CN101597588A - Produce the amino acid whose method of L-by the Escherichia bacterium - Google Patents
Produce the amino acid whose method of L-by the Escherichia bacterium Download PDFInfo
- Publication number
- CN101597588A CN101597588A CNA2009101594487A CN200910159448A CN101597588A CN 101597588 A CN101597588 A CN 101597588A CN A2009101594487 A CNA2009101594487 A CN A2009101594487A CN 200910159448 A CN200910159448 A CN 200910159448A CN 101597588 A CN101597588 A CN 101597588A
- Authority
- CN
- China
- Prior art keywords
- bacterium
- leu
- amino acid
- ala
- ile
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention provides a kind of use Escherichia bacterium and produce L-Threonine, L-Xie Ansuan, L-proline(Pro), L-leucine, the arginic production method of L-methionine(Met) and L-.The L-amino acid throughput of described bacterium is by raising b2682 and b2683 gene, or the b1242 gene, or the activity of proteins of b3434 genes encoding is enhanced.
Description
The application is for dividing an application, and the application number of original application is 02108086.0, and the applying date is on February 13rd, 2002, and denomination of invention is " producing the amino acid whose method of L-by the Escherichia bacterium ".
Technical field
The present invention relates to biotechnology, relate to by the method for fermentation producing L-amino-acid particularly and relate to more specifically available from colibacillary gene.These genes are useful for improving L-amino acid throughput, for example: L-Threonine, L-Xie Ansuan, L-proline(Pro), L-leucine, L-methionine(Met) and L-arginine.
Background technology
Traditional L-amino acid industrial process is a fermentation process, improves L-amino acid throughput by the mutant strain that is used to come from microorganism strains in the physical environment or specific modification.
The technology of a lot of raising L-amino acid throughput is disclosed, for example: transform microorganism (referring to for example US4,278,765) by recombinant DNA.The basis of these technology be improve the enzymic activity in amino acid bio is synthetic and/or make by the desensitization of the enzyme of L-amino acid product feedback inhibition (referring to for example, Japanese patent application No56-18596 (1981), WO95/16042 or U.S. Patent No. 5,661,012 and 6,040,160).
On the other hand, the throughput of the amino acid of raising secretion can enhancing L-amino acid preparation strain has the Escherichia bacterial isolates that strengthens expression L-Methionin secretory gene (lysE gene) and is disclosed (WO 9723597A2).In addition, coding is suitable for the encoding gene also open (U.S. Patent No. 5,972,663) of the secretory protein of L-halfcystine, L-Gelucystine, N-acetylserine or thiazolidine derivative.
Now, some codings bacillus coli gene that can improve the membranin of inferring of L-amino acid production is disclosed.It is preferred to the resistance of L-homoserine and the production (European patent application EP 994190A2) that improves L-homoserine, L-Threonine, L-L-Ala, L-Xie Ansuan and L-Isoleucine that the extra copy of rhtB gene has bacterium.In addition, the extra copy of rhtC gene makes bacterium have preferably the resistance of L-homoserine and L-Threonine and improves L-homoserine, L-Threonine and the leucic production of L-(European patent application EP 1013765A1).In addition, yahN, yeaS, the extra copy of yfiK and yggA gene improves the production (European patent application EP 1016710A2) of L-L-glutamic acid, L-Methionin, L-Threonine and L-L-Ala, L-Histidine, L-proline(Pro), L-arginine, L-Xie Ansuan and L-Isoleucine.Although the full-length gene group sequence of e. coli k-12 bacterial strain is described (Blattner F.R., Plunkett G., Bloch C.A.et al., Science, 227,1453-1474,1997; Ftp.genetics.wisc.edu/pub/sequence/ecolim52.seq.gz), but a lot of ORFs wherein, their function it be unclear that.
Summary of the invention
The objective of the invention is to improve the throughput of L-amino acid preparation strain and a kind of production L-is provided amino acid whose method, for example produce L-Threonine, L-by bacterial strain
Xie Ansuan, L-proline(Pro), L-leucine or L-methionine(Met) or L-arginine.
This purpose reaches by the proteinic gene of recognition coding, and these genes are not participated in the amino acid whose biosynthetic pathway of target L-but can be improved its output.An example is that this protein is the secreting active membranin of a kind of L-of having amino acid.On the basis of intestinal bacteria full-length gene group sequential analysis, have 4 or the protein of more a plurality of transmembrane segment of inferring (TMS) selected.Make great efforts results of screening, the inventor has therefrom discerned several genes, and they are: b2682, and b2683, b1242 and b3434, and carried out comprehensive research.Gene b2682 and b2683 have been considered to the CDS that inferred, the protein (being respectively the few nucleotide 92 to 829 and 819 to 1154 of GENE BANK registration number AE000353U00096 sequence) that it can encoding function the unknown.Gene b2683 also thinks ygaH.Gene b1242 has been considered to the CDS that infers, the protein (few nucleotide 8432 to 9079 of GENE BANK registration number AE000222U00096 sequence) that it can encoding function the unknown.Gene b1242 also thinks ychE.Gene b3434 has been considered to the CDS that infers, the protein (few nucleotide 1463 to 2056 of GENEBANK registration number AE000420 U00096 sequence) that it can encoding function the unknown.Gene b3434 also thinks yhgN.
The inventor also finds by raising b2682, b2683, and the activity of proteins of b1242 or b3434 genes encoding, the L-amino acid throughput of bacterial strain also is enhanced, and therefore, the present invention finishes.
The present invention is as follows:
1) the amino acid producing bacteria of a kind of L-, this bacterium belongs to Escherichia (Escherichia), this bacterium through modification so that the L-amino acid production of this bacterium can be by improving following A) or B), and C) or the activity of protein in bacterial cell that D) defines improve:
B) a kind ofly comprise the protein of aminoacid sequence shown in the SEQ ID NO:3 in the sequence table;
C) a kind ofly be included in the sequence table disappearance, displacement on the aminoacid sequence shown in the SEQ ID NO:3, insert, add one or more amino acid and the protein of the aminoacid sequence that forms, it has makes bacterium increase activity to the resistance of L-amino acid and/or its analogue;
D) a kind ofly comprise the protein of aminoacid sequence shown in the SEQ ID NO:5 in the sequence table;
E) a kind ofly be included in the sequence table disappearance, displacement on the aminoacid sequence shown in the SEQ ID NO:5, insert, increase one or more amino acid and the protein of the aminoacid sequence that forms, it has makes bacterium increase activity to the resistance of L-amino acid and/or its analogue;
(hereinafter) as above-mentioned A or B) and C) or D) protein of definition be called as " first embodiment of the invention protein ", the Escherichia bacterium that can improve above-mentioned protein active is called as " bacterium of first embodiment of the invention ")
2) according to above-mentioned bacterium, A) or B) and C) or D) activity of proteins of definition can be by the A that will encode) or B) and C) or the proteinic gene transformation that D) defines advance bacterium, or improve by the expression regulation sequence on the change bacterial chromosomal dna.
3) according to above-mentioned bacterium, conversion wherein is to use multi-copy vector to carry out.
4) the amino acid whose method of a kind of production L-is included in and cultivates above-mentioned bacterium in the substratum and collect the L-amino acid that produces and accumulate from substratum.
5) according to aforesaid method, described L-amino acid is the L-Threonine.
6) according to aforesaid method, to such an extent as to described bacterium is through modifying the expression that this bacterium can be improved threonine operon.
7) according to aforesaid method, described L-amino acid is L-
Xie Ansuan
8) according to aforesaid method, to such an extent as to described bacterium is through modifying the expression that this bacterium can be improved the ilv operon.
9) according to aforesaid method, described L-amino acid is the L-proline(Pro).
10) according to aforesaid method, to such an extent as to described bacterium is through modifying the expression that this bacterium can be improved the proline(Pro) biosynthesis gene.
11) according to aforesaid method, described L-amino acid is the L-leucine.
12) according to aforesaid method, to such an extent as to described bacterium is through modifying the expression that this bacterium can be improved the leu operon.
13) according to aforesaid method, described L-amino acid is the L-methionine(Met).
14) according to aforesaid method, to such an extent as to described bacterium is through modifying the expression that this bacterium can be improved the met regulon.
15) the amino acid producing bacteria of a kind of L-, this bacterium belongs to Escherichia, this bacterium through modification so that the L-amino acid production of this bacterium can be by improving following E) or the activity of protein in bacterial cell that F) defines improve:
F) a kind ofly comprise the protein of aminoacid sequence shown in the SEQ ID NO:11 in the sequence table;
G) a kind ofly be included in the sequence table disappearance, displacement on the aminoacid sequence shown in the SEQ ID NO:11, insert, increase one or more amino acid and the protein of the aminoacid sequence that forms, it has makes bacterium increase activity to the resistance of L-amino acid and/or its analogue;
(hereinafter, as above-mentioned E) or the protein that F) defines are referred to as " protein of second embodiment of the invention " sometimes, and the Escherichia bacterium that can improve above-mentioned protein active is meant " bacterium of second embodiment of the invention ")
16) according to above-mentioned bacterium, E) or F) activity of proteins of definition can be by the E that will encode) or the proteinic gene transformation that F) defines advance bacterium, or improve by the expression regulation sequence on the change bacterial chromosomal dna.
17) according to above-mentioned bacterium, conversion is to use multi-copy vector to carry out.
18) the amino acid whose method of a kind of production L-is included in and cultivates above-mentioned bacterium in the substratum and collect the L-amino acid that produces and accumulate from substratum.
19) according to aforesaid method, described L-amino acid is the L-Threonine.
20) according to aforesaid method, to such an extent as to described bacterium is through modifying the expression that this bacterium can be improved threonine operon.
21) according to aforesaid method, described L-amino acid is the L-Xie Ansuan.
22) according to aforesaid method, to such an extent as to described bacterium is through modifying the expression that this bacterium can be improved the ilv operon.
23) the amino acid producing bacteria of a kind of L-, this bacterium belongs to Escherichia, this bacterium through modification so that the L-amino acid production of this bacterium can be by improving following G) or the activity of protein in bacterial cell that H) defines improve:
H) a kind ofly comprise the protein of aminoacid sequence shown in the SEQ ID NO:15 in the sequence table;
I) a kind ofly be included in the sequence table disappearance, displacement on the aminoacid sequence shown in the SEQ ID NO:15, insert, increase one or more amino acid and the protein of the aminoacid sequence that forms, it has makes the activity of bacterium increase to the resistance of L-amino acid and/or its analogue, for example; DL-O-methyl Serine, 6-diazo-5-oxygen-L-nor-leucine and DL-beta-hydroxy-norvaline increases S-(2-aminoethyl) halfcystine susceptibility;
(hereinafter) as above-mentioned G or H) definition protein be referred to as " protein of third embodiment of the invention " sometimes, can improve E) or F) the Escherichia bacterium of protein active be meant " bacterium of third embodiment of the invention ")
24) according to above-mentioned bacterium, G) or H) activity of proteins of definition can advance bacterium by the proteinic gene transformation that will encode (G) or (H) define, or improve by the expression regulation sequence on the change bacterial chromosomal dna.
25) according to above-mentioned bacterium, conversion wherein is to use multi-copy vector to carry out.
26) the amino acid whose method of a kind of production L-is included in and cultivates above-mentioned bacterium in the substratum and collect the L-amino acid that produces and accumulate from substratum.
27) according to aforesaid method, described L-amino acid is the L-arginine.
28) according to aforesaid method, to such an extent as to described bacterium can strengthen the expression of arginine regulon through this bacterium of modification.
29) according to aforesaid method, described L-amino acid is the L-proline(Pro).
30) according to aforesaid method, to such an extent as to described bacterium can strengthen the expression of proline(Pro) biosynthesis gene through this bacterium of modification.
Produce the amino acid whose method of L-and comprise that the L-Threonine that uses the protein active that the present invention who contains aminoacid sequence shown in SEQ ID NO:3 and the SEQ ID NO:5 improved produces bacterium and produce the L-Threonine.The amino acid whose method of a kind of L-of production comprises that the L-Xie Ansuan production bacterium of the protein active that uses the present invention's raising that contains aminoacid sequence shown in SEQ ID NO:3 and the SEQ ID NO:5 produces the L-Xie Ansuan.In addition, produce the amino acid whose method of L-and comprise that the L-proline(Pro) production bacterium of the protein active that uses the present invention's raising that contains aminoacid sequence shown in SEQ ID NO:3 and the SEQ ID NO:5 produces the L-proline(Pro).In addition, produce the amino acid whose method of L-and comprise that the L-leucine production bacterium of the protein active that uses the present invention's raising that contains aminoacid sequence shown in SEQ ID NO:3 and the SEQ ID NO:5 produces the L-leucine.Produce the amino acid whose method of L-and comprise that the L-methionine(Met) production bacterium of the protein active that uses the present invention's raising that contains aminoacid sequence shown in SEQ ID NO:3 and the SEQ ID NO:5 produces the L-methionine(Met).
Further, produce the amino acid whose method of L-and comprise that the L-Threonine production bacterium of the protein active that uses the present invention's raising that contains aminoacid sequence shown in the SEQ ID NO:11 produces the L-Threonine.The amino acid whose method of a kind of L-of production comprises that the L-Xie Ansuan production bacterium of the protein active that uses the present invention's raising that contains aminoacid sequence shown in the SEQ ID NO:11 produces the L-Xie Ansuan.
Further, produce the amino acid whose method of L-and comprise that the L-arginine production bacterium of the protein active that uses the present invention's raising that contains aminoacid sequence shown in the SEQ ID NO:15 produces the L-arginine.In addition, produce the amino acid whose method of L-and comprise that the L-proline(Pro) production bacterium of the protein active that uses the present invention's raising that contains aminoacid sequence shown in the SEQ ID NO:15 produces the L-proline(Pro).
To explain the present invention in detail below
Bacterium of the present invention is the amino acid producing bacteria of a kind of L-, and this bacterium belongs to Escherichia, and this bacterium is through modifying so that the L-amino acid production of this bacterium can improve by improving the activity of protein of the present invention in bacterial cell.
Come among the present invention " the amino acid producing bacteria of L-" to be meant and have the ability to accumulate the amino acid whose bacterium of L-when bacterium is cultivated on substratum in substratum, the characteristic that the wild strain that this L-amino acid throughput can be bacterium has also can obtain or improves by cultivation.
Bacterium of the present invention is the amino acid producing bacteria of a kind of L-, this bacterium belongs to Escherichia, this bacterium has the protein active of raising, it can improve the amino acid whose throughput of target L-, concrete, bacterium of the present invention is the amino acid producing bacteria of a kind of L-, and this bacterium belongs to Escherichia, and this bacterium has at least a or two protein active of raising.
Term " protein active of raising " is meant that each cell activity will be higher than not modified bacterial strain, for example, and a kind of wild-type Escherichia bacterium.For example, with the example that the protein molecule number of mentioning each cell increases, example that each protein molecule given activity increases or the like.Further, a kind of wild-type Escherichia bacterium is used as contrast, and for example, the wild-type e. coli bacterial strain is with referred.
Concrete, the bacterium of first embodiment of the invention is loaded with the DNA that has b2682 and preferred the two overexpression of two genes of b2683 on chromosomal DNA of bacterium or plasmid at least, and have enhanced and produce the amino acid whose ability of L-, for example, L-Threonine, L-Xie Ansuan, L-proline(Pro), L-leucine and L-methionine(Met).The bacterium of second embodiment of the present invention is loaded with DNA on the chromosomal DNA of bacterium or plasmid, the b1242 gene overexpression is arranged on this DNA, and improves the amino acid whose ability of L-of producing, for example, and L-Threonine and/or L-Xie Ansuan.The bacterium of the 3rd embodiment of the present invention is docked in the DNA of overexpression b3434 gene on the chromosomal DNA of bacterium or the plasmid, and has enhanced and produce the amino acid whose ability of L-, for example, and L-arginine and/or L-proline(Pro).
The protein of first embodiment of the invention comprises following A) or B) and C) or D) definition protein in any:
J) a kind ofly comprise the protein of aminoacid sequence shown in the SEQ ID NO:3 in the sequence table;
K) a kind ofly be included in the sequence table disappearance, displacement on the aminoacid sequence shown in the SEQ ID NO:3, insert, increase one or more amino acid and the protein of the aminoacid sequence that forms, it has makes bacterium increase activity to the resistance of L-amino acid and/or its analogue;
L) a kind ofly comprise the protein of aminoacid sequence shown in the SEQ ID NO:5 in the sequence table;
M) a kind ofly be included in the sequence table disappearance, displacement on the aminoacid sequence shown in the SEQ ID NO:5, insert, increase one or more amino acid and the protein of the aminoacid sequence that forms, it has makes bacterium increase activity to the resistance of L-amino acid and/or its analogue;
" several " amino acid whose number is according to the type of amino-acid residue in site or the tertiary protein structure and difference.Respectively, be can be 2-24 to protein (A), preferably 2-12 is more preferably 2-5, is can 2-11, preferably 2-7, more preferably 2-5 to protein (C).
The protein of second embodiment of the invention comprises following E) or F) definition protein in any:
N) a kind ofly comprise the protein of aminoacid sequence shown in the SEQ ID NO:11 in the sequence table;
O) a kind ofly be included in the sequence table disappearance, displacement on the aminoacid sequence shown in the SEQ ID NO:11, insert, increase one or more amino acid and the protein of the aminoacid sequence that forms, it has makes bacterium increase activity to the resistance of L-amino acid and/or its analogue;
" several " amino acid whose number is according to the type of amino-acid residue in site or the tertiary protein structure and difference.To protein (E) is can be 2-22, and preferably 2-11 is more preferably 2-5.
The protein of third embodiment of the invention comprises following G) or H) definition protein in any:
P) a kind ofly comprise the protein of aminoacid sequence shown in the SEQ ID NO:15 in the sequence table;
Q) a kind ofly be included in the sequence table disappearance, displacement on the aminoacid sequence shown in the SEQ ID NO:15, insert, increase one or more amino acid and the protein of the aminoacid sequence that forms, it has makes the activity of bacterium increase to the resistance of L-amino acid and/or its analogue, for example; DL-o-methyl Serine, 6-diazo-5-oxygen-L-nor-leucine and DL--b-hydroxyl-remove the first Xie Ansuan, S-(2-aminoethyl) halfcystine susceptibility is increased;
" several " amino acid whose number is according to the type of amino-acid residue in site or the tertiary protein structure and difference.To protein (G) is can be 2-20, and preferably 2-10 is more preferably 2-5.
The resistance to L-amino acid and/or its analogue that increases means the bacterial strain that has at unmodified, or wild strain, or the parent strain of this bacterium can not grow, and contains the ability of growing on the substratum of L-amino acid and/or its analogue minimum concentration.Perhaps mean the bacterial strain that has than unmodified, or wild strain, or the parent strain of this bacterium the ability faster of on the substratum that contains L-amino acid and/or its analogue, growing.
More specifically, coli strain is cultivated on 37 ℃ of L-amino acid that contain conditions suitable and/or its analogue nutrient agar, if coli strain has formed 37 ℃ of ratios, coli strain was cultivated unmodified bacterial strain or the big bacterium colony of wild type strain that the back forms through 2-4 days on solid Adams substratum, we can say that then coli strain has increased the resistance to L-amino acid and/or its analogue.Term " appropriate condition " is meant temperature, pH, air feed or basic nutrition or other selection.
The L-amino acid analogue is by 3,4-dihydro proline(Pro), DL-sulfo-Isoleucine, DL-o-methyl Serine, 4-azepine leucine, nor-leucine, L-o-fluorobenzene L-Ala and DL-o-fluorobenzene L-Ala, homoserine, 6-diazo-5-oxygen-L-nor-leucine and DL-beta-hydroxy-norvaline come illustration.
Be mentioned to above under the condition of L-amino acid or their analogue existence, concentration when the bacterial strain of unmodified or wild type strain can not be grown, changing according to the structure difference of the compound that uses is noticeable (9600 μ g/ml from 0.5 μ g/ml of DL-sulfo-Isoleucine to DL-o-methyl Serine).For example, suppose it is 3,4-dihydro proline(Pro), general concentration is 7-70 μ g/ml, preferably 20-25 μ g/ml; Suppose it is DL-sulfo-Isoleucine, general concentration is 0.5-5 μ g/ml, preferably 0.9-1.1 μ g/ml; Suppose it is DL-o-methyl Serine, general concentration is 1100-9600 μ g/ml, preferably 3000-3500 μ g/ml; Suppose it is 4-azepine leucine, general concentration is 15-150 μ g/ml, preferably 40-50 μ g/ml; Suppose it is nor-leucine, general concentration is 150-1500 μ g/ml, preferably 450-550 μ g/ml; Suppose it is L-o-fluorobenzene L-Ala, general concentration is 0.6-6 μ g/ml, preferably 1.5-2 μ g/ml; Suppose it is DL-o-fluorobenzene L-Ala, general concentration is 2-20 μ g/ml, preferably 5-7 μ g/ml; Suppose it is homoserine, general concentration is 330-3300 μ g/ml, preferably 900-1100 μ g/ml; Suppose it is the 6-diazo-5-oxygen-L-nor-leucine, general concentration is 5-50 μ g/ml, preferably 12-18 μ g/ml; Suppose it is DL-beta-hydroxy-norvaline, general concentration is 25-250 μ g/ml, preferably 70-90 μ g/ml.
L-amino acid or their analogue sensitivity are meaned that bacterium has on the substratum of L-amino acid that contains minimum concentration or their analogue, have the ability of longer propagation phase without modifying bacterial strain or wild strain than it.Or, to L-amino acid or their analogue sensitivity mean bacterium it bacterial strain or wild strain can grow without modifying, contain on the substratum of the L-amino acid of minimum concentration or their analogue to grow.This L-amino acid analogue comes illustration by S-(2-aminoethyl) halfcystine.Above mentioned concentration hypothesis is for S-(2-aminoethyl) halfcystine, and general concentration is 0.2-2.0 μ g/ml, preferably 0.5-1.0 μ g/ml.
Bacterium of the present invention also comprises by the A that will encode) or B), and C) or D), or E) or F), or G) or H) the proteic DNA bacterial strain that changes bacterium over to or by changing the expression regulation sequence on the bacterial chromosomal dna activity of proteins of the present invention is improved.The membrane protein that the described dna encoding of in the present invention bacterium being modified is inferred.Further, this dna encoding has 4 or more a plurality of protein of striding the film sections.Such DNA can encode and have the secreting active protein of L-amino acid.Further, this DNA is by b2683, b2683, b1242 and b3434 gene representation.It must be noted that the coding region 728-738 of b2682 gene and the coding region 1-11 of b2683 gene are partly overlapping.These two genes can obtain by for example using SEQ ID NO:1 and the disclosed Nucleotide of SEQ ID NO:2 to carry out PCR as primer, are a PCR product.Gene b1242 can obtain by for example using SEQ ID NO:9 and the disclosed Nucleotide of SEQ ID NO:10 to carry out PCR as primer.Gene b3434 can obtain by for example using SEQ ID NO:13 and the disclosed Nucleotide of SEQ ID NO:14 to carry out PCR as primer.
Analysis can filter out to encode and have 4 or the proteinic gene of more a plurality of TMS that infer to the intestinal bacteria whole genome sequence.The protein of known function and translocator are at Paulsen I.T., Sliwinski M.I., (J.Mol.Biol., 1998,227 are described in the article of Saier M.H., 537) Linton K.J and HigginsC.F. (Molecular Microbiology, 1998,28 (1), 5) also it has been carried out exclude filter, in the result of remaining genescreen, several encoding is inferred film and is exported proteic gene and closed.Find b2682 and b2682 gene overexpression, or b1242 or b3434 gene overexpression have improved L-amino acid and produce the L-amino acid of bacterium and generate.
DNA of the present invention comprise be coded in a-protein) or sequence C) on one or more locational disappearance, displacement, insertion, the one or more amino acid of increase and the protein of the aminoacid sequence that forms, as long as they do not lose activity of proteins." several " amino acid whose number is according to the type of amino-acid residue in site or the tertiary protein structure and difference.Respectively, be can be 2-24 to protein (A), preferably 2-12 is more preferably 2-5, is can 2-11 to protein (C), and preferably 2-7 is more preferably 2-5.
Further, DNA of the present invention comprise be coded in a-protein) sequence on one or more locational disappearance, displacement, insertion, the one or more amino acid of increase and the protein of the aminoacid sequence that forms, as long as they do not lose activity of proteins." several " amino acid whose number is according to the type of amino-acid residue in site or the tertiary protein structure and difference.To protein (E) is can be 2-22, and preferably 2-11 is more preferably 2-5.Further, DNA of the present invention comprise be coded in protein E) sequence on one or more locational disappearance, displacement, insertion, the one or more amino acid of increase and the protein of the aminoacid sequence that forms, as long as they do not lose activity of proteins." several " amino acid whose number is according to the type of amino-acid residue in site or the tertiary protein structure and difference.To protein (G) is can be 2-20, and preferably 2-10 is more preferably 2-5.
Coding and A), C), E) or G) protein DNA that the protein of definition is substantially identical can be by for example to coding A), C), E) or G) the protein core nucleotide sequence of definition carries out site-directed mutagenesis and obtains, thus one or more amino-acid residue can be by disappearance, displacement, insert, increase.The DNA of this modification can generate the method acquisition of sudden change by conventional agent treated and condition.This processing comprises uses azanol to handle code book invention protein DNA or with ultraviolet radiation or for example N-methyl-N-nitro-N-nitrosoguanidine or nitrous acid are handled the DNA that bacterium contains.
DNA of the present invention is included in the different strains and isolating mutant in the natural various bacterial strain of Escherichia bacterium.The DNA of these mutant of encoding can obtain by DNA isolation, they can with gene b2862, b2863, b1242 or b3434 hybridization, or hybridize in rigorous condition lower section, and their encoded protein matter can improve the amino acid whose production of L-, and term " rigorous condition " is meant in the time of can forming so-called special hybridization, but not the condition of special hybridization when not forming.For example, rigorous condition comprises the hybridization conditions when DNA has high homology, when for example DNAs has 70% homology each other.Perhaps, rigorous condition can be come illustration by the usual terms when constituting Southern hybridization and clean, for example, 60 ℃, 1 * SSC, 0.1%SDS, 0.1 * SSC preferably, 0.1%SDS.Use the encoding mutant body, and can with gene b2862, b2863, the DNA of b1242 or b3434 hybridization is as probe, the DNA that contains the nucleotide sequence of part SEQ ID NO:3 or SEQ ID NO:5 also can be used as probe.Such probe can be by using based on SEQ ID NO:3,5,11,15 oligonucleotides of producing as primer, and the dna fragmentation that use contains SEQ ID NO:3,5,11,15 Nucleotide carries out PCR as template and prepares.When the dna fragmentation that is about 300bp when length was used as probe, the washing condition of hybridization was, for example, 50 ℃, 2 * SSC and 0.1%SDS.
Mean with the DNA transform bacteria of coded protein and to import DNA into bacterial cell, for example improve protein expression of the present invention and improve this activity of proteins in the bacterial cell by traditional method.
The technology that improves genetic expression comprises the increase gene copy number.In the Escherichia bacterium, import a carrier that contains gene into and can play the function that increases this gene copy number.For this purpose, preferably use multi-copy vector.Multi-copy vector is by pBr322, and pMW119, pUC19, pET22b or other similar carrier come illustration.
In addition, also can come on bacterial chromosome, to introduce the multiple copied of gene, and reach the purpose that improves genetic expression by for example homologous recombination or similarity method.
In order to have improved two or more expression of gene, gene can be positioned on the same plasmid together, also can be positioned on the different plasmids.A gene is positioned on the karyomit(e), and another is positioned on the plasmid and also is fine.
On the other hand, can improve genetic expression by changing the expression of gene regulating and controlling sequence.Change expression of gene regulating and controlling sequence is included in the original expression regulation sequence of gene and for example introduces sudden change in the promotor, and makes genetic expression improve (WO00/18935), also DNA of the present invention can be placed under the control of strong promoter.Lac promotor for example, the trp promotor, the trc promotor is considered to the P of strong promoter in the lambda particles phage
LPromotor, strong promoter can be united use with multi-copy gene.
Bacterium of the present invention can the above-mentioned DNA of introducing obtains in the amino acid whose bacterium of L-by producing to itself.Also can obtain described bacterium by giving this generation of the bacterium that contains DNA L-ability of amino acid.Be enhanced the parent strain of protein active of the present invention, the L-Threonine produces bacterial strain and belongs to Escherichia, for example, bacterial strain VL2054 (VKPM B-8067), VNIIGenetika 472T23 (United States Patent (USP) NO5,631,157), VKPM B-3996 (United States Patent (USP) NO5,175,107 and NO 5,976,843), KCCM-10132 (WO009660A1), KCCM-10133 (WO009661A1) or similar bacterial strain can use.Be enhanced the parent strain of protein active of the present invention, the L-Xie Ansuan produces bacterial strain and belongs to Escherichia, for example, bacterial strain H-81 (VKPMB-8066), NRRL B-12287 and NRRL B-12288 (United States Patent (USP) NO4,391,907), VKPMB-4411 (United States Patent (USP) NO5,658,766), VKPMB-7707 (European patent application EP 1016710A2) or similar bacterial strain can use.In addition, be enhanced the parent strain of protein active of the present invention, the L-proline(Pro) produces bacterial strain and belongs to Escherichia, for example, bacterial strain NRRL B-12403 and NRRLB-12404 (GB2075056), VKPM B-8012 (Russ P application NO2000124295), the mutant plasmid of describing among the patent DE3127361, (The 15 for the mutant plasmid that Bloom F.R.et al describes
ThMiami winter symposium, 1983, p.34) or similarly bacterial strain can use.Be enhanced the parent strain of protein active of the present invention, the L-leucine produces bacterial strain and belongs to Escherichia, for example, bacterial strain H-9070 (FERMBP-4704) and H-9072 (FERMBP-4706) are (US5744331), VKPMB-7368 and VKPM B-7388 (RU2140450), W1485atpA401/pMWdAR6, W1485lip2/pMWdAR6 and AJ12631/pMWdAR6 (EP0872547) or similar bacterial strain can use.Be enhanced the parent strain of protein active of the present invention, the L-methionine(Met) produces bacterial strain and belongs to Escherichia, for example, strains A J11539 (NRRLB-12399), AJ11540 (NRRLB-12400), AJ11541 (NRRLB-12401) AJ11542 (NRRLB-12402) (GB2075055), or similarly bacterial strain can use.
Further, be enhanced the parent strain of protein active of the present invention, the strain of L-arginine producing strain belongs to Escherichia, for example, strains A J11531 and AJ11538 (JP56106598A2), AJ11593 (FERMP-5616) and AJ11594 (FERMP-5617) (the open NO575693 of Japanese Patent) or similar bacterial strain can use.
Bacterium of the present invention can further improve be included in the L-amino acid bio synthetic in one or more expression of gene.Produce bacterial strain for the L-Threonine, this gene comes illustration by threonine operon, and it preferably comprises a coding by the aspartokinase of L-Threonine feedback inhibition-homoserine dehydrogenase gene (the open NO.1-29559 of Japanese Patent).Produce bacterial strain for the L-Xie Ansuan, this gene is by the ilv operon, and not fine expression Threonine deaminizating hydrogen enzyme, the repressed ilvGMEDA operon of its attenuation come (the open NO.8-47397 of Japanese Patent) of illustration.Produce bacterial strain for the L-proline(Pro), this gene comes illustration by L-proline(Pro) biosynthesis gene, and it is preferably used by the Glutamate kinase gene proB of L-proline(Pro) feedback inhibition and represents (DE3127361).Produce bacterial strain for the L-leucine, this gene is by leucine operon, and just the leu operon comes illustration, and it preferably comprises a coding by the isopropylmalate synthetase gene (Russ P discloses 99114325) of L-leucine feedback inhibition.Produce bacterial strain for the L-methionine(Met), this gene comes illustration by the methionine(Met) regulon.The methionine(Met) regulon contains coding and suppresses the active proteinic mutator gene that lowers of amino acid bio synthetic.This gene is the relevant arrestin of L-methionine(Met) biosynthesizing that obtains from intestinal bacteria by coding, comes (JP2000-157267A2) of illustration at the anomaly gene metJ that suppresses to lower on the biosynthetic activity of methionine(Met).Further; this gene is an arginine regulon illustration; its preferably comprise the N-ethanoyl NADPH-linked glutamate synthase gene that its L-arginine feedback inhibition desensitized (Apple.Environ.Microbiol.1998 such as Rajagopal B.S., U.64.No.5, P1805-1811).
The method of production L-Threonine of the present invention is included in the bacterium of cultivating first embodiment of the invention on the substratum, produces and accumulation L-Threonine the step of collecting the L-Threonine from substratum in substratum.The method of production L-Xie Ansuan of the present invention is included in cultivates bacterium of the present invention on the substratum, produce and accumulation L-Xie Ansuan the step of collecting the L-Xie Ansuan from substratum in substratum.In addition, the method for production L-proline(Pro) of the present invention is included in cultivates bacterium of the present invention on the substratum, produces and accumulation L-proline(Pro) the step of collecting the L-proline(Pro) from substratum in substratum.The leucic method of production L-of the present invention is included in cultivates bacterium of the present invention on the substratum, produce in substratum and accumulation L-leucine, collects the leucic step of L-from substratum.The method of production L-methionine(Met) of the present invention is included in cultivates bacterium of the present invention on the substratum, produce and accumulation L-methionine(Met) the step of collecting the L-methionine(Met) from substratum in substratum.
The method of production L-Threonine of the present invention is included in the bacterium of cultivating second embodiment of the invention on the substratum, produces and accumulation L-Threonine the step of collecting the L-Threonine from substratum in substratum.The method of production L-Xie Ansuan of the present invention is included in cultivates bacterium of the present invention on the substratum, produce and accumulation L-Xie Ansuan the step of collecting the L-Xie Ansuan from substratum in substratum.
The arginic method of production L-of the present invention is included in the bacterium of cultivating third embodiment of the invention on the substratum, produces in substratum and accumulation L-arginine, collects the arginic step of L-from substratum.The method of production L-proline(Pro) of the present invention is included in cultivates bacterium of the present invention on the substratum, produce and accumulation L-proline(Pro) the step of collecting the L-proline(Pro) from substratum in substratum.
Of the present inventionly in substratum and resemblance, cultivate, separation and purification L-amino acid, using microorganism to carry out amino acid when producing, can use with the traditional zymotic method in identical method carry out.The substratum that is used for cultivating can the time synthetic medium also can be natural medium, as long as substratum comprises carbon source, nitrogenous source and mineral substance, if necessary, can also comprise the nutritive substance of the suitable number of microorganism growth needs.Carbon source can comprise various carbohydrate, for example dextrose plus saccharose and various organic acid.The assimilation mode that depends on microorganism used therefor comprises that the alcohol of ethanol and glycerine also can use.As for nitrogenous source, various amine salt, for example ammonia and sulfuric acid amine comprise for example other nitrogen of amine, and natural nitrogenous source is peptone for example, and the digest of soybean hydrolyzate and microbial fermentation can use.
Cultivation is preferably in for example shaking culture, carries out under the aerobic conditions such as aerobic stir culture, and temperature is preferably in 30-38 ℃ at 20-40 ℃.PH value during cultivation is preferably in 6.5-7.2 generally at 5-9.The pH value can be used ammoniacal liquor, lime carbonate, and various acid, various alkali and damping fluid are regulated.Usually, cultivation caused purpose amino acid to accumulate in substratum in 1-5 days in the liquid medium within.
After cultivation, can the solid in the substratum be gone out by the method for centrifugal or membrane filtration, for example cell.Can concentrate and collection of crystalline method and purifying purpose amino acid by ion-exchange then.
Brief description of drawings
Fig. 1 has shown the structure of plasmid p Δ lacZ
Best mode for carrying out the invention
To explain the present invention more specifically by embodiment below, as undeclared, amino acid is the L-configuration in following example.
Embodiment 1: with b2682, and b2683, b1242 and b3434 gene clone are to p Δ lacZ
In order to clone b2682, b2683, b1242 and b3434 gene have used p Δ lacZ plasmid.Plasmid p Δ lacZ is derived from plasmid pET-22b (+) (Novagen, Madison.WI，USA)。Handle pET-22b (+) plasmid with enzyme BglII and Xba I, itself and plasmid pMB9-lac (Fuller F., Gene, 19,43-54,1982) are had P with identical restriction endonuclease processing
LacThe fragment of the PCR product of UV5 promotor connects.For amplification P
LacThe PCR primer that the UV5 promoter fragment can use SEQ ID NO:7 and SEQ ID NO:8 to describe.The fragment (DymakovaE.et al., Gene, 19,43-54,1982) of Sal I-BamH I by cloned plasmids pJEL250, last plasmid has increased the part-structure (237bp is not activated son) of lacZ gene.The collection of illustrative plates of the plasmid p Δ lacZ that obtains is represented in accompanying drawing 1.
It is the PCR fragment that clone intestinal bacteria b2682 and b2683 infer the initial substance of reading frame (b2682 and b2683 gene), this fragment is to obtain as template by the DNA that uses coli strain TG1, and two primers that amplified fragments is used are described in SEQ ID NO:1 and 2.PCR is reflected on the PE-2400, with 40sec.95 ℃, 40sec.47 ℃ 40sec.72 ℃, carries out under the 30 round-robin conditions.So, the linear dna fragmentation that has comprised b2682 and b2683 gene of 1158bp has just obtained.This fragment is handled and is inserted into Xba I and BamH I restriction endonuclease on the multi-copy vector plasmid p Δ lacZ that has handled with identical enzyme.
The segmental plasmid of PCR that contains that obtains is called as pYGAZH, and two genes of b2682 that carries and b2683 are in lactose promotor (P
LacUV5) under the control.
Similarly, it is the PCR fragment that clone intestinal bacteria b1242 infers the initial substance of reading frame (b1242 gene), this fragment is to obtain as template by the DNA that uses coli strain TG1, and two primers that amplified fragments is used are described in SEQ ID NO:9 and 10.The segmental plasmid of PCR that contains that obtains is called as pYCHE, and the b1242 gene that carries is in lactose promotor (P
LacUV5) under the control.It is the PCR fragment that clone intestinal bacteria b3434 infers the initial substance of reading frame (b3434 gene), this fragment is to obtain as template by the DNA that uses coli strain TG1, and two primers that amplified fragments is used are described in SEQ ID NO:13 and 14.The segmental plasmid of PCR that contains that obtains is called as pYHGN, and the b1242 gene that carries is in lactose promotor (P
LacUV5) under the control.
Embodiment 2: the b2862 of amplification, b2683 gene pairs coli strain TG1 is to the influence of amino acid and analogue resistance thereof.
Coli strain TG1 (pYGAZH) TG1 (pYCHE), TG1 (pYHGN) and contain the TG1 bacterial strain (control strain) that do not insert fragment plasmid overnight incubation on the LB substratum of additional 100 μ g/ml penbritins.Culture after the spending the night of all bacterial strains was cultivated 2 hours at 37 ℃ of aerobics with 25 times of the fresh LB substratum dilutions of additional 100 μ g/ml penbritins and IPTG0.5mM.The culture of logarithmic phase makes nearly 1000 cell inoculations on the Adams solid medium flat board of additional 100 μ g/ml penbritins and IPTG0.5mM and amino acid or its analogue with 0.9% NaCl solution dilution.37 ℃ cultivate 2-4 days after, record has between the TG1 bacterial strain of hybrid plasmid and the contrast TG1 bacterial strain different on the number that forms bacterium colony and bacterium colony size.Experimental result such as table 1.
Table 1:
No-does not have different with control strain
R-has more bacterium colony or bacterium colony bigger
S-compares the bacterium colony or the bacterium colony that have still less with control strain littler
Embodiment 3: produce Threonine with the bacterial strain that contains plasmid pYGAZH
Threonine is produced bacterial strain VL2054 and is in P by containing
LacThe b2682 under the control of UV5 promotor and the plasmid pYGAZH of b2683 gene change over to and obtain.The bacterial strain that obtains is named as VL2054 (pYGAZH).Bacterial strain VL2054 is the derivative of bacterial strain VKPM B-3996, on karyomit(e):
A)The target threonine operon is positioned at P
RUnder the control of promotor.
B)Wild-type rhtA gene
C)Gene (tdh gene) and Tn5 (tdh::Tn5, the Kan of reticent coding transhydrogenase on the karyomit(e)
S) upward reticent coding kalamycin resistance gene (kan)
D)Mutant ilvA
442
Bacterial strain VL2054 collects center (VKPM) preservation January 30 calendar year 2001 at the national industrial microorganism of Russia, and (Russia 113545, Moscow, 1 Dorozhny proezd, 1), preserving number is VKPMB-8067, and transfers international preservation according to budapest treaty by initial preservation in 2002.
With in contrast contain not that the bacterial strain VL2054 of the plasmid of insertion portion (p Δ lacZ) and bacterial strain VL2054 (pYGAZH) respectively get 5 bacterium colonies, in the test tube of 20-ml, be suspended in ((NH on the 2ml minimal medium
4)
2SO
4-11g/l; NaCl-0.4g/l; MgSO
4-0.4g/l; K
2HPO
3-1g/l; FeSO
4-10mg/l; MnSO
4-10mg/l; Thiamines-0.1mg/l; Yeast extract-0.5g/l; Sucrose-40g/l; Penbritin-300mg/l) if necessary, overnight incubation under 32 ℃ of ventilation conditions.The 0.2ml bacterium liquid of each culture is transferred in the test tube of three 20ml, adds the fresh fermention medium that 2ml adds or do not add IPTG, rotates shaking culture 48 or 72 hours at 32 ℃.
The fermentation culture based component:
(NH
4)
2SO
4-22g/l；
NaCl-0.8g/l；
MgSO
4-0.8g/l；
K
2HPO
3-2g/l；
FeSO
4-20mg/l；
MnSO
4-20mg/l；
Thiamines-0.2mg/l;
Yeast extract-1g/l;
CaCO
3-30g/l；
Sucrose-80g/l;
Penbritin-300mg/l, if necessary;
IPTG-0.5mM, if necessary;
After cultivation, record bacterium liquid by traditional method and determine the stable of plasmid at 540nm place absorbance value.Can be by the accumulation volume of Threonine in the tlc determination substratum.The liquid phase of tlc is as follows: Virahol-50ml, acetone-50ml, NH
4OH (30%)-12ml, water-8ml.The results are shown in Table 2.As what see, heterozygosis plasmid pYGAZH has improved the Threonine accumulation volume of Threonine generation bacterial strain VL2054.
Table 2:
Embodiment 4: produce Xie Ansuan with the bacterial strain that contains plasmid pYGAZH
Xie Ansuan is produced bacterial strain H-81 and is in P by containing
LacThe b2682 under the control of UV5 promotor and the plasmid pYGAZH of b2683 gene change over to and obtain.The bacterial strain that obtains is collected center (VKPM) preservation January 30 calendar year 2001 at the national industrial microorganism of Russia, and (Russia 113545, Moscow, 1 Dorozhny proezd, 1), preserving number is VKPM B-8066, and transfers international preservation according to budapest treaty by initial preservation in 2002.
With in contrast contain not that the bacterial strain H-81 of the plasmid of insertion portion (p Δ lacZ) and bacterial strain H-81 (pYGAZH) respectively get 5 bacterium colonies, in the test tube of 20-ml, be suspended in ((NH on the 2ml minimal medium
4)
2SO
4-18g/l; K
2HPO
4-1.8g/l; MgSO
4-1.2g/l; Thiamines-0.1mg/l; Yeast extract-0.5g/l; Sucrose-60g/l; Penbritin-300mg/l) if necessary, overnight incubation under ventilation condition.The 0.2ml bacterium liquid of each culture is transferred in the test tube of three 20ml, adds the fresh fermention medium that 2ml adds or do not add IPTG, rotates shaking culture 48 or 72 hours at 32 ℃.
The fermentation culture based component:
(NH
4)
2SO
4-18g/l；
K
2HPO
4-1.8g/l；
MgSO
4-1.2g/l；
CaCO
3-20g/l；
Thiamines-0.1mg/l;
Sucrose-60g/l;
Penbritin-300mg/l, if necessary;
IPTG-0.5mM, if necessary;
After cultivation, record bacterium liquid by traditional method and determine the stable of plasmid at 540nm place absorbance value.Can be by the accumulation volume of Xie Ansuan in the tlc determination substratum.The liquid phase of tlc is as follows: Virahol-80ml, ethyl acetate-80ml, NH
4OH (30%)-15ml, water-45ml.The results are shown in Table 3.As what see, heterozygosis plasmid pYGAZH has improved the Xie Ansuan accumulation volume of Xie Ansuan generation bacterial strain H-81.
Table 3:
Reference implementation 1: produce the L-proline(Pro) by ilvA defective type L-proline(Pro) bacterial strain
Wild-type e. coli K12 bacterial strain (VKPM B-7) cell, handled back 20 minutes through mutagenic compound N-methyl-N-nitro-N-nitrosoguanidines (0.1mg/ml) at 37 ℃, be coated on additional 1.25mg/ml peptone after the cleaning, 10mg/ml L-proline(Pro), 0.05mg/l 2,3, on the low agar M9 substratum of 5-triphenyltetrazolium chloride.At 37 ℃ of bacterium colonies that grow through the back great majority of cultivation in 3 days is red.Minority can not oxidation L-proline(Pro) bacterium colony be white.One of them bacterium colony is obtained (3,4-dihydro proline(Pro) and azetidine-2-carboxylate) are had the mutant of resistance with the parent, amino acid analogue is joined in the M9 nutrient agar by the concentration with 2mg/ml.
Some bacterial strains in the mutant that generates can be produced the L-proline(Pro).It is to be used in paraxin (Cm) resistant gene (Cm that the ilvA gene is inserted into that best L-proline(Pro) produces bacterial strain 702
r) the bacterial strain TG1 that interrupts goes up that the phage P1 of growth handles.The Cm resistance transductant that obtains, 702ilvA has become L-Isoleucine defective type, has higher L-proline(Pro) throughput (seeing Table 4) than the parent 702 of L-Isoleucine nutritional type.The fermentation culture based component comprises: 60g/l sucrose, 25g/l sulfuric acid amine, 2g/lK
2HPO
4, 1g/lMgSO
4, the 0.1mg/l thiamines, 50mg/L l-Isoleucine and 25g/l chalk (pH7.2), sucrose and chalk are sterilized separately.Put into the 2ml substratum in vitro, the tested microorganism of inoculation one ring was 37 ℃ of shaking culture 2 days.
Table 4:
| Bacterial strain | Phenotype | Proline(Pro) accumulation volume g/l |
| K12 | Wild-type | ＜0.1 |
| 702(VKPM B-8001) | L-proline(Pro) degraded defective type, the proline analogs resistance | 0.5 |
| 702ilvA(VKPM B-8012) | L-proline(Pro) degraded defective type, proline analogs resistance, L-Isoleucine auxotroph, Cm r | 8.0 |
(Russia 113545 respectively at collecting center (VKPM) preservation at the national industrial microorganism of Russia on June 25th, 2000 for bacterial strain 702 and bacterial strain 702ilvA, Moscow, 1 Dorozhny proezd, 1), preserving number is VKPMB-8011 and VKPMB-8012.
Embodiment 5: produce proline(Pro) with the bacterial strain that contains plasmid pYGAZH
Proline(Pro) is produced bacterial strain 702ilvA and is in P by containing
LacThe b2682 under the control of UV5 promotor and the plasmid pYGAZH of b2683 gene change over to and obtain.
With bacterial strain 702ilvA, in contrast contain not that the bacterial strain 702ilvA of the plasmid of insertion portion (p Δ lacZ) and bacterial strain 702ilvA (pYGAZH) respectively get 5 bacterium colonies, in the test tube of 20-ml, be suspended in ((NH on the 2ml minimal medium
4)
2SO
4-18g/l; K
2HPO
3-1.8g/l; MgSO
4-1.2g/l; Thiamines-0.1mg/l; Yeast extract-0.5g/l; Sucrose-60g/l; Isoleucine-50mg/l; Penbritin-300mg/l) if necessary, overnight incubation under 32 ℃ of ventilation conditions.The 0.2ml bacterium liquid of each culture is transferred in the test tube of three 20ml, adds the fresh fermention medium that 2ml adds or do not add IPTG, rotates shaking culture 40 hours at 32 ℃.
The fermentation culture based component:
(NH
4)
2SO
4-18g/l；
K
2HPO
4-1.8g/l；
MgSO
4-1.2g/l；
CaCO
3-20mg/l；
Thiamines-0.1mg/l;
Sucrose-60g/l;
Isoleucine-50mg/l;
Penbritin-300mg/l, if necessary;
IPTG-0.5mM, if necessary;
After cultivation, record bacterium liquid by traditional method and determine the stable of plasmid at 540nm place absorbance value.Can be by the accumulation volume of proline(Pro) in the tlc determination substratum.The liquid phase of tlc is as follows: Virahol-80ml, NH
4OH (30%)-5ml, water-25ml.The results are shown in Table 5.As what see, heterozygosis plasmid pYGAZH has improved the proline(Pro) accumulation volume of proline(Pro) generation bacterial strain 702ilvA.
Table 5:
Reference implementation 2: produce the L-leucine by ilvE defective type L-leucine bacterial strain
Wild-type e. coli K12 bacterial strain (VKPM B-7) cell, handled back 20 minutes through mutagenic compound N-methyl-N-nitro-N-nitrosoguanidines (0.05mg/ml) at 37 ℃, clean 4 times with physiological solution after after be coated on the additional leucic low agar M9 substratum of 4.0DL-azepine.37 ℃ cultivated through 5 days after, choose the bacterium colony that the grows purifying of on the L-agar plate, ruling.What obtain can resist the double auxotroph that a bacterium colony in the leucic mutant of DL-azepine is used to induce L-Isoleucine and L-Xie Ansuan.The bacterial strain great majority that obtain need L-Isoleucine and L-Xie Ansuan to grow, and this shows that the double auxotroph bacterial strain is that the sudden change of dependence and ilvE gene just forms.In the double auxotroph bacterial strain that obtains, to select best L-leucine and produced bacterial strain 505, output is the 1.8g/Ll-leucine.The fermentation culture based component comprises: 60g/l sucrose, 25g/l sulfuric acid amine, 2g/l K
2HPO
4, 1g/lMgSO
4, the 0.1mg/l thiamines, 100mg/L l-Isoleucine, 100mg/L l-Xie Ansuan and 25g/l chalk (pH7.2), sucrose and chalk are sterilized separately.Put into the 2ml substratum in vitro, the tested microorganism of inoculation one ring was 37 ℃ of shaking culture 2 days.
Coli strain 505 is respectively at collecting center (VKPM) preservation at the national industrial microorganism of Russia May 14 calendar year 2001, (Russia 113545, Moscow, 1 Dorozhny proezd, 1), preserving number is VKPM B-8011 and VKPM B-8124, and transfers international preservation according to budapest treaty by initial preservation in 2002.
Embodiment 6: produce leucine with the bacterial strain that contains plasmid pYGAZH
It is to be in P by containing that leucine is produced bacterial strain intestinal bacteria 505
LacThe b2682 under the control of UV5 promotor and the plasmid pYGAZH of b2683 gene change over to and obtain.
With bacterial strain 505, in contrast contain the not bacterial strain 505 of the plasmid of insertion portion (p Δ lacZ) and bacterial strain 505 (pYGAZH) are respectively got 20 colony inoculations one and are encircled in the test tube that is added with the 20-ml that L-meat soup additional or not additional penbritin cultivates overnight incubation under 32 ℃ of ventilation conditions.The 0.1ml bacterium liquid of each culture is transferred in the test tube of three 20ml (internal diameter 22mm), adds the fresh fermention medium that 2ml adds or do not add IPTG, rotates shaking culture 72 hours at 32 ℃.The fermentation culture based component:
(NH
4)
2SO
4-15g/l；
K
2HPO
4-1.5g/l；
MgSO
4X 7H
2O-1.0g/l；
CaCO
3-20g/l; (sterilization separately)
Thiamines-0.1mg/l;
Sucrose-60g/l; (sterilization separately)
Isoleucine-0.3g/l;
Xie Ansuan-0.3g/l;
Penbritin-150mg/l, if necessary;
IPTG-0.5mM, if necessary;
After cultivation, record bacterium liquid by traditional method and determine the stable of plasmid at 540nm place absorbance value.Can be by leucic accumulation volume in the tlc determination substratum.The liquid phase of tlc is as follows: Virahol-80ml, ethyl acetate-80ml, NH
4OH (30%)-25ml, water-50ml.The results are shown in Table 6.As what see, heterozygosis plasmid pYGAZH has improved the leucine accumulation volume of leucine generation bacterial strain 505.
Table 6:
Reference implementation 3: produce the L-methionine(Met) by the L-methionine(Met) bacterial strain that nor-leucine is had resistance
The Threonine of plasmid-free and leucine auxotrophy bacterial strain intestinal bacteria C600 are used as parent strain, at first, the leucine mutant of intestinal bacteria C600 bacterial strain can obtain by the transduction of the P1 phage that will grow on e. coli k12 strain, after 37 ℃ of process mutagenic compound N-methyl-N-nitro-N-nitrosoguanidines are handled, obtained to resist the mutants which had 44 of 8g/lL-homoserine.Mutants which had 44 is L-Threonine defective typies, and mutants which had 44 is collected center (VKPM) preservation at the national industrial microorganism of Russia, and preserving number is VKPM B-2175.
The bacterial strain that goes out with N-methyl-N-nitro-N-nitrosoguanidine mutagenesis from bacterial strain 44 is to the methionine(Met) analogue, and nor-leucine has resistance.Cell in the overnight culture of L-broth culture is got off by centrifugal, again suspend with the physiological solution (0.9%NaCl) that contains 50 μ g/mlN-methyl-N-nitro-N-nitrosoguanidine, cell is at 37 ℃, centrifugal after exposing 30 minutes in the N-methyl-N-nitro-N-nitrosoguanidine, be coated on the low agar M9 substratum of additional 0.5mg/ml Threonine and 2.5mg/ml or 5mg/ml nor-leucine after cleaning after 4 times with physiological solution.37 ℃ cultivated through 5 days after, choose the bacterium colony that the grows purifying of on the L-agar plate, ruling.Wherein best L-methionine production bacterin strain is a bacterial strain 218.The test tube of bacterial strain 218 is cultivated 32 ℃ of shaking culture 3 days, and the L-methionine(Met) has reached 1g/l in the substratum.Minimum M9 substratum as fermention medium comprises: sucrose (4%), sulfuric acid amine (2.5%), Threonine (0.5g/l), CaCO
3(25g/l).Sucrose and chalk are sterilized separately.
Bacterial strain 218 is collected center (VKPM) preservation May 14 calendar year 2001 at the national industrial microorganism of Russia, and preserving number is VKPM B-8125, and transfers international preservation according to budapest treaty by initial preservation in 2002.
Further, the pycA gene (Russ P application 99121636) that derives from the bacillus subtilis Pseudomonas by integration is incorporated into bacterial strain 218 with the ppc genetically deficient that phage P1 mediates, and results strain 218pycA has lost and aligned leucic resistance.So, can give bacterial strain again with aforesaid method and align leucic resistance.In the bacterial strain that obtains, best L-methionine production bacterin strain coli strain 73, under these conditions, output is about the 1.0g/lL-methionine(Met).
Coli strain 73 is collected center (VKPM) preservation May 14 calendar year 2001 at the national industrial microorganism of Russia, and preserving number is VKPM B-8126, and transfers international preservation according to budapest treaty by initial preservation in 2002.
Embodiment 7: produce methionine(Met) with the bacterial strain that contains plasmid pYGAZH
Methionine production bacterin strain intestinal bacteria 73 are to be in P by containing
LacThe b2682 under the control of UV5 promotor and the plasmid pYGAZH of b2683 gene change over to and obtain.
With bacterial strain 73, in contrast contain the not bacterial strain 73 of the plasmid of insertion portion (p Δ lacZ) and bacterial strain 73 (pYGAZH) are respectively got 5 bacterium colonies, are suspended in ((NH on the 2ml minimal medium in the test tube of 20-ml
4)
2SO
4-18g/l; K
2HPO
3-1.8g/l; MgSO
4-1.2g/l; Thiamines-0.1mg/l; Yeast extract-10g/l; Sucrose-60g/l; Threonine-400mg/l; Penbritin-300mg/l) if necessary, overnight incubation under 32 ℃ of ventilation conditions.The 0.2ml bacterium liquid of each culture is transferred in the test tube of three 20ml, adds the fresh fermention medium that 2ml adds or do not add IPTG, rotates shaking culture 48 hours at 32 ℃.
The fermentation culture based component:
(NH
4)
2SO
4-18g/l；
K
2HPO
4-1.8g/l；
MgSO
4-1.2g/l；
CaCO
3-20mg/l；
Thiamines-0.1mg/l;
Sucrose-60g/l;
Threonine-400mg/l;
Yeast extract-10g/l;
Penbritin-300mg/l, if necessary;
IPTG-0.5mM, if necessary;
After cultivation, record bacterium liquid by traditional method and determine the stable of plasmid at 540nm place absorbance value.Can be by the accumulation volume of methionine(Met) in the tlc determination substratum.The liquid phase of tlc is as follows: Virahol-80ml, ethyl acetate-80ml, NH
4OH (30%)-15ml, water-45ml.The results are shown in Table 7.As what see, heterozygosis plasmid pYGAZH has improved the methionine(Met) accumulation volume of methionine(Met) generation bacterial strain 73.
Table 7:
Embodiment 8: produce Threonine with the bacterial strain that contains plasmid pYCHE
Threonine is produced bacterial strain intestinal bacteria VL2054 and is in P by containing
LacThe plasmid pYCHE of the b1242 gene under the control of UV5 promotor changes over to and obtains.The bacterial strain that obtains is named as VL2054pYCHE.
With bacterial strain VL2054, in contrast contain not that the bacterial strain VL2054 of the plasmid of insertion portion (p Δ lacZ) and bacterial strain VL2054 (pYCHE) respectively get 5 bacterium colonies, in the test tube of 20-ml, be suspended in ((NH on the 2ml minimal medium
4)
2SO
4-11g/l; NaCl-0.4g/l; MgSO
4-0.4g/l; K
2HPO
3-1g/l; FeSO
4-10mg/l; MnSO
4-10mg/l; Thiamines-0.1mg/l; Yeast extract-0.5g/l; Sucrose-40g/l; Penbritin-300mg/l) if necessary, overnight incubation under 32 ℃ of ventilation conditions.The 0.2ml bacterium liquid of each culture is transferred in the test tube of three 20ml, adds the fresh fermention medium that 2ml adds or do not add IPTG, rotates shaking culture 45 hours at 32 ℃.
The fermentation culture based component:
(NH
4)
2SO
4-22g/l；
NaCl-0.8g/l；
MgSO
4-0.8g/l；
K
2HPO
3-2g/l；
FeSO
4-20mg/l；
MnSO
4-20mg/l；
Thiamines-0.2mg/l;
Yeast extract-1g/l;
CaCO
3-30g/l；
Sucrose-80g/l;
Penbritin-300mg/l, if necessary;
IPTG-0.5mM, if necessary;
After cultivation, record bacterium liquid by traditional method and determine the stable of plasmid at 540nm place absorbance value.Can be by the accumulation volume of Threonine in the tlc determination substratum.The liquid phase of tlc is as follows: Virahol-50ml, acetone-50ml, NH
4OH (30%)-12ml, water-8ml.The results are shown in Table 8.As what see, heterozygosis plasmid pYCHE has improved the Threonine accumulation volume of Threonine generation bacterial strain VL2054.
Table 8:
| The VL2054 that plasmid is arranged | IPTG | OD 540 | Thr，g/l | Thr/OD |
| NO | - + | 21 20 | 4.8 4.7 | 0.23 0.24 |
| pΔlacZ | - + | 16 13 | 4.6 3.0 | 0.29 0.23 |
| pYCHE | - + | 20 20 | 6.2 7.0 | 0.31 0.35 |
Embodiment 9: produce Xie Ansuan with the bacterial strain that contains plasmid pYCHE
Xie Ansuan is produced bacterial strain H-81 and is in P by containing
LacThe plasmid pYCHE of the b1242 gene under the control of UV5 promotor changes over to and obtains.
With in contrast contain not that the bacterial strain H-81 of the plasmid of insertion portion (p Δ lacZ) and bacterial strain H-81 (pYCHE) respectively get 5 bacterium colonies, in the test tube of 20-ml, be suspended in ((NH on the 2ml minimal medium
4)
2SO
4-18g/l; K
2HPO
4-1.8g/l; MgSO
4-1.2g/l; Thiamines-0.1mg/l; Yeast extract-0.5g/l; Sucrose-60g/l; Penbritin-300mg/l) if necessary, overnight incubation under ventilation condition.The 0.2ml bacterium liquid of each culture is transferred in the test tube of three 20ml, adds the fresh fermention medium that 2ml adds or do not add IPTG, rotates shaking culture 45 hours at 32 ℃.
The fermentation culture based component:
(NH
4)
2SO
4-18g/l；
K
2HPO
4-1.8g/l；
MgSO
4-1.2g/l；
CaCO
3-20g/l；
Thiamines-0.1mg/l;
Sucrose-60g/l;
Penbritin-300mg/l, if necessary;
IPTG-0.5mM, if necessary;
After cultivation, record bacterium liquid by traditional method and determine the stable of plasmid at 540nm place absorbance value.Can be by the accumulation volume of Xie Ansuan in the tlc determination substratum.The liquid phase of tlc is as follows: Virahol-80ml, ethyl acetate-80ml, NH
4OH (30%)-15ml, water-45ml.The results are shown in Table 9.As what see, heterozygosis plasmid pYCHE has improved the Xie Ansuan accumulation volume of Xie Ansuan generation bacterial strain H-81.
Table 9:
| The H-81 that plasmid is arranged | IPTG | OD 540 | Val，g/l | Val/OD |
| NO | - + | 34 34 | 11.6 11.7 | 0.34 0.34 |
| pΔlacZ | - + | 34 20 | 10.5 7.8 | 0.31 0.39 |
| pYCHE | - + | 32 30 | 14.0 13.9 | 0.44 0.46 |
Embodiment 10: produce arginine with the bacterial strain that contains plasmid pYHGN
It is to be in P by containing that arginine is produced bacterial strain 382
LacThe plasmid pYHGN of the b3434 gene under the control of UV5 promotor changes over to and obtains.The bacterial strain 382 that obtains is collected center (VKPM) preservation on April 10th, 2000 at the national industrial microorganism of Russia, and (Russia 113545, Moscow, 1Dorozhny proezd, 1), preserving number is VKPM B-7962.
With bacterial strain 382, in contrast contain the not bacterial strain 382 of the plasmid of insertion portion (p Δ lacZ) and bacterial strain 382 (pYHGN) are respectively got 5 bacterium colonies, are suspended in ((NH on the 2ml minimal medium in the test tube of 20-ml
4)
2SO
4-25g/l; K
2HPO
4-2.0g/l; MgSO
47H
2O-1.0g/l; Thiamines-0.2mg/l; Yeast extract-5g/l; Sucrose-60g/l; Penbritin-300mg/l) if necessary, overnight incubation under ventilation condition.The 0.2ml bacterium liquid of each culture is transferred in the test tube of three 20ml, adds the fresh fermention medium that 2ml adds or do not add IPTG, rotates shaking culture 72 hours at 32 ℃.
The fermentation culture based component:
(NH
4)
2SO
4-25g/l；
K
2HPO
4-2.0g/l；
MgSO
4 7H
2O-1.0g/l；
Thiamines-0.2mg/l;
Yeast extract-5g/l;
Sucrose-60g/l;
CaCO
3-20g/l；
Penbritin-100mg/l, if necessary;
IPTG-0.5mM, if necessary;
After cultivation, record bacterium liquid by traditional method and determine the stable of plasmid at 540nm place absorbance value.Can be by arginic accumulation volume in the tlc determination substratum.The liquid phase of tlc is as follows: Virahol-80ml, and ethyl acetate-40ml,, NH
4OH (30%)-25ml, water-50ml.The results are shown in Table 10.As what see, heterozygosis plasmid pYHGN has improved the Xie Ansuan accumulation volume of Xie Ansuan generation bacterial strain 382.
Table 10:
| The intestinal bacteria 382 that plasmid is arranged | IPTG | OD 540 | Arg，g/l | Arg/OD |
| NO | - + | 20 22 | 8.5 6.7 | 0.43 0.31 |
| pΔlacZ | - + | 28 26 | 6.3 5.4 | 0.23 0.21 |
| pYHGN | - + | 24 26 | 5.8 9.3 | 0.24 0.36 |
Embodiment 11: produce proline(Pro) with the bacterial strain that contains plasmid pYHGN
Proline(Pro) is produced bacterial strain 702ilvA and is in P by containing
LacThe plasmid pYHGN of the b3434 gene under the control of UV5 promotor changes over to and obtains.
With bacterial strain 702ilvA, in contrast contain not that the bacterial strain 702ilvA of the plasmid of insertion portion (p Δ lacZ) and bacterial strain 702ilvA (pYHGN) respectively get 5 bacterium colonies, in the test tube of 20-ml, be suspended in ((NH on the 2ml minimal medium
4)
2SO
4-18g/l; K
2HPO
3-1.8g/l; MgSO
4-1.2g/l; Thiamines-0.1mg/l; Yeast extract-0.5g/l; Sucrose-60g/l; Isoleucine-50mg/l; Penbritin-300mg/l) if necessary, overnight incubation under 32 ℃ of ventilation conditions.The 0.2ml bacterium liquid of each culture is transferred in the test tube of three 20ml, adds the fresh fermention medium that 2ml adds or do not add IPTG, rotates shaking culture 40 hours at 32 ℃.
The fermentation culture based component:
(NH
4)
2SO
4-18g/l；
K
2HPO
4-1.8g/l；
MgSO
4-1.2g/l；
CaCO
3-20mg/l；
Thiamines-0.1mg/l;
Sucrose-60g/l;
Isoleucine-50mg/l;
Penbritin-300mg/l, if necessary;
IPTG-0.5mM, if necessary;
After cultivation, record bacterium liquid by traditional method and determine the stable of plasmid at 540nm place absorbance value.Can be by the accumulation volume of proline(Pro) in the tlc determination substratum.The liquid phase of tlc is as follows: Virahol-80ml, NH
4OH (30%)-5ml, water-25ml.The results are shown in Table 11.As what see, heterozygosis plasmid pYHGN has improved the proline(Pro) accumulation volume of proline(Pro) generation bacterial strain 702ilvA.
Table 11:
Sequence table
＜110〉Ajincomoto Co., Inc
＜120〉produce the amino acid whose method of L-by colibacillus bacteria
<130>P-8206F
<140>
<141>2002--
<150>RU 2001103865
<151>2001-02-13
<150>RU 2001104998
<151>2001-02-26
<150>RU 2001104999
<151>2001-02-26
<150>RU 2001117632
<151>2001-06-28
<150>RU 2001117633
<151>2001-06-28
<160>16
<170>PatentIn Ver.2.0
<210>1
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: primer
<400>1
ggtctagaca atcgttaagc gtacac 26
<210>2
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: primer
<400>2
ccggatccga tatagtaacg acagtg 26
<210>3
<211>738
<212>DNA
＜213〉intestinal bacteria (Escherichia coli)
<220>
<221>CDS
<222>(1)..(735)
<400>3
atg gaa agc cct act cca cag cct gct cct ggt tcg gcg acc ttc atg 48
Met Glu Ser Pro Thr Pro Gln Pro Ala Pro Gly Ser Ala Thr Phe Met
1 5 10 15
gaa gga tgc aaa gac agt tta ccg att gtt att agt tat att ccg gtg 96
Glu Gly Cys Lys Asp Ser Leu Pro Ile Val Ile Ser Tyr Ile Pro Val
20 25 30
gcc ttt gcg ttc ggt ctg aat gcg acc cgt ctg gga ttc tct cct ctc 144
Ala Phe Ala Phe Gly Leu Asn Ala Thr Arg Leu Gly Phe Ser Pro Leu
35 40 45
gaa agc gtt ttt ttc tcc tgc atc att tat gca ggc gcg agc cag ttc 192
Glu Ser Val Phe Phe Ser Cys Ile Ile Tyr Ala Gly Ala Ser Gln Phe
50 55 60
gtc att acc gcg atg ctg gca gcc ggg agt agt ttg tgg att gct gca 240
Val Ile Thr Ala Met Leu Ala Ala Gly Ser Ser Leu Trp Ile Ala Ala
65 70 75 80
ctg acc gtc atg gca atg gat gtt cgc cat gtg ttg tat ggc ccg tca 288
Leu Thr Val Met Ala Met Asp Val Arg His Val Leu Tyr Gly Pro Ser
85 90 95
ctg cgt agc cgt att att cag cgt ctg caa aaa tcg aaa acc gcc ctg 336
Leu Arg Ser Arg Ile Ile Gln Arg Leu Gln Lys Ser Lys Thr Ala Leu
100 105 110
tgg gcg ttt ggc ctg acg gat gag gtt ttt gcc gcc gca acc gca aaa 384
Trp Ala Phe Gly Leu Thr Asp Glu Val Phe Ala Ala Ala Thr Ala Lys
115 120 125
ctg gta cgc aat aat cgc cgc tgg agc gag aac tgg atg atc ggc att 432
Leu Val Arg Asn Asn Arg Arg Trp Ser Glu Asn Trp Met Ile Gly Ile
130 135 140
gcc ttc agt tca tgg tca tcg tgg gta ttt ggt acg gta ata ggg gca 480
Ala Phe Ser Ser Trp Ser Ser Trp Val Phe Gly Thr Val Ile Gly Ala
145 150 155 160
ttc tcc ggc agc ggc ttg ctg caa ggt tat ccc gcc gtt gaa gct gca 528
Phe Ser Gly Ser Gly Leu Leu Gln Gly Tyr Pro Ala Val Glu Ala Ala
165 170 175
tta ggt ttt atg ctt ccg gca ctc ttt atg agt ttc ctg ctc gcc tct 576
Leu Gly Phe Met Leu Pro Ala Leu Phe Met Ser Phe Leu Leu Ala Ser
180 185 190
ttc cag cgc aaa caa tct ctt tgc gtt acc gca gcg tta gtt ggt gcc 624
Phe Gln Arg Lys Gln Ser Leu Cys Val Thr Ala Ala Leu Val Gly Ala
195 200 205
ctt gca ggc gta acg cta ttt tct att ccc gtc gcc att ctg gca ggc 672
Leu Ala Gly Val Thr Leu Phe Ser Ile Pro Val Ala Ile Leu Ala Gly
210 215 220
att gtc tgt ggc tgc ctc act gcg tta atc cag gca ttc tgg caa gga 720
Ile Val Cys Gly Cys Leu Thr Ala Leu Ile Gln Ala Phe Trp Gln Gly
225 230 235 240
gcg ccc gat gag cta tga 738
Ala Pro Asp Glu Leu
245
<210>4
<211>245
<212>PRT
＜213〉intestinal bacteria (Escherichia coli)
<400>4
Met Glu Ser Pro Thr Pro Gln Pro Ala Pro Gly Ser Ala Thr Phe Met
1 5 10 15
Glu Gly Cys Lys Asp Ser Leu Pro Ile Val Ile Ser Tyr Ile Pro Val
20 25 30
Ala Phe Ala Phe Gly Leu Asn Ala Thr Arg Leu Gly Phe Ser Pro Leu
35 40 45
Glu Ser Val Phe Phe Ser Cys Ile Ile Tyr Ala Gly Ala Ser Gln Phe
50 55 60
Val Ile Thr Ala Met Leu Ala Ala Gly Ser Ser Leu Trp Ile Ala Ala
65 70 75 80
Leu Thr Val Met Ala Met Asp Val Arg His Val Leu Tyr Gly Pro Ser
85 90 95
Leu Arg Ser Arg Ile Ile Gln Arg Leu Gln Lys Ser Lys Thr Ala Leu
100 105 110
Trp Ala Phe Gly Leu Thr Asp Glu Val Phe Ala Ala Ala Thr Ala Lys
115 120 125
Leu Val Arg Asn Asn Arg Arg Trp Ser Glu Asn Trp Met Ile Gly Ile
130 135 140
Ala Phe Ser Ser Trp Ser Ser Trp Val Phe Gly Thr Val Ile Gly Ala
145 150 155 160
Phe Ser Gly Ser Gly Leu Leu Gln Gly Tyr Pro Ala Val Glu Ala Ala
165 170 175
Leu Gly Phe Met Leu Pro Ala Leu Phe Met Ser Phe Leu Leu Ala Ser
180 185 190
Phe Gln Arg Lys Gln Ser Leu Cys Val Thr Ala Ala Leu Val Gly Ala
195 200 205
Leu Ala Gly Val Thr Leu Phe Ser Ile Pro Val Ala Ile Leu Ala Gly
210 215 220
Ile Val Cys Gly Cys Leu Thr Ala Leu Ile Gln Ala Phe Trp Gln Gly
225 230 235 240
Ala Pro Asp Glu Leu
245
<210>5
<211>336
<212>DNA
＜213〉intestinal bacteria (Escherichia coli)
<220>
<221>CDS
<222>(1)..(333)
<400>5
atg agc tat gag gtt ctg ctg ctt ggg tta cta gtt ggc gtg gcg aat 48
Met Ser Tyr Glu Val Leu Leu Leu Gly Leu Leu Val Gly Val Ala Asn
1 5 10 15
tat tgc ttc cgc tat ttg ccg ctg cgc ctg cgt gtg ggt aat gcc cgc 96
Tyr Cys Phe Arg Tyr Leu Pro Leu Arg Leu Arg Val Gly Asn Ala Arg
20 25 30
cca acc aaa cgt ggc gcg gta ggt att ttg ctc gac acc att ggc atc 144
Pro Thr Lys Arg Gly Ala Val Gly Ile Leu Leu Asp Thr Ile Gly Ile
35 40 45
gcc tcg ata tgc gct ctg ctg gtt gtc tct acc gca cca gaa gtg atg 192
Ala Ser Ile Cys Ala Leu Leu Val Val Ser Thr Ala Pro Glu Val Met
50 55 60
cac gat aca cgc cgt ttc gtg ccc acg ctg gtc ggc ttc gcg gta ctg 240
His Asp Thr Arg Arg Phe Val Pro Thr Leu Val Gly Phe Ala Val Leu
65 70 75 80
ggt gcc agt ttc tat aaa aca cgc agc att atc atc cca aca ctg ctt 288
Gly Ala Ser Phe Tyr Lys Thr Arg Ser Ile IleIle Pro Thr Leu Leu
85 90 95
agt gcg ctg gcc tat ggg ctc gcc tgg aaa gtg atg gcg att ata taa 336
Ser Ala Leu Ala Tyr Gly Leu Ala Trp Lys Val Met Ala Ile Ile
100 105 110
<210>6
<211>111
<212>PRT
＜213〉intestinal bacteria (Escherichia coli)
<400>6
Met Ser Tyr Glu Val Leu Leu Leu Gly Leu Leu Val Gly Val Ala Asn
1 5 10 15
Tyr Cys Phe Arg Tyr Leu Pro Leu Arg Leu Arg Val Gly Asn Ala Arg
20 25 30
Pro Thr Lys Arg Gly Ala Val Gly Ile Leu Leu Asp Thr Ile Gly Ile
35 40 45
Ala Ser Ile Cys Ala Leu Leu Val Val Ser Thr Ala Pro Glu Val Met
50 55 60
His Asp Thr Arg Arg Phe Val Pro Thr Leu Val Gly Phe Ala Val Leu
65 70 75 80
Gly Ala Ser Phe Tyr Lys Thr Arg Ser Ile Ile Ile Pro Thr Leu Leu
85 90 95
Ser Ala Leu Ala Tyr Gly Leu Ala Trp Lys Val Met Ala Ile Ile
100 105 110
<210>7
<211>37
<212>DNA
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: primer
<400>7
cctttggtac cagatctgcg ggcagtgagc gcaacgc 37
<210>8
<211>34
<212>DNA
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: primer
<400>8
ctgtttctag atcctgtgtg aaattgttat ccgc 34
<210>9
<211>28
<212>DNA
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: primer
<400>9
ggtctagata tggctaacat tatccggc 28
<210>10
<211>28
<212>DNA
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: primer
<400>10
ccggatccaa acggagcatg gcagctcc 28
<210>11
<211>648
<212>DNA
＜213〉intestinal bacteria (Escherichia coli)
<220>
<221>CDS
<222>(1)..(645)
<400>11
gtg att cag acc ttt ttt gat ttt ccc gtt tac ttc aaa ttt ttc atc 48
Met Ile Gln Thr Phe Phe Asp Phe Pro Val Tyr Phe Lys Phe Phe Ile
1 5 10 15
ggg tta ttt gcg ctg gtc aac ccg gta ggg att att ccc gtc ttt atc 96
Gly Leu Phe Ala Leu Val Asn Pro Val Gly Ile Ile Pro Val Phe Ile
20 25 30
agc atg acc agt tat cag aca gcg gca gcg cga aac aaa act aac ctt 144
Ser Met Thr Ser Tyr Gln Thr Ala Ala Ala Arg Asn Lys Thr Asn Leu
35 40 45
aca gcc aac ctg tct gtg gcc att atc ttg tgg atc tcg ctt ttt ctc 192
Thr Ala Asn Leu Ser Val Ala Ile Ile Leu Trp Ile Ser Leu Phe Leu
50 55 60
ggc gac acg att cta caa ctt ttt ggt ata tca att gat tcg ttc cgt 240
Gly Asp Thr Ile Leu Gln Leu Phe Gly Ile Ser Ile Asp Ser Phe Arg
65 70 75 80
atc gcc ggg ggt atc ctg gtg gtg aca ata gcg atg tcg atg atc agc 288
Ile Ala Gly Gly Ile Leu Val Val Thr Ile Ala Met Ser Met Ile Ser
85 90 95
ggc aag ctt ggc gag gat aaa cag aac aag caa gaa aaa tca gaa acc 336
Gly Lys Leu Gly Glu Asp Lys Gln Asn Lys Gln Glu Lys Ser Glu Thr
100 105 110
gcg gta cgt gaa agc att ggt gtg gtg cca ctg gcg ttg ccg ttg atg 384
Ala Val Arg Glu Ser Ile Gly Val Val Pro Leu Ala Leu Pro Leu Met
115 120 125
gcg ggg cca ggg gcg atc agt tct acc atc gtc tgg ggt acg cgt tat 432
Ala Gly Pro Gly Ala Ile Ser Ser Thr Ile Val Trp Gly Thr Arg Tyr
130 135 140
cac agc att agc tat ctg ttt ggt ttc ttt gtg gct att gca ttg ttc 480
His Ser Ile Ser Tyr Leu Phe Gly Phe Phe Val Ala Ile Ala Leu Phe
145 150 155 160
gct tta tgt tgt tgg gga ttg ttc cgc atg gca ccg tgg ctg gta cgg 528
Ala Leu Cys Cys Trp Gly Leu Phe Arg Met Ala Pro Trp Leu Val Arg
165 170 175
gtt tta cgc cag acc ggc atc aac gtg att acg cgt att atg ggg cta 576
Val Leu Arg Gln Thr Gly Ile Asn Val Ile Thr Arg Ile Met Gly Leu
180 185 190
ttg ctg atg gca ttg ggg att gaa ttt atc gtt act ggt att aag ggg 624
Leu Leu Met Ala Leu Gly Ile Glu Phe Ile Val Thr Gly Ile Lys Gly
195 200 205
att ttc ccc ggc ctg ctt aat taa 648
Ile Phe Pro Gly Leu Leu Asn
210 215
<210>12
<211>215
<212>PRT
＜213〉intestinal bacteria (Escherichia coli)
<400>12
Met Ile Gln Thr Phe Phe Asp Phe Pro Val Tyr Phe Lys Phe Phe Ile
1 5 10 15
Gly Leu Phe Ala Leu Val Asn Pro Val Gly Ile Ile Pro Val Phe Ile
20 25 30
Ser Met Thr Ser Tyr Gln Thr Ala Ala Ala Arg Asn Lys Thr Asn Leu
35 40 45
Thr Ala Asn Leu Ser Val Ala Ile Ile Leu Trp Ile Ser Leu Phe Leu
50 55 60
Gly Asp Thr Ile Leu Gln Leu Phe Gly Ile Ser Ile Asp Ser Phe Arg
65 70 75 80
Ile Ala Gly Gly Ile Leu Val Val Thr Ile Ala Met Ser Met Ile Ser
85 90 95
Gly Lys Leu Gly Glu Asp Lys Gln Asn Lys Gln Glu Lys Ser Glu Thr
100 105 110
Ala Val Arg Glu Ser Ile Gly Val Val Pro Leu Ala Leu Pro Leu Met
115 120 125
Ala Gly Pro Gly Ala Ile Ser Ser Thr Ile Val Trp Gly Thr Arg Tyr
130 135 140
His Ser Ile Ser Tyr Leu Phe Gly Phe Phe Val Ala Ile Ala Leu Phe
145 150 155 160
Ala Leu Cys Cys Trp Gly Leu Phe Arg Met Ala Pro Trp Leu Val Arg
165 170 175
Val Leu Arg Gln Thr Gly Ile Asn Val Ile Thr Arg Ile Met Gly Leu
180 185 190
Leu Leu Met Ala Leu Gly Ile Glu Phe Ile Val Thr Gly Ile Lys Gly
195 200 205
Ile Phe Pro Gly Leu Leu Asn
210 215
<210>13
<211>28
<212>DNA
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: primer
<400>13
ggtctagagt ccgcggcaat tatcaggg 28
<210>14
<211>29
<212>DNA
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: primer
<400>14
ccagatctgg tagttgtgac gctaccggg 29
<210>15
<211>594
<212>DNA
＜213〉intestinal bacteria (Eseherichia coli)
<220>
<221>CDS
<222>(1)..(591)
<400>15
atg aat gaa atc att tct gca gca gtt tta ttg atc ctg att atg gat 48
Met Asn Glu Ile Ile Ser Ala Ala Val Leu Leu Ile Leu Ile Met Asp
1 5 10 15
ccg ctc gga aac cta cct att ttc atg tcc gta ctg aaa cat act gaa 96
Pro Leu Gly Asn Leu Pro Ile Phe Met Ser Val Leu Lys His Thr Glu
20 25 30
ccg aaa aga cgg cgg gca atc atg gtg cga gag ttg ctt att gct ctc 144
Pro Lys Arg Arg Arg Ala Ile Met Val Arg Glu Leu Leu Ile Ala Leu
35 40 45
ctg gtg atg ctg gtg ttc ctg ttt gcg ggt gag aaa att ctg gca ttt 192
Leu Val Met Leu Val Phe Leu Phe Ala Gly Glu Lys Ile Leu Ala Phe
50 55 60
ctt agc cta cga gca gaa acc gtc tcc att tct ggc ggc atc att ctg 240
Leu Ser Leu Arg Ala Glu Thr Val Ser Ile Ser Gly Gly Ile Ile Leu
65 70 75 80
ttt ctg atc gcc att aaa atg att ttc ccc agc gct tca gga aat agc 288
Phe Leu Ile Ala Ile Lys Met Ile Phe Pro Ser Ala Ser Gly Asn Ser
85 90 95
agc ggg ctt ccg gca ggt gaa gag cca ttt atc gtg ccg ttg gca att 336
Ser Gly Leu Pro Ala Gly Glu Glu Pro Phe Ile Val Pro Leu Ala Ile
100 105 110
ccg tta gtc gcc ggg ccg act att ctc gcc acg ctg atg ttg ttg tct 384
Pro Leu Val Ala Gly Pro Thr Ile Leu Ala Thr Leu Met Leu Leu Ser
115 120 125
cat cag tac ccg aat cag atg ggg cat ctg gtg att gct ctg ctg ctg 432
His Gln Tyr Pro Asn Gln Met Gly His Leu Val Ile Ala Leu Leu Leu
130 135 140
gcc tgg ggc ggc acc ttt gtc atc ctg cta cag tct tcg cta ttt tta 480
Ala Trp Gly Gly Thr Phe Val Ile Leu Leu Gln Ser Ser Leu Phe Leu
145 150 155 160
cgt ctg ctg ggc gag aaa ggg gtg aac gca ctt gaa cgc ctg atg gga 528
Arg Leu Leu Gly Glu Lys Gly Val Asn Ala Leu Glu Arg Leu Met Gly
165 170 175
ttg att ctg gtg atg atg gca acc cag atg ttc ctc gac ggc att cga 576
Leu Ile Leu Val Met Met Ala Thr Gln Met Phe Leu Asp Gly Ile Arg
180 185 190
atg tgg atg aag ggg taa 594
Met Trp Met Lys Gly
195
<210>16
<211>197
<212>PRT
＜213〉intestinal bacteria (Escherichia coli)
<400>16
Met Asn Glu Ile Ile Ser Ala Ala Val Leu Leu Ile Leu Ile Met Asp
1 5 10 15
Pro Leu Gly Asn Leu Pro Ile Phe Met Ser Val Leu Lys His Thr Glu
20 25 30
Pro Lys Arg Arg Arg Ala Ile Met Val Arg Glu Leu Leu Ile Ala Leu
35 40 45
Leu Val Met Leu Val Phe Leu Phe Ala Gly Glu Lys Ile Leu Ala Phe
50 55 60
Leu Ser Leu Arg Ala Glu Thr Val Ser Ile Ser Gly Gly Ile Ile Leu
65 70 75 80
Phe Leu Ile Ala Ile Lys Met Ile Phe Pro Ser Ala Ser Gly Asn Ser
85 90 95
Ser Gly Leu Pro Ala Gly Glu Glu Pro Phe Ile Val Pro Leu Ala Ile
100 105 110
Pro Leu Val Ala Gly Pro Thr Ile Leu Ala Thr Leu Met Leu Leu Ser
115 120 125
His Gln Tyr Pro Asn Gln Met Gly His Leu Val Ile Ala Leu Leu Leu
130 135 140
Ala Trp Gly Gly Thr Phe Val Ile Leu Leu Gln Ser Ser Leu Phe Leu
145 150 155 160
Arg Leu Leu Gly Glu Lys Gly Val Asn Ala Leu Glu Arg Leu Met Gly
165 170 175
Leu Ile Leu Val Met Met Ala Thr Gln Met Phe Leu Asp Gly Ile Arg
180 185 190
Met Trp Met Lys Gly
195
Claims (8)
1. amino acid producing bacteria of the L-that belongs to Escherichia, wherein this bacterium through modification so that the L-amino acid production of this bacterium by improving following A) activity of protein in described bacterial cell that define improve:
A) comprise the protein of aminoacid sequence shown in the SEQ ID NO:11 in the sequence table.
2. according to the bacterium of claim 1, A wherein) activity of proteins of definition is by the A that will encode) protein DNA that defines transforms into bacterium, or improves by the expression regulation sequence of the above DNA of change bacterial chromosome.
3. according to the bacterium of claim 2, conversion is wherein undertaken by multi-copy vector.
4. produce the amino acid whose method of L-for one kind, be included in the bacterium of cultivating among the claim 1-3 each in the substratum and from substratum, collect and produce and the L-amino acid of accumulation.
5. according to the method for claim 4, wherein said L-amino acid is the L-Threonine.
6. according to the method for claim 5, wherein described bacterium is modified, so that this bacterium has been improved the expression of threonine operon by copy number that increases threonine operon or the expression regulation sequence that changes threonine operon.
7. according to claim 4 method, described L-amino acid is the L-Xie Ansuan.
8. according to claim 7 method, wherein described bacterium is modified, so that this bacterium has been improved the expression of ilv operon by copy number that increases the ilv operon or the expression regulation sequence that changes the ilv operon.
Applications Claiming Priority (10)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| RU2001103865 | 2001-02-13 | ||
| RU2001103865 | 2001-02-13 | ||
| RU2001104999 | 2001-02-26 | ||
| RU2001104998 | 2001-02-26 | ||
| RU2001104998A RU2215782C2 (en) | 2001-02-26 | 2001-02-26 | Method for preparing l-amino acids, strain escherichia coli as producer of l-amino acid (variants) |
| RU2001104999 | 2001-02-26 | ||
| RU2001117633A RU2215785C2 (en) | 2001-06-28 | 2001-06-28 | Method for preparing l-amino acids, strain escherichia coli as producer of l-amino acid (variants) |
| RU2001117632 | 2001-06-28 | ||
| RU2001117632A RU2215784C2 (en) | 2001-06-28 | 2001-06-28 | Method for preparing l amino acids, strain escherichia coli as producer of l-amino acid (variants) |
| RU2001117633 | 2001-06-28 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB021080860A Division CN100529057C (en) | 2001-02-13 | 2002-02-13 | Method for producing L-amino acid by colibacillus bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101597588A true CN101597588A (en) | 2009-12-09 |
| CN101597588B CN101597588B (en) | 2011-04-06 |
Family
ID=41419186
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009101594504A Expired - Lifetime CN101597589B (en) | 2001-02-13 | 2002-02-13 | Method for producing l-amino acid using bacteria belonging to the genus escherichia |
| CN2009101594487A Expired - Lifetime CN101597588B (en) | 2001-02-13 | 2002-02-13 | Method for producing l-amino acid using bacteria belonging to the genus escherichia |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009101594504A Expired - Lifetime CN101597589B (en) | 2001-02-13 | 2002-02-13 | Method for producing l-amino acid using bacteria belonging to the genus escherichia |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN101597589B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103003437A (en) * | 2010-06-03 | 2013-03-27 | 味之素株式会社 | A method for producing an L-amino acid using a bacterium of the enterobacteriaceae family, having attenuated expression of gene(s) encoding peptidase |
| CN107109359A (en) * | 2014-09-01 | 2017-08-29 | 代谢探索者公司 | Method and microorganism for the fermenting and producing methionine by being discharged with improved methionine |
| CN114729012A (en) * | 2019-09-09 | 2022-07-08 | Cj第一制糖株式会社 | L-threonine exporter variants and methods of producing L-threonine using the same |
| CN117683802A (en) * | 2024-02-02 | 2024-03-12 | 中国农业科学院北京畜牧兽医研究所 | Ralstonia engineering strain for producing isoleucine through methyl malic acid pathway, construction method and production method thereof |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BR112018017227A2 (en) * | 2016-02-25 | 2019-02-05 | Ajinomoto Kk | method to produce an l-amino acid |
| CN109554322B (en) * | 2018-12-03 | 2020-08-04 | 江南大学 | Recombinant Escherichia coli with high L-threonine yield and construction method thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000197490A (en) * | 1998-11-02 | 2000-07-18 | Ajinomoto Co Inc | Production of l-arginine |
| RU2148642C1 (en) * | 1998-12-23 | 2000-05-10 | ЗАО "Научно-исследовательский институт АДЖИНОМОТО-Генетика" (ЗАО "АГРИ") | Dna rhtc fragment encoding synthesis of rhtc protein that determines enhanced resistance of bacterium escherichia coli to l-threonine and method of l-amino acid producing |
| RU2175351C2 (en) * | 1998-12-30 | 2001-10-27 | Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО "АГРИ") | Escherichia coli dna fragment determining enhanced production of l-amino acids (variants) and method of l-amino acid producing |
-
2002
- 2002-02-13 CN CN2009101594504A patent/CN101597589B/en not_active Expired - Lifetime
- 2002-02-13 CN CN2009101594487A patent/CN101597588B/en not_active Expired - Lifetime
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103003437A (en) * | 2010-06-03 | 2013-03-27 | 味之素株式会社 | A method for producing an L-amino acid using a bacterium of the enterobacteriaceae family, having attenuated expression of gene(s) encoding peptidase |
| CN103003437B (en) * | 2010-06-03 | 2015-05-20 | 味之素株式会社 | A method for producing an L-amino acid using a bacterium of the enterobacteriaceae family, having attenuated expression of gene(s) encoding peptidase |
| CN107109359A (en) * | 2014-09-01 | 2017-08-29 | 代谢探索者公司 | Method and microorganism for the fermenting and producing methionine by being discharged with improved methionine |
| CN107109359B (en) * | 2014-09-01 | 2021-06-29 | 赢创运营有限公司 | Method and microorganism for producing methionine by fermentation with improved methionine excretion |
| CN114729012A (en) * | 2019-09-09 | 2022-07-08 | Cj第一制糖株式会社 | L-threonine exporter variants and methods of producing L-threonine using the same |
| CN114729012B (en) * | 2019-09-09 | 2024-02-06 | Cj第一制糖株式会社 | L-threonine export protein variants and methods of producing L-threonine using the same |
| CN117683802A (en) * | 2024-02-02 | 2024-03-12 | 中国农业科学院北京畜牧兽医研究所 | Ralstonia engineering strain for producing isoleucine through methyl malic acid pathway, construction method and production method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101597589A (en) | 2009-12-09 |
| CN101597588B (en) | 2011-04-06 |
| CN101597589B (en) | 2011-08-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100529057C (en) | Method for producing L-amino acid by colibacillus bacteria | |
| CN1914328B (en) | L-thereonine producing bacterium belonging to the genus escherichia and method for producing l-threonine | |
| US6887691B2 (en) | DNA coding for protein which confers on bacterium Escherichia coli resistance to L-homoserine, and method for producing L-amino acids | |
| ES2314076T3 (en) | CORINEFORM BACTERIES THAT PRODUCE CHEMICAL COMPOUNDS II. | |
| EP1149911B1 (en) | Amino acid producing strains belonging to the genus Escherichia and method for producing an amino acid | |
| CN1954065A (en) | Method for producing L-amino acid by fermentation | |
| CN1280184A (en) | Corgnebaclerium for producing L-lysine and method for preparing L-lysine | |
| CN101760441B (en) | A method for producing l-arginine using a bacterium of enterobacteriaceae family, having attenuated expression of a gene encoding an l-arginine transporter | |
| FR2844277A1 (en) | Escherichia strains that produce L-amino acids, particularly L-threonine, at increased levels, contain an inactivated mlc gene | |
| KR100815041B1 (en) | Metabolic engineering of amino acid production | |
| RU2215782C2 (en) | Method for preparing l-amino acids, strain escherichia coli as producer of l-amino acid (variants) | |
| CN101597588B (en) | Method for producing l-amino acid using bacteria belonging to the genus escherichia | |
| CN111763699A (en) | Recombinant DNA (deoxyribonucleic acid) for producing 1, 5-pentanediamine through fermentation, strain and application of recombinant DNA | |
| JP2007513601A (en) | L-threonine producing bacteria belonging to the genus Escherichia and method for producing L-threonine | |
| RU2215784C2 (en) | Method for preparing l amino acids, strain escherichia coli as producer of l-amino acid (variants) | |
| KR100793215B1 (en) | Mutant bacterial strains l-lysine production | |
| US20060040364A1 (en) | DNA coding for a protein which imparts L-homoserine resistance to Escherichia coli bacterium, and a method for producing L-amino acids | |
| RU2215785C2 (en) | Method for preparing l-amino acids, strain escherichia coli as producer of l-amino acid (variants) | |
| WO2020138178A1 (en) | Method for producing basic l-amino acids or salts thereof by fermentation of an enterobacteriaceae bacterium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term |
Granted publication date: 20110406 |
|
| CX01 | Expiry of patent term |