each bacterial strain in a suitable manner.See " bacteriology universal method handbook " (Washington, DC D.C., 1981) of U.S.'s bacteriology meeting about the elaboration of various microbiological culture medias.
Spendable carbon source comprises sugar and carbohydrate, glucose for example, sucrose, lactose, fructose, maltose, molasses, starch and Mierocrystalline cellulose, oil ﹠ fat such as soya-bean oil, Trisun Oil R 80, Peanut oil and Oleum Cocois, lipid acid such as palmitinic acid, stearic acid and linolic acid, alcohol is as glycerine and ethanol, and organic acid such as acetate.These materials can be used alone or as a mixture.
Spendable nitrogenous source comprises nitrogenous organic compound such as peptone, yeast extract, meat extract, malt extract, corn immersion liquid, soyflour and urea, or inorganicization thing such as ammonium sulfate, ammonium chloride, ammonium phosphate, volatile salt and ammonium nitrate.Nitrogenous source can be used alone or as a mixture.
Spendable phosphorus source comprises phosphoric acid, potassium primary phosphate or dipotassium hydrogen phosphate, or corresponding sodium salt.Substratum also must contain in addition and is required metal-salt such as sal epsom or the ferric sulfate of growing.At last, except above-mentioned substance, can use the essential material of growth such as amino acid and VITAMIN.In addition, suitable precursor can be added in the substratum, above-mentioned substance can singly be criticized form or suitably add in the training period.
Can add basic cpd such as NaOH in a suitable manner, KOH, ammonia or ammoniacal liquor, or acidic cpd such as phosphoric acid or sulfuric acid are to regulate the pH value of culture.Foam reducing composition for example fatty acid polyglycol ester can be used for the control foam generation.The suitable selectivity working substance is microbiotic for example, can add in the substratum to keep the stability of plasmid.Oxygen or contain oxygen gas mixture, for example air can charge in the culture to maintain oxygen condition.Culture temperature is usually at 20 ℃～45 ℃, preferred 25 ℃-40 ℃.Continue to cultivate and form maximum until required product.This purpose reached in the scope at 10～160 hours usually.
Have been found that bar shaped bacteria of the present invention, especially produce the bar shaped bacteria of L-Methionin, have unexpected high stability.They are at 10-20 at least, 20-30, and 30-40,40-50, preferred 50-60 at least, 60-70 keeps in a 70-80 and 80-90 generation or the cell division cycle process stablizing.
The preservation of following microorganism:
Corynebacterium glutamicum strain DSM12866glu::lysC is deposited in Germany microorganism and cell culture preservation center (DSMZ, Braunschweig, Germany), preserving number DSM15039 on June 5th, 2002 according to budapest treaty with the pure growth form.
Plasmid pK18mobsacBglu1_1 is with coli strain DH5 α mcr/pK18mobsacBglu1_1 (=DH5alphamcr/pK18mobsacBglu1_1) pure growth form, be deposited in Germany microorganism and cell culture preservation center (DSMZ April 20 calendar year 2001 according to budapest treaty, Braunschweig, Germany), preserving number DSM14243.
Plasmid pK18mobsacBaecD1_1 is with coli strain DH5 α mcr/pK18mobsacBaecD1_1 (=DH5alphamcr/pK18mobsacBaecD1_1) pure growth form, be deposited in Germany microorganism and cell culture preservation center (DSMZ on June 5th, 2002 according to budapest treaty, Braunschweig, Germany), preserving number DSM15040.
Embodiment 1:With lysC
FBRAllelic second copy mixes in the karyomit(e) of strain DSM 13994 and strain DSM 12866
Corynebacterium glutamicum strain DSM13994 selects to produce by repeatedly non-directional mutagenesis, selection and mutant from Corynebacterium glutamicum ATCC13032.This bacterial strain has resistance to lysine analogues S-(2-amino-ethyl)-L-halfcystine, and has a kind of feedback resistance E.C. 2.7.2.4., and the restraining effect of its mixture to Methionin and Threonine (each 25mM) is insensitive.The lysC of this bacterial strain
FBRAllelic nucleotides sequence is shown in SEQ ID NO:3.Be also referred to as lysC T311I hereinafter.The proteic aminoacid sequence of coded E.C. 2.7.2.4. is shown in SEQ ID NO:4.The pure growth of this bacterial strain is deposited in Germany microorganism and cell culture preservation center (DSMZ, Braunschweig, Germany) according to budapest treaty January 16 calendar year 2001.
Strain DSM 12866 produces by the mutant that non-directional mutagenesis and selection have an accumulation of best L-Methionin from Corynebacterium glutamicum ATCC13032, and it is a methionine(Met) susceptibility.Growth on the minimum medium that comprises the L-methionine(Met) can be set up by adding Threonine again.This bacterial strain has the lysC gene of wild-type form, is shown in SEQ IDNO:1.The proteic corresponding aminoacid sequence of wild-type E.C. 2.7.2.4. is shown in SEQ ID NO:2.The pure growth of this bacterial strain is preserved in Germany microorganism and cell culture preservation center (DSMZ, Braunschweig, Germany) according to budapest treaty on June 10th, 1999.
1.1 the allelic DNA of lysC of separation and order-checking strain DSM 13994
From strain DSM 13994, separate chromosomal DNA (Eikmanns etc., microbiology 140:1817-1828 (1994)) by ordinary method.By means of polymeric enzyme reaction, a lysC gene or an allelic DNA sections are carried in amplification.Sequence (Kalinowski etc., molecular microbiology 5 (5), 1197-1204 (1991) based on known Corynebacterium glutamicum lysC gene; Registration number X57226), select following Oligonucleolide primers to carry out PCR:
lysC1beg(SEQ ID No：5)：
5’TA(G GAT CC)T CCG GTG TCT GAC CAC GGT G3’
lysC2end：(SEQ ID NO：6)：
5’AC(G GAT CC)G CTG GGA AAT TGCGCT CTT CC3’
Shown in primer synthetic by MWG Biotech, the PCR reaction is carried out (PCR scheme, method and application directs, 1990, academic press) by described standard pcrs such as Innis.Described primer can amplification length be approximately the DNA sections of 1.7kb, and it carries lysC gene or allelotrope.Described primer also contains the sequence of the cleavage site of restriction enzyme BamHI in addition, and it indicates with bracket in nucleotide sequence shown in above.
The allelic length of lysC of carrying strain DSM 13994 is approximately the dna fragmentation of the amplification of 1.7kb, differentiate by electrophoresis in 0.8% sepharose, from gel, separate and by the ordinary method purifying (the QIA PhastGel extracts test kit, Qiagen, Hilden).
Utilize Topo TA clone test kit (Invitrogen, Leek, The Netherlands, Cat.Number K4600-01) then, in carrier pCRII-TOPO, carry out fragment and connect.To connect mixture transforms among the coli strain TOP10 (Invitrogen, Leek, Holland).Transformation mixture is plated on the X-Gal that has that contains kantlex (50mg/l), and (5-bromo-4-chloro-3-indyl-β-D-galactopyranoside, the cell of plasmid is carried in selection on LB agar 64mg/l).
The plasmid that obtains detects by restriction cutting after DNA isolation, and in sepharose electrophoresis.The gained plasmid is called pCRIITOPOlysC.
The dna fragmentation of amplification or the nucleotide sequence of PCR product are measured (institute of American Academy of Sciences reports 74:5463-5467 (1977)) by described dideoxy-chain terminating methods such as Sanger, use the ABI Prism377 sequencing equipment (Weiterstadt, Germany) of PE Applied Biosystems.The coding region sequence of described PCR product is shown in SEQ ID No:3.The relevant proteic aminoacid sequence of E.C. 2.7.2.4. is shown in SEQ ID NO:4.
The base thymus pyrimidine is at the lysC of strain DSM 13994
FBRThe 932nd discovery (SEQ ID NO:3) of the nucleotide sequence of allelotrope coding region.The base cytosine(Cyt) is found (SEQ ID NO:1) in the corresponding position of wild type gene.
Isoleucine is in the 311st discovery (SEQ ID No:4) of the proteic aminoacid sequence of E.C. 2.7.2.4. of strain DSM 13994.Threonine is found (SEQ ID No:2) at the proteic corresponding position of wild-type albumen.
The 932nd contains thymus pyrimidine and therefore is coded in the proteic lysC allelotrope of E.C. 2.7.2.4. that the 311st of aminoacid sequence contains Isoleucine in the coding region, is called lysC hereinafter
FBRAllelotrope or lysC T311I.
Carry lysC
FBRThe plasmid pCRIITOPOlysC of allelotrope lysC T311I, be preserved in Germany microorganism and cell culture preservation center (DSMZ April 20 calendar year 2001 with the pure growth form of coli strain TOP10/pCRIITOPOlysC according to budapest treaty, Braunschweig, Germany), preserving number DSM14242.
1.2 make up replacement vector pK18mobsacBglu1_1
Corynebacterium glutamicum strain ATCC13032 is as the donor of chromosomal DNA.Use ordinary method from strains A TCC13032, to separate chromosomal DNA (Eikmanns etc., microbiology 140:1817-1828 (1994)).By means of polymerase chain reaction, the dna fragmentation of gluB gene and peripheral region is carried in amplification.Based on the sequence (Kronemeyer etc., bacteriology magazine 177:1152-1158 (1995)) (registration number X81191) of known Corynebacterium glutamicum gluABCD gene cluster, select following Oligonucleolide primers to carry out PCR:
gluBgl1(SEQ ID NO：7)：
5’TA(A GAT CT)G TGT TGG ACG TCA TGG CAA G3’
gluBgl2(SEQ ID NO：8)：
5’AC(A GAT CT)T GAA GCC AAG TAC GGC CAA G3’
Shown in primer synthetic by MWG Biotech, and carry out PCR (PCR scheme, methods and applications instruct, 1990, academic press) by the described standard pcr of Innis.Can the increase dna fragmentation of about 1.7kb of described primer, it carries gluB gene and peripheral region.Described peripheral region be represent the gluA gene 3 ' the terminal about 0.33kb that is positioned at the gluB upstream region of gene the sequence sections and represent the sequence sections of about 0.44kb in gluB gene downstream of 5 ' end of gluC gene.Described primer also contains the cleavage site sequence of restriction enzyme BglII, is indicated with bracket in above-mentioned nucleotide sequence.
Carry the dna fragmentation of amplification of about 1.7kb of gluB gene and peripheral region, separate by electrophoresis in 0.8% sepharose and from gel and by the ordinary method purifying differentiate (the QIAquick gel extraction kit, Qiagen, Hilden).
Utilizing TOPO TA clone's test kit (Invitrogen, Leek, Holland, Cat.Number K4600-01) to carry out fragment then in carrier pCRII-TOPO connects.To connect mixture transforms among the coli strain TOP10 (Invitrogen, Leek, Holland).(5-bromo-4-chloro-3-indyl-β-D-galactopyranoside, the cell of plasmid is carried in selection on LB agar 64mg/l) by conversion product being plated on the X-Gal that has that contains kantlex (50mg/l).
The plasmid that obtains detects by the restriction cutting after DNA isolation, and differentiates in sepharose.The gained plasmid is called pCRII-TOPOglu.
With plasmid pCRII-TOPOglu restriction enzyme BglII (Amersham-Pharmacia, Freiburg, Germany) cutting, and in sepharose (0.8%) by means of QIAquick gel extraction kit (Qiagen, Hilden, Germany) separate after, the gluB fragment of from this sepharose, separating about 1.7kb, be used for
Described removable cloning vector pK18mobsacB (gene 14:69-73 (1994)) connects.In advance with this carrier with restriction enzyme BamHI cutting and with alkaline phosphatase dephosphorylation (alkaline phosphatase, BoehringerMannheim), mix, and mixture is handled (Amersham-Pharmacia with the T4DNA ligase enzyme with the gluB fragment of about 1.7kb, Freiburg, Germany).
Then coli strain DH5 α (Grant etc., newspaper 87 (1990) 4645-4649 of institute of American Academy of Sciences) is transformed (Hanahan, dna clone practical plan, the 1st volume, ILR-press, cold spring port, New York, 1989) with connecting mixture.By conversion product being plated on the cell (Sambrook etc., molecular cloning experiment instruction, the 2nd edition, cold spring port, New York, 1989) of selecting to carry plasmid on the LB agar of adding the 50mg/l kantlex.
By means of the QIAprep Spin Miniprep test kit isolated plasmid dna from transformant that derives from Qiagen, and the agarose gel electrophoresis that reaches subsequently by the restriction cutting detects.Described plasmid is called pK18mobsacBglu1.
Isolated plasmid dna from the strain DSM 14242 that carries plasmid pCRIITOPOlysC (seeing embodiment 1.1), with restriction enzyme BamHI cutting (Amersham-Pharmacia, Freiburg, Germany), by means of QIAquick gel extraction kit (Qiagen, Hilden, Germany) in sepharose (0.8%), separate after, with the lySC that contains of about 1.7kb
FBRDna fragmentation from sepharose, separate, and be used for being connected with above-mentioned carrier pK18mobsacBglu1.This carrier is in advance with restriction enzyme BamHI cutting, with alkaline phosphatase dephosphorylation (alkaline phosphatase, Boehringer Mannheim, Germany), with the lysC of about 1.7kb
FBRFragment is mixed, and described mixture is handled with T4DNA ligase enzyme (Amersham-Pharmacia, Freiburg, Germany).
Then coli strain DH5 α mcr (Life Technologies GmbH, Karlsruhe, Germany) is transformed (Hanahan, dna clone practical plan, the 1st volume, ILR-press, cold spring port, New York, 1989) with connecting mixture.By conversion product being plated on the cell (Sambrook etc., molecular cloning experiment instruction, the 2nd edition, cold spring port, New York, 1989) of selecting to carry plasmid on the LB agar of adding the 50mg/l kantlex.
By means of the QIAprep Spin Miniprep test kit isolated plasmid dna from transformant that derives from Qiagen, and the agarose gel electrophoresis that reaches subsequently by the restriction cutting detects.Described plasmid is called pK18mobsacBglu1_1.This plasmid is illustrated in Fig. 1.
Plasmid pK18mobsacBglu1_1 is according to budapest treaty, (=DH5alphamcr/pK18mobsacBglu1_1) pure growth form is preserved in Germany microorganism and cell culture preservation center (DSMZ with coli strain DH5 α mcr/pK18mobsacBglu1_1 April 20 calendar year 2001, Braunschweig, Germany), preserving number DSM14243.
1.3 utilize replacement vector pK18mobsacBglu1_1 with lySC
FBRSecond copy of allelotrope lysC T311I mixes (target site: the gluB gene) in the karyomit(e) of strain DSM 13994
By described methods such as Schafer (JOURNAL OF MICROBIOLOGY 172:1663-1666 (1990)), embodiment 1.2 described carrier pK18mobsacBglu1_1 are advanced among the Corynebacterium glutamicum strain DSM13994 by conjugal transfer.Described carrier can not be independently duplicated in DSM13994, only is integrated in the karyomit(e) just to be retained in the cell.Be plated on the LB agar (Sambrook etc. that add 15mg/l kantlex and 50mg/ Nalidixic Acid with engaging mixture, the molecular cloning experiment instruction, the 2nd edition, the cold spring port, New York, 1989) go up clone or the transconjugant of the pK18mobsacBglu1_1 that selects to have integration.The kalamycin resistance transconjugant is plated on the LB agar with 25mg/l kantlex, 33 ℃ of incubations 48 hours.
, will be cloned in the liquid substratum of LB and cultivate 20 hours because the mutant of described plasmid has been excised in reorganization for the second time for selecting, be plated on the LB agar with 10% sucrose then and incubation 48 hours.
Similar to initial plasmid pK18mobsacB, plasmid pK18mobsacBglu1_1 also contains the copy of sacB gene of the levansucrase of coding Bacillus subtilis except kalamycin resistance gene.Can cause levansucrase to form by the expression of sucrase inductive, its catalysis be synthetic to the virose product Polylevulosan of Corynebacterium glutamicum.Therefore have only because those of pK18mobsacBglu1_1 of integration have been excised in reorganization for the second time is cloned on the LB agar and grows.According to the position of the recombination event second time, after excision, lysC
FBRAllelic second copy demonstrates at karyomit(e) gluB locus, and perhaps host's original gluB locus is kept.
About 40-50 bacterium colony test " in growth in the presence of the sucrose " reached the phenotype of " in the presence of kantlex, not growing ".To being shown, " in growth in the presence of the sucrose " reach " not growing " phenotype in the presence of kantlex about 20 bacterium colonies study by means of polymerase chain reaction.The dna fragmentation of gluB gene and peripheral region is carried in amplification from the chromosomal DNA of described bacterium colony.Select the primer identical to carry out PCR with structure integrated plasmid among the embodiment 1.2.
gluBgl1(SEQ ID NO：7)：
5’TA(A GAT CT)G TGT TGG ACG TCA TGG CAA G3’
gluBgl2(SEQ ID NO：8)：
5′AC(A GATCT)T GAA GCCAAG TAC GGC CAA G3’
Described primer can amplify the dna fragmentation of about 1.7kb in having the contrast clone of original gluB locus.Has lysC at karyomit(e) gluB locus
FBRAmong the clone of second copy of allelotrope, approximately the dna fragmentation of 3.4kb is amplified.
The dna fragmentation of amplification is differentiated by electrophoresis in 0.8% sepharose.
Differentiate a kind of clone by this way, described clone also has lysC at karyomit(e) gluB locus except the copy that is present in the lysC locus
FRBSecond copy of the lysC gene of allelotrope lysC T311I form.This clone is called strain DSM 13994glu::lysC.
1.4 pass through replacement vector pK18mobsacBglu1_1 with lysC
FBRSecond copy of the lysC gene of allelotrope lysC T311I form mixes (target site: the gluB gene) in the karyomit(e) of strain DSM 12866
As described in embodiment 1.3, plasmid pK18mobsacBglu1_1 is advanced among the Corynebacterium glutamicum strain DSM12866 by conjugal transfer.Differentiate a clone in embodiment 1.3 described modes, described clone also has lysC except existing at the lysC locus the copy of wild type gene at chromosomal gluB locus
FBRSecond copy of the lysC gene of allelotrope lysC T311I form.This clone is called strain DSM 12866glu::lysC.
Be carried at the lysC in the gluB gene
FBRThe Corynebacterium glutamicum strain of the present invention of second copy of allelotrope, be preserved in Germany microorganism and cell culture preservation center (DSMZ on June 5th, 2002 with the pure growth form of Corynebacterium glutamicum strain DSM12866glu::lysC according to budapest treaty, Braunschweig, Germany), preserving number DSM15039.
1.5 make up replacement vector pK18mobsacBpck1_1
Corynebacterium glutamicum strain ATCC13032 is as the donor of chromosomal DNA.Use ordinary method from strains A TCC13032, to separate chromosomal DNA (Eikmanns etc., microbiology 140:1817-1828 (1994)).By means of polymerase chain reaction, the dna fragmentation of pck gene and peripheral region is carried in amplification.Based on the sequence (EP1094111 and Riedel etc., molecule and microbial technique magazine 3:573-583 (2001)) (accession number AJ269506) of known Corynebacterium glutamicum pck gene, select following Oligonucleolide primers to carry out PCR:
pck_beg(SEQ ID NO：9)：
5’TA(AGAT CT)G CCG GCA TGA CTT CAG TTT3’
pck_end(SEQ ID NO：10)：
5’AC(A GAT CT)G GTG GGA GCC TTT CTT GTTATT3’
Shown in primer synthetic by MWG Biotech, carry out PCR (PCR scheme, methods and applications instruct, 1990, academic press) by the described standard pcr of Innis.Described primer can amplify the dna fragmentation of about 2.9kb, and it carries pck gene and peripheral region.Described primer also contains the cleavage site sequence of restriction enzyme BglII, is indicated with bracket in above-mentioned nucleotide sequence.
Carry the dna fragmentation of amplification of about 2.9kb of pck gene and peripheral region, separate by electrophoresis in 0.8% sepharose and from gel and by the ordinary method purifying differentiate (the QIAquick gel extraction kit, Qiagen, Hilden).
Utilizing TOPO TA clone's test kit (Invitrogen, Leek, Holland, Cat.Number K4600-01) to carry out fragment then in carrier pCRII-TOPO connects.To connect mixture transforms among the coli strain TOP10 (Invitrogen, Leek, Holland).By conversion product being plated on the cell of selecting to carry plasmid on the LB agar that contains kantlex (50mg/l) with X-Gal (64mg/l).
The plasmid that obtains detects by the restriction cutting after DNA isolation, and differentiates in sepharose.The gained plasmid is called pCRII-TOPOpck.
With plasmid pCRII-TOPOpck restriction enzyme BglII (Amersham-Pharmacia, Freiburg, Germany) cutting, and in sepharose (0.8%) by means of QIAquick gel extraction kit (Qiagen, Hilden, Germany) separate after, the pck fragment of from this sepharose, separating about 2.9kb, be used for
Described removable cloning vector pK18mobsacB (gene 14:69-73 (1994)) connects.In advance with this carrier with restriction enzyme BamHI cutting and with alkaline phosphatase dephosphorylation (alkaline phosphatase, BoehringerMannheim), mix, and mixture is handled (Amersham-Pharmacia with the T4 dna ligase with the pck fragment of about 2.9kb, Freiburg, Germany).
Then coli strain DH5 α (Grant etc., newspaper 87 (1990) 4645-4649 of institute of American Academy of Sciences) is transformed (Hanahan, dna clone practical plan, the 1st volume, ILR-press, cold spring port, New York, 1989) with connecting mixture.By conversion product being plated on the cell (Sambrook etc., molecular cloning experiment instruction, the 2nd edition, cold spring port, New York, 1989) of selecting to carry plasmid on the LB agar of adding the 50mg/l kantlex.
By means of the QIAprep Spin Miniprep test kit isolated plasmid dna from transformant that derives from Qiagen, and the agarose gel electrophoresis that reaches subsequently by the restriction cutting detects.Described plasmid is called pK18mobsacBpck1.
Isolated plasmid dna from the strain DSM 14242 that carries plasmid pCRIITOPOlysC (seeing embodiment 1.1), with restriction enzyme BamHI cutting (Amersham-Pharmacia, Freiburg, Germany), by means of QIAquick gel extraction kit (Qiagen, Hilden, Germany) in sepharose (0.8%), separate after, with the lySC that contains of about 1.7kb
FBRDna fragmentation from sepharose, separate, and be used for being connected with above-mentioned carrier pK18mobsacBpck1.This carrier in advance with restriction enzyme BamHI cutting, is used alkaline phosphatase dephosphorylation (alkaline phosphatase, Boehringer Mannheim, Germany), with the lysC of about 1.7kb
FBRFragment is mixed, and described mixture is handled with T4 dna ligase (Amersham-Pharmacia, Freiburg, Germany).
Then coli strain DH5 α mcr (Life Technologies GmbH, Karlsruhe, Germany) is transformed (Hanahan, dna clone practical plan, the 1st volume, ILR-press, cold spring port, New York, 1989) with connecting mixture.By conversion product being plated on the cell (Sambrook etc., molecular cloning experiment instruction, the 2nd edition, cold spring port, New York, 1989) of selecting to carry plasmid on the LB agar of adding the 50mg/l kantlex.
By means of the QIAprep Spin Miniprep test kit isolated plasmid dna from transformant that derives from Qiagen, and the agarose gel electrophoresis that reaches subsequently by the restriction cutting detects.Described plasmid is called pK18mobsacBpck1_1.This plasmid is illustrated in Fig. 3.
1.6 second copy of the lysC gene of lysCFBR allelotrope lysCT311I form mixed the karyomit(e) (target site: the pck gene) of strain DSM 12866 by replacement vector pK18mobsacBpck1_1
As described in embodiment 1.3, embodiment 1.5 described carrier pK18mobsacBpck1_1 are advanced among the Corynebacterium glutamicum strain DSM12866 by conjugal transfer.As described in embodiment 1.3, in the karyomit(e) of Corynebacterium glutamicum DSM12866, select at directed recombination event.According to the position of the recombination event second time, after excision, lysC
FBRThe pck locus of allelic second copy in karyomit(e) demonstrates, and perhaps host's original pck locus is kept.
About 40-50 bacterium colony test " in growth in the presence of the sucrose " reached the phenotype of " in the presence of kantlex, not growing ".To being shown, " in growth in the presence of the sucrose " reach " not growing " phenotype in the presence of kantlex about 20 bacterium colonies study by means of polymerase chain reaction.The dna fragmentation of pck gene and peripheral region is carried in amplification from the chromosomal DNA of described bacterium colony.Select the primer identical to carry out PCR with structure integrated plasmid among the embodiment 1.5.
pck_beg(SEQ ID NO：9)：
5’TA(AGAT CT)G CCG GCA TGA CTT CAG TTT3’
pck_end(SEQ ID NO：10)：
5’AC(A GAT CT)G GTG GGA GCC TTT CTT GTTATT3’
Described primer can amplify the dna fragmentation of about 2.9kb in having the contrast clone of original pck locus.Has lysC at karyomit(e) pck locus
FBRAmong the clone of second copy of allelotrope, approximately the dna fragmentation of 4.6kb is amplified.
The dna fragmentation of amplification is differentiated by electrophoresis in 0.8% sepharose.
Differentiate a kind of clone by this way, described clone also has lysC except existing at the lysC locus the copy of wild type gene at karyomit(e) pck locus
FRBSecond copy of the lysC gene of allelotrope lysC T311I form.This clone is called strain DSM 12866pck::lysC.
1.7 make up replacement vector pK18mobsacBaecD1_1
Corynebacterium glutamicum strain ATCC 13032 is as the donor of chromosomal DNA.Use ordinary method from strains A TCC13032, to separate chromosomal DNA (Eikmanns etc., microbiology 140:1817-1828 (1994)).By means of polymerase chain reaction, the dna fragmentation of aecD gene and peripheral region is carried in amplification.Based on the sequence (Kronemeyer etc., bacteriology magazine 177:1152-1158 (1995)) (registration number X81191) of known Corynebacterium glutamicum aecD gene, select following Oligonucleolide primers to carry out PCR:
aecD_beg(SEQ ID NO：11)：
5’GAA CTT ACG CCA AGC TGT TC3’
aecD_end(SEQ ID NO：12)：
5’AGC ACC ACA ATC AAC GTG AG3’
Shown in primer synthetic by MWG Biotech, carry out PCR (PCR scheme, methods and applications instruct, 1990, academic press) by the described standard pcr of Innis.Described primer can amplify the dna fragmentation of about 2.1kb, and it carries aecD gene and adjacent domain.
The dna fragmentation of the amplification of this about 2.1kb, separate by electrophoresis in 0.8% sepharose and from gel and by the ordinary method purifying differentiate (the QIAquick gel extraction kit, Qiagen, Hilden).
The dna fragmentation of purifying is cut (AmershamPharmacia, Freiburg, Germany) with restriction enzyme BamHI and EcoRV.In carrier pUC18, carry out fragment then and connect (Norrander etc., gene 26:101-106 (1983)).In advance this carrier is cut with restriction enzyme BglII and SmaI, dephosphorylation mixes with the fragment of carrying aecD of about 1.5kb, and mixture is handled (Amersham-Pharmacia, Freiburg, Germany) with the T4 dna ligase.To connect mixture transforms in coli strain TOP10 (Invitrogen, Leek, Holland).By conversion product being plated on the cell of selecting to carry plasmid on the LB agar that contains the 50mg/l kantlex with X-Gal (64mg/l).
The plasmid that obtains detects by the restriction cutting after DNA isolation, and differentiates in sepharose.The gained plasmid is called pUC18aecD.
Isolated plasmid dna from the strain DSM 14242 that carries plasmid pCRIITOPOlysC (seeing embodiment 1.1) with restriction enzyme BamHI cutting (Amersham-Pharmacia, Freiburg, Germany), is handled with the Klenow polysaccharase then.After separating in sepharose (0.8%) by means of QIAquick gel extraction kit (Qiagen, Hilden, Germany), with the lySC that contains of about 1.7kb
FBRDna fragmentation from sepharose, separate, and be used for being connected with above-mentioned carrier pUC18aecD.This carrier in advance with restriction enzyme StuI cutting, is used alkaline phosphatase dephosphorylation (alkaline phosphatase, Boehringer Mannheim, Germany), with the lysC of about 1.7kb
FBRFragment is mixed, and described mixture is handled with T4 dna ligase (Amersham-Pharmacia, Freiburg, Germany).
Then coli strain DH5 α mcr (Life Technologies GmbH, Karlsruhe, Germany) is transformed (Hanahan, dna clone practical plan, the 1st volume, ILR-press, cold spring port, New York, 1989) with connecting mixture.By conversion product being plated on the cell (Sambrook etc., molecular cloning experiment instruction, the 2nd edition, cold spring port, New York, 1989) of selecting to carry plasmid on the LB agar of adding the 50mg/l kantlex.
By means of the QIAprep Spin Miniprep test kit isolated plasmid dna from transformant that derives from Qiagen, and the agarose gel electrophoresis that reaches subsequently by the restriction cutting detects.Described plasmid is called pUC18aecD1.
Plasmid pUC18 aecD1 is handled with the Klenow polysaccharase then with restriction enzyme KpnI cutting.This plasmid is with restriction enzyme SalI (Amersham-Pharmacia then, Freiburg, Germany) cutting, by means of QIAquick gel extraction kit (Qiagen, Hilden, Germany) in sepharose (0.8%), after the separation, the fragment of carrying aecD and lysC of about 3.2kb is separated from sepharose, and be used for being connected with the described removable cloning vector pK18mobsacB of people (gene 14:69-73 (1994)) such as Schafer.This carrier is cut with restriction enzyme SmaI and SalI in advance, with alkaline phosphatase dephosphorylation (alkaline phosphatase, BoehringerMannheim, Germany), mix with this fragment of carrying aecD and lysC of about 3.2kb, described mixture is handled with T4 dna ligase (Amersham-Pharmacia, Freiburg, Germany).
Then coli strain DH5 α (people such as Grant, newspaper 87 (1990) 4645-4649 of institute of American Academy of Sciences) is transformed (Hanahan, dna clone practical plan, the 1st volume, ILR-press, cold spring port, New York, 1989) with connecting mixture.By conversion product being plated on the cell (Sambrook etc., molecular cloning experiment instruction, the 2nd edition, cold spring port, New York, 1989) of selecting to carry plasmid on the LB agar of adding the 50mg/l kantlex.
By means of the QIAprep Spin Miniprep test kit isolated plasmid dna from transformant that derives from Qiagen, and the agarose gel electrophoresis that reaches subsequently by the restriction cutting detects.Described plasmid is called pK18mobsacBaecD1_1.This plasmid is illustrated in Fig. 2.
Plasmid pK18mobsacBaecD1_1 is according to budapest treaty, (=DH5alphamcr/pK18mobsacBaecD1_1) pure growth form was preserved in Germany microorganism and cell culture preservation center (DSMZ with coli strain DH5 α mcr/pK18mobsacBaecD1_1 on June 5th, 2002, Braunschweig, Germany), preserving number DSM15040.
1.8 by replacement vector pK18mobsacBaecD1_1, with lysC
FBRSecond copy of the lysC gene of allelic form mixes (target site: the aecD gene) in the karyomit(e) of strain DSM 12866
As described in embodiment 1.3, embodiment 1.4 described plasmid pK18mobsacBaecD1_1 are advanced among the Corynebacterium glutamicum strain DSM12866 by conjugal transfer.As described in embodiment 1.3, in the karyomit(e) of Corynebacterium glutamicum DSM12866, select at directed recombination event.According to the position of the recombination event second time, after excision, lysC
FBRAllelic second copy demonstrates in karyomit(e) aecD locus, and perhaps host's original aecD locus is kept.
About 40-50 bacterium colony test " in growth in the presence of the sucrose " reached the phenotype of " in the presence of kantlex, not growing ".To being shown, " in growth in the presence of the sucrose " reach " not growing " phenotype in the presence of kantlex about 20 bacterium colonies study by means of polymerase chain reaction.The dna fragmentation of aecD gene and peripheral region is carried in amplification from the chromosomal DNA of described bacterium colony.Select the primer identical to carry out PCR with structure integrated plasmid among the embodiment 1.7.
aecD_beg(SEQ ID NO：11)：
5’GAA CTT ACG CCA AGC TGT TC3’
aecD_end(SEQ ID NO：12)：
5’AGC ACC ACA ATC AAC GTG AG3’
Described primer can amplify the dna fragmentation of about 2.1kb in having the contrast clone of original aecD locus.Has lysC at karyomit(e) aecD locus
FBRAmong the clone of second copy of allelotrope, approximately the dna fragmentation of 3.8kb is amplified.
The dna fragmentation of amplification is differentiated by electrophoresis in 0.8% sepharose.
Differentiate a kind of clone by this way, described clone also has lysC except existing at the lysC locus the copy of wild type gene at karyomit(e) aeeD locus
FRBSecond copy of the lysC gene of allelotrope lysC T311I form.This clone is called strain DSM 12866aecD::lysC.
Embodiment 2: second copy of ddh gene mixed (target site: gluB) in the karyomit(e) of strain DSM 12866
2.1 make up replacement vector pK18mobsacBglu2_1
Corynebacterium glutamicum strain ATCC13032 is as the donor of chromosomal DNA.Use ordinary method from strains A TCC13032, to separate chromosomal DNA (Eikmanns etc., microbiology 140:1817-1828 (1994)).By means of polymerase chain reaction, the dna fragmentation of gluB gene and peripheral region is carried in amplification.Based on the sequence (Kronemeyer etc., bacteriology magazine 177:1152-1158 (1995)) (registration number X81191) of known Corynebacterium glutamicum gluABCD gene cluster, select following Oligonucleolide primers to carry out PCR:
gluA_beg(SEQ ID NO：13)：
5’CAC GGT TGC TCA TTG TAT CC3’
gluD_end(SEQ ID NO：14)：
5’CGA GGC GAA TCA GAC TTC TT3’
Shown in primer synthetic by MWG Biotech, and carry out PCR (PCR scheme, methods and applications instruct, 1990, academic press) by the described standard pcr of Innis.Described primer can amplify the dna fragmentation of about 4.4kb, and it carries gluB gene and peripheral region.
The dna fragmentation of amplification, separate by electrophoresis in 0.8% sepharose and from gel and by the ordinary method purifying differentiate (the QIAquick gel extraction kit, Qiagen, Hilden).
Utilizing TOPO TA clone's test kit (Invitrogen, Leek, Holland, Cat.Number K4600-01) to carry out fragment then in carrier pCRII-TOPO connects.To connect mixture transforms among the coli strain TOP 10 (Invitrogen, Leek, Holland).By conversion product being plated on the cell of selecting to carry plasmid on the LB agar that contains kantlex (50mg/l) with X-Gal (64mg/l).
The plasmid that obtains detects by the restriction cutting after DNA isolation, and differentiates in sepharose.The gained plasmid is called pCRII-TOPOglu2.
With plasmid pCRII-TOPOglu2 limiting enzyme EcoRI and SalI (Amersham-Pharmacia, Freiburg, Germany) cutting, and in sepharose (0.8%) by means of QIAquick gel extraction kit (Qiagen, Hilden, Germany) separate after, the gluB fragment of from this sepharose, separating about 3.7kb, be used for
Described removable cloning vector pK18mobsacB (gene 14:69-73 (1994)) connects.In advance with this carrier with limiting enzyme EcoRI and SalI cutting and with alkaline phosphatase dephosphorylation (alkaline phosphatase, Boehringer Mannheim), mix with the gluB fragment of about 3.7kb, and with mixture T4 dna ligase processing (Amersham-Pharmacia, Freiburg, Germany).
Then coli strain DH5 α (Grant etc., newspaper 87 (1990) 4645-4649 of institute of American Academy of Sciences) is transformed (Hanahan, dna clone practical plan, the 1st volume, ILR-press, cold spring port, New York, 1989) with connecting mixture.By conversion product being plated on the cell (Sambrook etc., molecular cloning experiment instruction, the 2nd edition, cold spring port, New York, 1989) of selecting to carry plasmid on the LB agar of adding the 50mg/l kantlex.
By means of the QIAprep Spin Miniprep test kit isolated plasmid dna from transformant that derives from Qiagen, and the agarose gel electrophoresis that reaches subsequently by the restriction cutting detects.Described plasmid is called pK18mobsacBglu2.
As described in embodiment 2.1, carry the dna fragmentation of ddh gene and peripheral region by means of the polymeric enzyme reaction amplification.Based on the sequence (Ishino etc., nucleic acids research 15,3917 (1987)) (accession number Y00151) of known Corynebacterium glutamicum ddh gene cluster, select following primer to carry out PCR:
ddh_beg(SEQ ID NO：15)：
5’CTG AAT CAA AGG CGG ACA TG3′
ddh_end(SEQ ID NO：16)：
5’TCG AGC TAA ATT AGA CGT CG3’
Shown in primer synthetic by MWG Biotech, and carry out PCR (PCR scheme, methods and applications instruct, 1990, academic press) by the described standard pcr of Innis.Described primer can amplify the dna fragmentation of about 1.6kb, and it carries the ddh gene.
Carry the dna fragmentation of amplification of about 1.6kb of ddh gene, separate by electrophoresis in 0.8% sepharose and from gel and by the ordinary method purifying differentiate (the QIAquick gel extraction kit, Qiagen, Hilden).
Behind purifying, the fragment of carrying the ddh gene is connected among the described carrier pK18mobsacBglu2.Described carrier partly cuts with restriction enzyme BamHI in advance.Using Klenow polysaccharase (Amersham-Pharmacia, Freiburg, Germany) behind the processing carrier, the overhang of incision tip becomes flush end fully, then the dna fragmentation of this carrier with the about 1.6kb that carries the ddh gene mixed, mixture is handled with T4 dna ligase (Amersham-Pharmacia, Freiburg, Germany).By using Vent polysaccharase (New England Biolabs, Frankfurt, Germany) to carry out the PCR reaction, produce a dna fragmentation that carries ddh, it has flush end and is suitable for being connected among the pretreated carrier pK18mobsacBglu2.
Then coli strain DH5 α mcr ((Life Technologies GmbH, Karlsruhe, Germany)) is transformed (Hanahan, dna clone practical plan, the 1st volume, ILR-press, cold spring port, New York, 1989) with connecting mixture.By conversion product being plated on the cell (Sambrook etc., molecular cloning experiment instruction, the 2nd edition, cold spring port, New York, 1989) of selecting to carry plasmid on the LB agar of adding the 50mg/l kantlex.
By means of the QIAprep Spin Miniprep test kit isolated plasmid dna from transformant that derives from Qiagen, and the agarose gel electrophoresis that reaches subsequently by the restriction cutting detects.Described plasmid is called pK18mobsacBglu2_1.This plasmid is illustrated in Fig. 4.
2.2 second copy of ddh gene mixed (target site: the gluB gene) in the karyomit(e) of strain DSM 12866 by replacement vector pK18mobsacBglu2_1
As described in embodiment 1.3, embodiment 2.1 described carrier pK18mobsacBglu2_1 are advanced among the Corynebacterium glutamicum strain DSM12866 by conjugal transfer.As described in embodiment 1.3, in the karyomit(e) of Corynebacterium glutamicum DSM12866, select at directed recombination event.According to the position of the recombination event second time, after excision, second copy of ddh gene shows that in karyomit(e) gluB locus perhaps host's original gluB locus is kept.
About 40-50 bacterium colony test " in growth in the presence of the sucrose " reached the phenotype of " in the presence of kantlex, not growing ".To being shown, " in growth in the presence of the sucrose " reach " not growing " phenotype in the presence of kantlex about 20 bacterium colonies study by means of polymerase chain reaction.The dna fragmentation in glu zone is carried in amplification from the chromosomal DNA of described bacterium colony.Select the primer identical to carry out PCR with structure displacement plasmid among the embodiment 2.1.
gluA_beg(SEQ ID NO：13)：
5’CAC GGT TGC TCA TTG TAT CC3’
gluD_end(SEQ ID NO：14)：
5’CGA GGC GAA TCA GAC TTC TT3’
Described primer can amplify the dna fragmentation of about 4.4kb in having the contrast clone of original glu locus.Have among the clone of second copy of ddh gene at karyomit(e) gluB locus, approximately the dna fragmentation of 6kb is amplified.
The dna fragmentation of amplification is differentiated by electrophoresis in 0.8% sepharose.
Differentiate a kind of clone by this way, described clone also has second copy of ddh gene at karyomit(e) gluB locus except the copy that exists at the ddh locus.This clone is called strain DSM 12866glu::ddh.
Embodiment 3: second copy of dapA gene mixed (target site: the aecD gene) in the karyomit(e) of strain DSM 12866
3.1 make up replacement vector pK18mobsacBaecD2_1
Corynebacterium glutamicum strain ATCC13032 is as the donor of chromosomal DNA.Use ordinary method from strains A TCC13032, to separate chromosomal DNA (Eikmanns etc., microbiology 140:1817-1828 (1994)).By means of polymerase chain reaction, the dna fragmentation of aecD gene and peripheral region is carried in amplification.Based on the sequence (Rossol etc., bacteriology magazine 174:2968-2977 (1992)) (accession number M89931) of known Corynebacterium glutamicum aecD gene, select following Oligonucleolide primers to carry out PCR:
aecD_beg(SEQ ID NO：11)：
5’GAA CTT ACG CCA AGC TGT TC3’
aecD_end(SEQ ID NO：12)：
5’AGC ACC ACA ATC AAC GTG AG 3’
Shown in primer synthetic by MWG Biotech, and carry out PCR (PCR scheme, methods and applications instruct, 1990, academic press) by the described standard pcr of Innis.Described primer can amplify the dna fragmentation of about 2.1kb, and it carries aecD gene and adjacent domain.
The about dna fragmentation of the amplification of 2.1kb, separate by electrophoresis in 0.8% sepharose and from gel and by the ordinary method purifying differentiate (the QIAquick gel extraction kit, Qiagen, Hilden).
The dna fragmentation of purifying is cut (Amersham-Pharmacia, Freiburg, Germany) with restriction enzyme BglII and EcoRV.In carrier pUC18, carry out fragment then and connect (Norrander etc., gene 26:101-106 (1983)).Described carrier with restriction enzyme BamHI and SmaI cutting and dephosphorylation, mixes with the fragment of carrying the aecD gene of about 1.5kb, and mixture is handled (Amersham-Pharmacia, Freiburg, Germany) with the T4 dna ligase in advance.To connect mixture transforms among the coli strain TOP10 (Invitrogen, Leek, Holland).By conversion product being plated on the cell of selecting to carry plasmid on the LB agar that contains the 50mg/l kantlex with X-Gal (64mg/l).
The gained plasmid detects by the restriction cutting after DNA isolation, and differentiates in sepharose.The gained plasmid is called pUC18aecD.
By means of polymeric enzyme reaction, another dna fragmentation of dapA gene and peripheral region is carried in amplification.Based on the sequence (Bonassi etc., nucleic acids research 18,6421 (1990)) (accession number X53993 and AX127149) of known Corynebacterium glutamicum dapA gene, select following Oligonucleolide primers to carry out PCR:
dapA_beg(SEQ ID NO：17)：
5’AGA GCC AGT GAA CAT GCA GA3’
dapA_end(SEQ D NO：18)：
5’CTT GAG CAC CTT GCG CAG CA3’
Shown in primer synthetic by MWG Biotech, and carry out PCR (PCR scheme, methods and applications instruct, 1990, academic press) by the described standard pcr of Innis.Described primer can amplify the dna fragmentation of about 1.4kb, and it carries dapA gene and adjacent domain.
The about dna fragmentation of the amplification of 1.4kb, separate by electrophoresis in 0.8% sepharose and from gel and by the ordinary method purifying differentiate (the QIAquick gel extraction kit, Qiagen, Hilden).
Behind purifying, the dna fragmentation that contains the dapA gene of about 1.4kb is connected in above-mentioned carrier pUC18aecD.Described carrier with restriction enzyme StuI cutting, mixes with the dna fragmentation of about 1.4kb in advance, and mixture is handled with T4DNA ligase enzyme (Amersham-Pharmacia, Freiburg, Germany).
Then coli strain DH5 α mcr (Life Technologies GmbH, Karlsruhe, Germany) is transformed (Hanahan, dna clone practical plan, the 1st volume, ILR-press, cold spring port, New York, 1989) with connecting mixture.By conversion product being plated on the cell (Sambrook etc., molecular cloning experiment instruction, the 2nd edition, cold spring port, New York, 1989) of selecting to carry plasmid on the LB agar of adding the 50mg/l kantlex.
By means of the QIAprep Spin Miniprep test kit isolated plasmid dna from transformant that derives from Qiagen, and the agarose gel electrophoresis that reaches subsequently by the restriction cutting detects.Described plasmid is called pUC18aecD2.
Plasmid pUC18 aecD2 is partly cut (Amersham-Pharmacia with restriction enzyme SalI cutting and with EcoRI, Freiburg, Germany), and in sepharose (0.8%) by means of QIAquick gel extraction kit (Qiagen, Hilden, Germany) separate after, the fragment of carrying aecD and dapA of from this sepharose, separating about 2.7kb, be used for
Described removable cloning vector pK18mobsacB (gene 14:69-73 (1994)) connects.In advance with this carrier with limiting enzyme EcoRI and SalI cutting and with alkaline phosphatase dephosphorylation (alkaline phosphatase, Boehringer Mannheim), mix with the fragment of carrying aecD and dapA of about 2.7kb, and with mixture T4 dna ligase processing (Amersham-Pharmacia, Freiburg, Germany).
Then coli strain DH5 α (Grant etc., newspaper 87 (1990) 4645-4649 of institute of American Academy of Sciences) is transformed (Hanahan, dna clone practical plan, the 1st volume, ILR-press, cold spring port, New York, 1989) with connecting mixture.By conversion product being plated on the cell (Sambrook etc., molecular cloning experiment instruction, the 2nd edition, cold spring port, New York, 1989) of selecting to carry plasmid on the LB agar of adding the 50mg/l kantlex.
By means of the QIAprep Spin Miniprep test kit isolated plasmid dna from transformant that derives from Qiagen, and the agarose gel electrophoresis that reaches subsequently by the restriction cutting detects.Described plasmid is called pK18mobsacBaecD2_1.This plasmid is illustrated in Fig. 5.
3.2 by replacement vector pK18mobsacBaecD2_1, second copy of dapA gene mixed (target site: the aecD gene) in the karyomit(e) of strain DSM 12866
As described in embodiment 1.3, embodiment 3.1 described carrier pK18mobsacBaecD2_1 are advanced among the Corynebacterium glutamicum strain DSM12866 by conjugal transfer.As described in embodiment 1.3, in the karyomit(e) of Corynebacterium glutamicum DSM12866, select at directed recombination event.According to the position of the recombination event second time, after excision, second copy of dapA gene demonstrates in karyomit(e) aecD locus, and perhaps host's original aecD locus is kept.
About 40-50 bacterium colony test " in growth in the presence of the sucrose " reached the phenotype of " in the presence of kantlex, not growing ".To being shown, " in growth in the presence of the sucrose " reach " not growing " phenotype in the presence of kantlex about 20 bacterium colonies study by means of polymerase chain reaction.The dna fragmentation of aecD gene and peripheral region is carried in amplification from the chromosomal DNA of described bacterium colony.Select the Oligonucleolide primers identical to carry out PCR with structure integrated plasmid among the embodiment 3.1.
aecD_beg(SEQ ID NO：11)：
5’GAA CTT ACG CCA AGC TGT TC3’
aecD_end(SEQ ID NO：12)：
5’AGC ACC ACA ATC AAC GTG AG3’
Described primer can amplify the dna fragmentation of about 2.1kb in having the contrast clone of original aecD locus.Have among the clone of second copy of dapA gene at karyomit(e) aecD locus, approximately the dna fragmentation of 3.6kb is amplified.
The dna fragmentation of amplification is differentiated by electrophoresis in 0.8% sepharose.
Differentiate a kind of clone by this way, described clone also has second copy of dapA gene at karyomit(e) aecD locus except the copy that exists at the dapA locus.This clone is called strain DSM 12866aecD::dapA.
Embodiment 4: second copy of the pyc gene of pyc allelotrope pycP458S form mixed (target site: the pck gene) in the karyomit(e) of strain DSM 12866
4.1 make up replacement vector pK18mobsacBpck1_3
Embodiment 1.5 described replacement vector pK18mobsacBpck1 are used as the allelic carrier is carrier of insertion pyc.
As described in embodiment 2.1, carry a dna fragmentation of pyc gene and peripheral region by means of PCR amplification.Based on the sequence (Peters-Wendisch etc., JOURNAL OF MICROBIOLOGY 144:915-927 (1998)) (registration number Y09548) of known Corynebacterium glutamicum pyc gene cluster, select following Oligonucleolide primers to carry out PCR:
pyc_beg(SEQ ID NO：19)：
5’TC(A CGC GT)C TTG AAG TCG TGC AGG TCA G3’
pyc_end(SEQ ID NO：20)：
5’TC(A CGC GT)C GCC TCC TCC ATG AGG AAG A3’
Shown in primer synthetic by MWG Biotech, and carry out PCR (PCR scheme, methods and applications instruct, 1990, academic press) by the described standard pcr of Innis.What described primer can amplify about 3.6kb carries the pyc gene DNA fragment.Described primer also contains the sequence of the cleavage site of restriction enzyme MluI in addition, is indicated with bracket in above-mentioned nucleotide sequence.
The dna fragmentation of amplification of about 3.6kb that carries the pyc gene is with restriction enzyme MluI cutting, separate by electrophoresis in 0.8% sepharose and from gel and differentiate (QIAquick gel extraction kit by the ordinary method purifying, Qiagen, Hilden).
Behind purifying, the dna fragmentation that carries the pyc gene is connected in above-mentioned carrier pK18mobsacBpck1.Described carrier is in advance with restriction enzyme BssHII cutting, with alkaline phosphatase dephosphorylation (alkaline phosphatase, Boehringer Mannheim), mix with the dna fragmentation that carries the pyc gene of about 3.6kb, with mixture T4DNA ligase enzyme (Amersham-Pharmacia, Freiburg, Germany) handle.
Then coli strain DH5 α mcr (Life Technologies GmbH, Karlsruhe, Germany) is transformed (Hanahan, dna clone practical plan, the 1st volume, ILR-press, cold spring port, New York, 1989) with connecting mixture.By conversion product being plated on the cell (Sambrook etc., molecular cloning experiment instruction, the 2nd edition, cold spring port, New York, 1989) of selecting to carry plasmid on the LB agar of adding the 50mg/l kantlex.
By means of the QIAprep Spin Miniprep test kit isolated plasmid dna from transformant that derives from Qiagen, and the agarose gel electrophoresis that reaches subsequently by the restriction cutting detects.Described plasmid is called pK18mobsacBpck1_2.
4.2 by the gene constructed pyc allelotrope of site-specific mutagenesis wild-type pyc pyc P458S
(Stratagene, La Jolla, the U.S.) carries out site-directed mutagenesis with QuikChange site-directed mutagenesis test kit.EP-A-1108790 has described Corynebacterium glutamicum pyc gene mutations, and it is improved the production of L-Methionin.Be replaced into the point mutation in the nucleotide sequence of thymus pyrimidine based on the 1372nd cytosine(Cyt) of pyc gene, cause therefore that proline(Pro) is replaced into Serine in its aminoacid sequence.This equipotential gene is called pyc P458S.For producing described sudden change, select following Oligonucleolide primers to carry out linear amplification:
P458S-1(SEQ ID NO：21)：
5′GGATTCATTGCCGATCAC(TCG)CACCTCCTTCAGGCTCCA3′
P458S-2(SEQID NO：22)：
5′GTGGAGGAAGTCCGAGGT(CGA)GTGATCGGCAATGAATCC3′
Described primer is synthetic by MWG Biotech.The Serine codon of replacing the 458th proline(Pro) indicates with bracket in above-mentioned nucleotide sequence.Embodiment 4.1 described plasmid pK18mobsacBpck1_2 and two primers one are used from by Pfu Turbo archaeal dna polymerase carry out linear amplification, described primer all is complementary to described plasmid chain.By this extension of primer, form the mutant plasmid of endless chain with fracture.The product of linear amplification is handled with DpnI, and this restriction endonuclease specificity cutting methylates and hemimethylated template DNA.The mutational vector DNA of new synthetic fracture is transformed among the coli strain XL1 Blue (Bullock, Fernandez and Short, biotechnology (5) 376-379 (1987)).After conversion, the fracture part in the XL1 Blue cell repairing mutant plasmid.Select transformant having on the LB substratum of 50mg/l kantlex.The plasmid that obtains detects by restricted cutting after DNA isolation, and differentiates in sepharose.The segmental dna sequence dna of mutant DNA detects by order-checking.The sequence of PCR product is consistent with (2002) described sequences such as Ohnishi.The gained plasmid is called pK18mobsacBpck1_3.This plasmid is illustrated in Fig. 6..
4.3 by replacement vector pK18mobsacspck1_3, second copy of the pyc gene of pyc allelotrope pycP458S form mixed (target site: the pck gene) in the karyomit(e) of strain DSM 12866
Embodiment 4.2 described plasmid pK18mobsacBpck1_3 are advanced among the Corynebacterium glutamicum strain DSM12866 by conjugal transfer as described in embodiment 1.3.As described in embodiment 1.3, in the karyomit(e) of Corynebacterium glutamicum DSM12866, select at directed recombination event.According to the position of the recombination event second time, after excision, allelic second copy of pyc pck locus in karyomit(e) demonstrates, and perhaps host's original pck locus is kept.
About 40-50 bacterium colony test " in growth in the presence of the sucrose " reached the phenotype of " in the presence of kantlex, not growing ".To being shown, " in growth in the presence of the sucrose " reach " not growing " phenotype in the presence of kantlex about 20 bacterium colonies study by means of polymerase chain reaction.The dna fragmentation of pck gene and peripheral region is carried in amplification from the chromosomal DNA of described bacterium colony.Select the Oligonucleolide primers identical to carry out PCR with structure displacement plasmid among the embodiment 1.5.
pck_beg(SEQ ID NO：9)：
5’TA(A GATCT)G CCG GCA TGA CTT CAG TTT3’
pck_end(SEQ ID NO：10)：
5’AC(A GATCT)G GTG GGA GCC TTT CTT GTTATT3’
Described primer can amplify the dna fragmentation of about 2.9kb in having the contrast clone of original pck locus.Have among the clone of second copy of pyc gene at karyomit(e) pck locus, approximately the dna fragmentation of 6.5kb is amplified.
The dna fragmentation of amplification is differentiated by electrophoresis in 0.8% sepharose.
Differentiate a kind of clone by this way, described clone also has second copy of the pyc gene of pyc allelotrope pycP458S form at karyomit(e) pck locus except the copy of the wild type gene that exists at the pyc locus.This clone is called strain DSM 12866pck::pyc.
Embodiment 5: produce Methionin
Will be at embodiment 1,2, the Corynebacterium glutamicum strain DSM13994glu::lysC that obtains in 3 and 4, DSM12866glu::lysC, DSM12866pck::lysC, DSM12866aecD::lysC, DSM12866glu::ddh, DSM12866aecD::dapA and DSM12866pck::pyc cultivate in the nutritional medium that is suitable for Methionin production, measure lysine content in the culture supernatant.
At first, at 33 ℃ culture was gone up incubation 24 hours at brain heart agar plate (Merck, Darmstadt, Germany).This agar plate culture is used to inoculate pre-culture (10ml cultivates based on the 100ml Erlenmeyer flask).Being used for pre-incubated substratum is the MM substratum.On shaking table in 33 ℃ with the pre-culture of 240rpm incubation 24 hours.With these pre-culture inoculation master cultures, the initial OD of master culture (wavelength 660nm) is 0.1 thus.Substratum MM is also as master culture.
Substratum MM:
CSL 5g/l
MOPS 20g/l
Glucose (autoclaving separately) 50g/l
(NH
4)
2SO
4 25g/l
KH
2PO
4 0.1g/l
MgSO
4·7H
2O 1.0g/l
CaCl
2·2H
2O 10mg/l
FeSO4·7H
2O 10mg/l
MnSO
4·H
2O 5.0mg/l
Vitamin H (filtration sterilization) 0.3mg/l
VitB1 HCl (filtration sterilization) 0.2mg/l
CaCO
3 25g/l
With CSL (corn steep liquor), the pH regulator of MOPS (morpholino propanesulfonic acid) and described salts solution is 7, and autoclaving with ammoniacal liquor.Add aseptic substrate and vitamin solution then, and under drying regime autoclaved CaCO
3
In having the 100ml Erlenmeyer flask of baffle plate, cultivate with the 10ml volume.Under 33 ℃ and 80% humidity, cultivate.
After 48 hours, (Beckmann Instruments GmbH Munich) measures OD under 660nm mensuration wavelength with Biomek1000.Amino acidanalyser (hamburger, Germany) with Eppendorf-BioTronik company detects post-column derivatization and determines the Methionin growing amount through ion exchange chromatography with triketohydrindene hydrate.
Experimental result is shown in table 10.
Table 10
| Bacterial strain | OD(660nm) | Lysine hydrochloride g/l |
| DSM13994 | 12.0 | 19.1 |
| DSM13994glu::lysC | 9.9 | 20.0 |
| DSM12866 | 12.5 | 14.9 |
| DSM15039 | 11.4 | 16.2 |
| DSM12866pck::lysC | 12.6 | 16.5 |
| DSM12866aecD::lysC | 12.0 | 15.9 |
| DSM12866glu::ddh | 11.0 | 15.5 |
| DSM12866aecD::dapA | 11.1 | 16.2 |
| DSM12866pck::pyc | 10.9 | 16.9 |
The accompanying drawing summary:
Specified base pair number is the approximation that obtains in replication.
Fig. 1: plasmid pK18mobsacBglu1_1 figure.
Used abbreviation and title have following implication:
KanR: kalamycin resistance gene
HindIII: the cleavage site of restriction enzyme HindIII
BamHI: the cleavage site of restriction enzyme BamHI
LysC:lysC
FBRAllelotrope, lysCT311I
3 ' terminal fragment of ' gluA:gluA gene
GluB ': 5 ' terminal fragment of gluB gene
3 ' terminal fragment of ' gluB:gluB gene
GluC ': 5 ' terminal fragment of gluC gene
The sacB:sacB gene
RP4mob: mob zone with the replication orgin (oriT) that is used to shift
OriV: replication orgin V
Fig. 2: plasmid pK18mobsacBaecD1_1 figure.
Used abbreviation and title have following implication:
KanR: kalamycin resistance gene
SalI: the cleavage site of restriction enzyme SalI
LysC:lysC allelotrope, lysC T311I
AecD ': 5 ' end of aecD gene
3 ' end of ' aecD:aecD gene
The sacB:sacB gene
RP4mob: mob zone with the replication orgin (oriT) that is used to shift
OriV: replication orgin V
Fig. 3: plasmid pK18mobsacBpck1_1 figure.
Used abbreviation and title have following implication:
KanR: kalamycin resistance gene
BamHI: the cleavage site of restriction enzyme BamHI
LysC:lysC
FBRAllelotrope, lysC T311I
Pck ': 5 ' terminal fragment of pck gene
3 ' terminal fragment of ' pck:pck gene
The sacB:sacB gene
RP4mob: mob zone with the replication orgin (oriT) that is used to shift
OriV: replication orgin V
Fig. 4: plasmid pK18mobsacBgluB2_1 figure.
Used abbreviation and title have following implication:
KanR: kalamycin resistance gene
SalI: the cleavage site of restriction enzyme SalI
EcoRI: the cleavage site of limiting enzyme EcoRI
BamHI: the cleavage site of restriction enzyme BamHI
The ddh:ddh gene
The gluA:gluA gene
GluB ': 5 ' terminal fragment of gluB gene
3 ' terminal fragment of ' gluB:gluB gene
The gluC:gluC gene
GluD ': 5 ' terminal fragment of gluD gene
The sacB:sacB gene
RP4mob: mob zone with the replication orgin (oriT) that is used to shift
OriV: replication orgin V
Fig. 5: plasmid pK18mobsacBaecD2_1 figure.
Used abbreviation and title have following implication:
KanR: kalamycin resistance gene
EcoRI: the cleavage site of limiting enzyme EcoRI
SalI: the cleavage site of restriction enzyme SalI
The dapA:dapA gene
AecD ': 5 ' terminal fragment of aecD gene
3 ' terminal fragment of ' aecD:aecD gene
The sacB:sacB gene
RP4mob: mob zone with the replication orgin (oriT) that is used to shift
OriV: replication orgin V
Fig. 6: plasmid pK18mobsacBpck1_3 figure.
Used abbreviation and title have following implication:
KanR: kalamycin resistance gene
Pyc:pyc allelotrope, pyc P458S
Pck ': 5 ' terminal fragment of pck gene
3 ' terminal fragment of ' pck:pck gene
The sacB:sacB gene
RP4mob: mob zone with the replication orgin (oriT) that is used to shift
OriV: replication orgin V
Sequence table
＜110〉Degussa
＜120〉produce L-Methionin by the corynebacterium glutamicum strain of genetic modification
<130>DEDEG0213
<160>22
<170>PatentIn version 3.1
<210>1
<211>1263
<212>DNA
<213>Corynebacterium glutamicum
<220>
<221>CDS
<222>(1)..(1263)
＜223〉lysC wild type gene
<400>1
gtg gcc ctg gtc gta cag aaa tat ggc ggt tcc tcg ctt gag agt gcg 48
Met Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala
1 5 10 15
gaa cgc att aga aac gtc gct gaa cgg atc gtt gcc acc aag aag gct 96
Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Val Ala Thr Lys Lys Ala
20 25 30
gga aat gat gtc gtg gtt gtc tgc tcc gca atg gga gac acc acg gat 144
Gly Asn Asp Val Val Val Val Cys Ser Ala Met Gly Asp Thr Thr Asp
35 40 45
gaa ctt cta gaa ctt gca gcg gca gtg aat ccc gtt ccg cca gct cgt 192
Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Val Pro Pro Ala Arg
50 55 60
gaa atg gat atg ctc ctg act gct ggt gag cgt att tct aac gct ctc 240
Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu
65 70 75 80
gtc gcc atg gct att gag tcc ctt ggc gca gaa gcc caa tct ttc acg 288
Val Ala Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr
85 90 95
ggc tct cag gct ggt gtg ctc acc acc gag cgc cac gga aac gca cgc 336
Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg
100 105 110
att gtt gat gtc act cca ggt cgt gtg cgt gaa gca ctc gat gag ggc 384
Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly
115 120 125
aag atc tgc att gtt gct ggt ttc cag ggt gtt aat aaa gaa acc cgc 432
Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg
130 135 140
gat gtc acc acg ttg ggt cgt ggt ggt tct gac acc act gca gtt gcg 480
Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala
145 150 155 160
ttg gca gct gct ttg aac gct gat gtg tgt gag att tac tcg gac gtt 528
Leu Ala Ala Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val
165 170 175
gac ggt gtg tat acc gct gac ccg cgc atc gtt cct aat gca cag aag 576
Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys
180 185 190
ctg gaa aag ctc agc ttc gaa gaa atg ctg gaa ctt gct gct gtt ggc 624
Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala Ala Val Gly
195 200 205
tcc aag att ttg gtg ctg cgc agt gtt gaa tac gct cgt gca ttc aat 672
Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn
210 215 220
gtg cca ctt cgc gta cgc tcg tct tat agt aat gat ccc ggc act ttg 720
Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu
225 230 235 240
att gcc ggc tct atg gag gat att cct gtg gaa gaa gca gtc ctt acc 768
Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr
245 250 255
ggt gtc gca acc gac aag tcc gaa gcc aaa gta acc gtt ctg ggt att 816
Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly Ile
260 265 270
tcc gat aag cca ggc gag gct gcg aag gtt ttc cgt gcg ttg gct gat 864
Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg Ala Leu Ala Asp
275 280 285
gca gaa atc aac att gac atg gtt ctg cag aac gtc tct tct gta gaa 912
Ala Glu Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu
290 295 300
gac ggc acc acc gac atc acc ttc acc tgc cct cgt tcc gac ggc cgc 960
Asp Gly Thr Thr Asp Ile Thr Phe Thr Cys Pro Arg Ser Asp Gly Arg
305 310 315 320
cgc gcg atg gag atc ttg aag aag ctt cag gtt cag ggc aac tgg acc 1008
Arg Ala Met Glu Ile Leu Lys Lys Leu Gln Val Gln Gly Asn Trp Thr
325 330 335
aat gtg ctt tac gac gac cag gtc ggc aaa gtc tcc ctc gtg ggt gct 1056
Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala
340 345 350
ggc atg aag tct cac cca ggt gtt acc gca gag ttc atg gaa gct ctg 1104
Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu
355 360 365
cgc gat gtc aac gtg aac atc gaa ttg att tcc acc tct gag att cgt 1152
Arg Asp Val Asn Val Asn Ile Glu Leu Ile Ser Thr Ser Glu Ile Arg
370 375 380
att tcc gtg ctg atc cgt gaa gat gat ctg gat gct gct gca cgt gca 1200
Ile Ser Val Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala
385 390 395 400
ttg cat gag cag ttc cag ctg ggc ggc gaa gac gaa gcc gtc gtt tat 1248
Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu Ala Val Val Tyr
405 410 415
gca ggc acc gga cgc 1263
Ala Gly Thr Gly Arg
420
<210>2
<211>421
<212>PRT
<213>Corynebacterium glutamicum
<400>2
Met Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala
1 5 10 15
Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Val Ala Thr Lys Lys Ala
20 25 30
Gly Asn Asp Val Val Val Val Cys Ser Ala Met Gly Asp Thr Thr Asp
35 40 45
Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Val Pro Pro Ala Arg
50 55 60
Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu
65 70 75 80
Val Ala Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr
85 90 95
Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg
100 105 110
Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly
115 120 125
Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg
130 135 140
Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala
145 150 155 160
Leu Ala Ala Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val
165 170 175
Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys
180 185 190
Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala Ala Val Gly
195 200 205
Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn
210 215 220
Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu
225 230 235 240
Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr
245 250 255
Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly Ile
260 265 270
Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg Ala Leu Ala Asp
275 280 285
Ala Glu Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu
290 295 300
Asp Gly Thr Thr Asp Ile Thr Phe Thr Cys Pro Arg Ser Asp Gly Arg
305 310 315 320
Arg Ala Met Glu Ile Leu Lys Lys Leu Gln Val Gln Gly Asn Trp Thr
325 330 335
Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala
340 345 350
Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu
355 360 365
Arg Asp Val Asn Val Asn Ile Glu Leu Ile Ser Thr Ser Glu Ile Arg
370 375 380
Ile Ser Val Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala
385 390 395 400
Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu Ala Val Val Tyr
405 410 415
Ala Gly Thr Gly Arg
420
<210>3
<211>1263
<212>DNA
<213>Corynebacterium glutamicum
<220>
<221>CDS
<222>(1)..(1263)
<223>lysC-fbr allele lysC T311I
<400>3
gtg gcc ctg gtc gta cag aaa tat ggc ggt tcc tcg ctt gag agt gcg 48
Met Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala
1 5 10 15
gaa cgc att aga aac gtc gct gaa cgg atc gtt gcc acc aag aag gct 96
Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Val Ala Thr Lys Lys Ala
20 25 30
gga aat gat gtc gtg gtt gtc tgc tcc gca atg gga gac acc acg gat 144
Gly Asn Asp Val Val Val Val Cys Ser Ala Met Gly Asp Thr Thr Asp
35 40 45
gaa ctt cta gaa ctt gca gcg gca gtg aat ccc gtt ccg cca gct cgt 192
Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Val Pro Pro Ala Arg
50 55 60
gaa atg gat atg ctc ctg act gct ggt gag cgt att tct aac gct ctc 240
Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu
65 70 75 80
gtc gcc atg gct att gag tcc ctt ggc gca gaa gcc caa tct ttc acg 288
Val Ala Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr
85 90 95
ggc tct cag gct ggt gtg ctc acc acc gag cgc cac gga aac gca cgc 336
Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg
100 105 110
att gtt gat gtc act cca ggt cgt gtg cgt gaa gca ctc gat gag ggc 384
Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly
115 120 125
aag atc tgc att gtt gct ggt ttc cag ggt gtt aat aaa gaa acc cgc 432
Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg
130 135 140
gat gtc acc acg ttg ggt cgt ggt ggt tct gac acc act gca gtt gcg 480
Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala
145 150 155 160
ttg gca gct gct ttg aac gct gat gtg tgt gag att tac tcg gac gtt 528
Leu Ala Ala Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val
165 170 175
gac ggt gtg tat acc gct gac ccg cgc atc gtt cct aat gca cag aag 576
Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys
180 185 190
ctg gaa aag ctc agc ttc gaa gaa atg ctg gaa ctt gct gct gtt ggc 624
Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala Ala Val Gly
195 200 205
tcc aag att ttg gtg ctg cgc agt gtt gaa tac gct cgt gca ttc aat 672
Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala phe Asn
210 215 220
gtg cca ctt cgc gta cgc tcg tct tat agt aat gat ccc ggc act ttg 720
Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu
225 230 235 240
att gcc ggc tct atg gag gat att cct gtg gaa gaa gca gtc ctt acc 768
Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr
245 250 255
ggt gtc gca acc gac aag tcc gaa gcc aaa gta acc gtt ctg ggt att 816
Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly Ile
260 265 270
tcc gat aag cca ggc gag gct gcg aag gtt ttc cgt gcg ttg gct gat 864
Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg Ala Leu Ala Asp
275 280 285
gca gaa atc aac att gac atg gtt ctg cag aac gtc tct tct gta gaa 912
Ala Glu Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu
290 295 300
gac ggc acc acc gac atc atc ttc acc tgc cct cgt tcc gac ggc cgc 960
Asp Gly Thr Thr Asp Ile Ile Phe Thr Cys Pro Arg Ser Asp Gly Arg
305 310 315 320
cgc gcg atg gag atc ttg aag aag ctt cag gtt cag ggc aac tgg acc 1008
Arg Ala Met Glu Ile Leu Lys Lys Leu Gln Val Gln Gly Asn Trp Thr
325 330 335
aat gtg ctt tac gac gac cag gtc ggc aaa gtc tcc ctc gtg ggt gct 1056
Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala
340 345 350
ggc atg aag tct cac cca ggt gtt acc gca gag ttc atg gaa gct ctg 1104
Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu
355 360 365
cgc gat gtc aac gtg aac atc gaa ttg att tcc acc tct gag att cgt 1152
Arg Asp Val Asn Val Asn Ile Glu Leu Ile Ser Thr Ser Glu Ile Arg
370 375 380
att tcc gtg ctg atc cgt gaa gat gat ctg gat gct gct gca cgt gca 1200
Ile Ser Val Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala
385 390 395 400
ttg cat gag cag ttc cag ctg ggc ggc gaa gac gaa gcc gtc gtt tat 1248
Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu Ala Val Val Tyr
405 410 415
gca ggc acc gga cgc 1263
Ala Gly Thr Gly Arg
420
<210>4
<211>421
<212>PRT
<213>Corynebacterium glutamicum
<400>4
Met Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala
1 5 10 15
Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Val Ala Thr Lys Lys Ala
20 25 30
Gly Asn Asp Val Val Val Val Cys Ser Ala Met Gly Asp Thr Thr Asp
35 40 45
Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Val pro Pro Ala Arg
50 55 60
Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu
65 70 75 80
Val Ala Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr
85 90 95
Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg
100 105 110
Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly
115 120 125
Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg
130 135 140
Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala
145 150 155 160
Leu Ala Ala Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val
165 170 175
Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys
180 185 190
Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala Ala Val Gly
195 200 205
Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn
210 215 220
Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu
225 230 235 240
Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr
245 250 255
Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly Ile
260 265 270
Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg Ala Leu Ala Asp
275 280 285
Ala Glu Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu
290 295 300
Asp Gly Thr Thr Asp Ile Ile Phe Thr Cys Pro Arg Ser Asp Gly Arg
305 310 315 320
Arg Ala Met Glu Ile Leu Lys Lys Leu Gln Val Gln Gly Asn Trp Thr
325 330 335
Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala
340 345 350
Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu
355 360 365
Arg Asp Val Asn Val Asn Ile Glu Leu Ile Ser Thr Ser Glu Ile Arg
370 375 380
Ile Ser Val LeuIle Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala
385 390 395 400
Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu Ala Val Val Tyr
405 410 415
Ala Gly Thr Gly Arg
420
<210>5
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(28)
＜223〉primer lysC1beg
<400>5
taggatcctc cggtgtctga ccacggtg 28
<210>6
<211>29
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(29)
＜223〉primer lysC2end
<400>6
acggatccgc tgggaaattg cgctcttcc 29
<210>7
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(28)
＜223〉primer gluBgl1
<400>7
taagatctgt gttggacgtc atggcaag 28
<210>8
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(28)
＜223〉primer gluBgl2
<400>8
acagatcttg aagccaagta cggccaag 28
<210>9
<211>27
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(27)
＜223〉primer pck_beg
<400>9
taagatctgc cggcatgact tcagttt 27
<210>10
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(30)
＜223〉primer pck_end
<400>10
acagatctgg tgggagcctt tcttgttatt 30
<210>11
<211>20
<212>DNA
<213>Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer aecD_beg
<400>11
gaacttacgc caagctgttc 20
<210>12
<211>20
<212>DNA
<213>Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer aecD_end
<400>12
agcaccacaa tcaacgtgag 20
<210>13
<211>20
<212>DNA
<213>Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer gluA_beg
<400>13
cacggttgct cattgtatcc 20
<210>14
<211>20
<212>DNA
<213>Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer gluD_end
<400>14
cgaggcgaat cagacttctt 20
<210>15
<211>20
<212>DNA
<213>Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer ddh_beg
<400>15
ctgaatcaaa ggcggacatg 20
<210>16
<211>20
<212>DNA
<213>Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer ddh_end
<400>16
tcgagctaaa ttagacgtcg 20
<210>17
<211>20
<212>DNA
<213>Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer dapA_beg
<400>17
cgagccagtg aacatgcaga 20
<210>18
<211>20
<212>DNA
<213>Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer dapA_end
<400>18
cttgagcacc ttgcgcagca 20
<210>19
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(28)
＜223〉primer pyc_beg
<400>19
tcacgcgtct tgaagtcgtg caggtcag 28
<210>20
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(28)
＜223〉primer pyc_end
<400>20
tcacgcgtcg cctcctccat gaggaaga 28
<210>21
<211>39
<212>DNA
<213>Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(39)
＜223〉primer P458S-1
<400>21
ggattcattg ccgatcactc gcacctcctt caggctcca 39
<210>22
<211>39
<212>DNA
<213>Corynebacterium glutamicum
<220>
<221>misc_feature
<222>(1)..(39)
＜223〉primer P458S-2
<400>22
gtggaggaag tccgaggtcg agtgatcggc aatgaatcc 39
Claims (15)
1. bar shaped bacteria of producing the conversion of L-Methionin, wherein these bacteriums produce the protein or one or more gene of RNA synthetic or allelic at least one copy of L-Methionin except have coding at natural site (locus), at second, randomly the 3rd or the 4th site also have and are integrated into chromosomal this open reading frame, gene or allelic second, randomly the 3rd or the 4th copy, wherein said site is different from natural site, do not exist can/make can be in microorganism the nucleotide sequence of episomal replication or swivel base, and described second, randomly the 3rd or the 4th site and bacterial growth and desirable compound are produced necessary open reading frame, gene or allelotrope are uncorrelated, wherein
A) described conversion is undertaken by being used in the carrier that does not duplicate in the bar shaped bacteria,
B) described bacterium does not show the antibiotics resistance that is shifted by described carrier,
C) be integrated into maximum 24 Nucleotide that chromosomal described second, the 3rd or the 4th site keep described carrier sequence residue,
D) and described gene or allelotrope be selected from next group: accBC, accDA, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dapA, dapB, dapC, dapD, dapE, dapF, ddh, dps, eno, gap, gap2, gdh, gnd, lysC, lysC
FBR, lysE, msiK, opcA, oxyR, ppc, ppc
FBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwal, zwf and zwfA213T.
2. the bar shaped bacteria of claim 1, wherein said gene or the allelotrope that is used for Methionin production is one or more gene or the allelotrope that is selected from next group: dapA, ddh, lysC
FBRWith pyc P458S.
3. the bar shaped bacteria of claim 1, wherein said gene or the allelotrope that is used for Methionin production is lysC
FBRAllelotrope, its encoder feedback resistance form E.C. 2.7.2.4..
4. the bar shaped bacteria of claim 3, wherein lysC
FBRThe feedback resistance form E.C. 2.7.2.4. of allelotrope coding contains aminoacid sequence shown in the SEQ ID NO:2, and SEQ IDNO:2 contains one or more amino-acid substitution A279T that is selected from next group, A279V, S301F, T308I, S301Y, G345D, R320G, T311I and S381F.
5. the bar shaped bacteria of claim 3 is wherein by lysC
FBRThe E.C. 2.7.2.4. of the feedback resistance form of allelotrope coding comprises aminoacid sequence shown in the SEQ ID NO:4.
6. the bar shaped bacteria of claim 3, wherein lysC
FBRAllelic coding region comprises nucleotide sequence shown in the SEQ ID NO:3.
7. the bar shaped bacteria of the production L-Methionin of claim 1, wherein this specific second, randomly the 3rd or the 4th site are gene: aecD who is selected from next group, ccpA1, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, menE, mqo, pck, pgi and poxB.
8. the bar shaped bacteria of claim 1, wherein second, randomly the 3rd or the 4th site are the aecD gene locuss.
9. the bar shaped bacteria of claim 1, wherein second, randomly the 3rd or the 4th site are the gluB gene locuss.
10. the bar shaped bacteria of claim 1, wherein second, randomly the 3rd or the 4th site are the pck gene locuss.
11. the bar shaped bacteria of claim 1, wherein said bar shaped bacteria belongs to Corynebacterium.
12. the bar shaped bacteria of the production L-Methionin of the Corynebacterium of claim 11, wherein these bacteriums belong to the Corynebacterium glutamicum bacterial classification.
13. prepare the method for L-Methionin, wherein carry out following steps:
A) each bar shaped bacteria of fermentation claim 1-12,
B) the L-Methionin in gathering fermenting broth and/or the bacterial cell.
14. the method for claim 13 is further comprising the steps of:
C) separate L-Methionin, randomly
D) with component and/or biomass greater than 0 to 100wt% fermenting broth.
15. the method for claim 13 or 14, described method comprises:
A) at least a gene or the allelic nucleotide sequence of separation claim 8 from bar shaped bacteria randomly comprise and expressing and/or conditioning signal,
B) nucleotide sequence with target site offers described open reading frame, gene or allelic 5 ' and 3 ' end,
C) described gene or allelic nucleotide sequence with target site nucleotide sequence mixed in the carrier, described carrier does not duplicate in bar shaped bacteria,
D) with b) or nucleotides sequence column jump c) advance in the bar shaped bacteria,
E) separate such bar shaped bacteria, wherein nucleotide sequence a) is incorporated in target site, this target site do not have can/make can be in microorganism the nucleotide sequence of episomal replication or swivel base, and do not authorize the nucleotide sequence of antibiotics resistance, and
F) the described bar shaped bacteria that ferments.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US30987801P | 2001-08-06 | 2001-08-06 | |
| US60/309,878 | 2001-08-06 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1539015A CN1539015A (en) | 2004-10-20 |
| CN100554426C true CN100554426C (en) | 2009-10-28 |
Family
ID=23200067
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB028154460A Expired - Fee Related CN100554426C (en) | 2001-08-06 | 2002-07-30 | Corynebacterium glutamicum with genetic modification produces L-Methionin |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP1414970A2 (en) |
| CN (1) | CN100554426C (en) |
| AU (1) | AU2002325923A1 (en) |
| BR (1) | BR0211723A (en) |
| CA (1) | CA2455878A1 (en) |
| WO (1) | WO2003040373A2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111471638A (en) * | 2020-05-22 | 2020-07-31 | 江南大学 | Construction and application of corynebacterium glutamicum mutant strain capable of producing L-homoserine |
Families Citing this family (51)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7160711B2 (en) | 2001-08-06 | 2007-01-09 | Degussa Ag | Coryneform bacteria which produce chemical compounds I |
| US6844176B1 (en) | 2001-10-16 | 2005-01-18 | Degussa Ag | Alleles of the lysC gene from corynebacteria |
| DE10359594A1 (en) | 2003-12-18 | 2005-07-28 | Basf Ag | PEF TU-expression units |
| DE102004035067A1 (en) * | 2004-07-20 | 2006-02-16 | Basf Ag | P1-35 expression units |
| DE102005013676A1 (en) | 2005-03-24 | 2006-09-28 | Degussa Ag | Alleles of the zwf gene from coryneform bacteria |
| DE102005023829A1 (en) | 2005-05-24 | 2006-11-30 | Degussa Ag | Alleles of the opcA gene from coryneform bacteria |
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| DE102006026328A1 (en) * | 2006-06-02 | 2008-01-03 | Evonik Degussa Gmbh | Process for the preparation of a feed additive containing L-lysine |
| KR100830289B1 (en) * | 2007-01-18 | 2008-05-16 | 씨제이제일제당 (주) | Corynebacterium glutamicum variety producing l-arginine and method for fabricating the same |
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| JP2011067095A (en) | 2008-01-10 | 2011-04-07 | Ajinomoto Co Inc | Method for producing target substance by fermentation process |
| KR100987281B1 (en) * | 2008-01-31 | 2010-10-12 | 씨제이제일제당 (주) | Enhanced promoter and method for producing L-lysine using the same |
| DE102008001874A1 (en) | 2008-05-20 | 2009-11-26 | Evonik Degussa Gmbh | Process for the preparation of L-amino acids |
| DE102009030342A1 (en) | 2009-06-25 | 2010-12-30 | Evonik Degussa Gmbh | Process for the fermentative production of organic chemical compounds |
| DE102010003419B4 (en) | 2010-03-30 | 2019-09-12 | Evonik Degussa Gmbh | Process for the fermentative production of L-ornithine |
| DE102010025124A1 (en) | 2010-06-25 | 2011-12-29 | Forschungszentrum Jülich GmbH | Process for the preparation of D-amino acids, microorganism, and vector |
| EP2479279A1 (en) | 2011-01-20 | 2012-07-25 | Evonik Degussa GmbH | Method for producing sulphuric amino acids by means of fermentation |
| EP2678421B1 (en) * | 2011-02-22 | 2018-04-11 | Basf Se | Processes and recombinant microorganisms for the production of cadaverine |
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| PL2700715T3 (en) | 2012-08-20 | 2019-03-29 | Evonik Degussa Gmbh | Method for manufacturing L-amino acids using improved strains of the enterobacteriaceae family by means of fermentation |
| DE102012016716A1 (en) | 2012-08-22 | 2014-02-27 | Forschungszentrum Jülich GmbH | A process for the preparation of vectors comprising a gene coding for an enzyme which has been reduced or eliminated in its feedback inhibition and the use thereof for the production of amino acids and nucleotides |
| EP2762571A1 (en) | 2013-01-30 | 2014-08-06 | Evonik Industries AG | Microorganism and method for the production of amino acids by fermentation |
| ES2623161T3 (en) | 2013-06-03 | 2017-07-10 | Evonik Degussa Gmbh | Procedure for the preparation of L-valine using recombinant corinebacteria containing the propionate inducible ilvBN operon |
| CN103374540B (en) * | 2013-08-13 | 2015-04-22 | 廊坊梅花生物技术开发有限公司 | Corynebacterium glutamicum and application thereof |
| PL2940143T3 (en) | 2014-04-30 | 2020-06-29 | Evonik Operations Gmbh | Method for the production of l-amino acids using an alkaliphilic bacterium |
| DE102014208199A1 (en) | 2014-04-30 | 2015-11-05 | Evonik Degussa Gmbh | Process for the production of L-amino acids using an alkaliphilic bacterium |
| EP2940039A1 (en) | 2014-04-30 | 2015-11-04 | Evonik Degussa GmbH | Method for the production of l-amino acids in coryne bacteria using a glycine splitting system |
| EP2940144A1 (en) | 2014-04-30 | 2015-11-04 | Evonik Degussa GmbH | Method for the production of l-lysine using an alkaliphilic bacterium |
| BR112018011503A2 (en) | 2015-12-07 | 2018-12-04 | Zymergen Inc | corynebacterium glutamicum promoters |
| US9988624B2 (en) | 2015-12-07 | 2018-06-05 | Zymergen Inc. | Microbial strain improvement by a HTP genomic engineering platform |
| US11208649B2 (en) | 2015-12-07 | 2021-12-28 | Zymergen Inc. | HTP genomic engineering platform |
| KR101756338B1 (en) * | 2016-01-15 | 2017-07-10 | 고려대학교 산학협력단 | Variant Microorganism for Producing L-Cystein and Method for Preparing L-Cystein Using thereof |
| CN105838740B (en) * | 2016-05-03 | 2020-11-06 | 湖北大学 | Method for preparing nano red element selenium by using tea tree endophytic asplenium |
| JP2019519241A (en) | 2016-06-30 | 2019-07-11 | ザイマージェン インコーポレイテッド | Method for producing a glucose permease library and its use |
| WO2018005655A2 (en) | 2016-06-30 | 2018-01-04 | Zymergen Inc. | Methods for generating a bacterial hemoglobin library and uses thereof |
| CN109415417A (en) * | 2016-07-08 | 2019-03-01 | 赢创德固赛有限公司 | Methionine or the method for its hydroxy analogs form are produced by the microbial fermentation of the gene containing coding sugar phosphotransferase system (PTS) |
| CN106635944A (en) * | 2016-12-29 | 2017-05-10 | 廊坊梅花生物技术开发有限公司 | Glutamic acid corynebacterium and construction method and application thereof |
| US20200239897A1 (en) | 2017-06-07 | 2020-07-30 | Zymergen Inc. | Promoters from corynebacterium glutamicum and uses thereof in regulating ancillary gene expression |
| EP3415622A1 (en) | 2017-06-14 | 2018-12-19 | Evonik Degussa GmbH | Method for production of fine chemicals using a corynebacterium secreting modified alpha-1,6-glucosidases |
| KR102011994B1 (en) | 2017-06-30 | 2019-08-20 | 씨제이제일제당 주식회사 | Novel aspartokinase variant and method of producing L-amino acid using thereof |
| CN109536428B (en) * | 2018-12-07 | 2022-08-30 | 武汉远大弘元股份有限公司 | Genetically engineered bacterium for producing L-isoleucine and construction method and application thereof |
| CN113994002B (en) * | 2021-05-07 | 2022-06-03 | Cj第一制糖株式会社 | Novel promoter and use thereof |
| CN114181288B (en) * | 2022-02-17 | 2022-05-03 | 北京中科伊品生物科技有限公司 | Process for producing L-valine, gene used therefor and protein encoded by the gene |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19931317A1 (en) * | 1999-07-07 | 2001-01-11 | Degussa | L-lysine-producing coryneform bacteria and method for producing L-lysine |
| AU3837400A (en) * | 1999-04-19 | 2000-11-02 | Kyowa Hakko Kogyo Co. Ltd. | Novel desensitized aspartokinase |
| DE19950409A1 (en) * | 1999-10-20 | 2001-04-26 | Degussa | New nucleotide sequences coding for the pck gene |
| JP4623825B2 (en) * | 1999-12-16 | 2011-02-02 | 協和発酵バイオ株式会社 | Novel polynucleotide |
-
2002
- 2002-07-30 CN CNB028154460A patent/CN100554426C/en not_active Expired - Fee Related
- 2002-07-30 BR BR0211723-1A patent/BR0211723A/en not_active Application Discontinuation
- 2002-07-30 EP EP02760293A patent/EP1414970A2/en not_active Withdrawn
- 2002-07-30 CA CA002455878A patent/CA2455878A1/en not_active Abandoned
- 2002-07-30 AU AU2002325923A patent/AU2002325923A1/en not_active Abandoned
- 2002-07-30 WO PCT/EP2002/008464 patent/WO2003040373A2/en not_active Application Discontinuation
Non-Patent Citations (2)
| Title |
|---|
| Structure of the gluABCD cluster encoding the glutamateuptake system of Corynebacterium glutamicum.. Kronemeyer.W等.J.Bacteriology,Vol.177 No.5. 1995 |
| Structure of the gluABCD cluster encoding the glutamateuptake system of Corynebacterium glutamicum.. Kronemeyer.W等.J.Bacteriology,Vol.177 No.5. 1995 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111471638A (en) * | 2020-05-22 | 2020-07-31 | 江南大学 | Construction and application of corynebacterium glutamicum mutant strain capable of producing L-homoserine |
| CN111471638B (en) * | 2020-05-22 | 2021-11-23 | 江南大学 | Construction and application of corynebacterium glutamicum mutant strain capable of producing L-homoserine |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2003040373A2 (en) | 2003-05-15 |
| CA2455878A1 (en) | 2003-05-15 |
| WO2003040373A3 (en) | 2003-12-18 |
| EP1414970A2 (en) | 2004-05-06 |
| BR0211723A (en) | 2004-09-21 |
| CN1539015A (en) | 2004-10-20 |
| AU2002325923A1 (en) | 2003-05-19 |
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