CN101223279A

CN101223279A - Methionine producing recombinant microorganisms

Info

Publication number: CN101223279A
Application number: CNA200680026101XA
Authority: CN
Inventors: O·策尔德尔; S·哈夫纳; C·克洛普罗格; H·施罗德; A·赫罗尔德; T·A·帕特森; T·赫尔曼; R·R·约库姆; M·K·威廉姆斯; J·G·佩罗
Original assignee: BASF SE
Priority date: 2005-07-18
Filing date: 2006-07-18
Publication date: 2008-07-16
Anticipated expiration: 2026-07-18
Also published as: ZA200801497B; CN101223279B

Abstract

This invention relates to methionine producing recombinant microorganisms. Specifically, this invention relates to recombinant strains of Corynebacterium that produce increased levels of methionine compared to their wild-type counterparts and further to methods of generating such microorganisms.

Description

Produce the recombinant microorganism of methionine(Met)

Related application

The application requires the U.S. Provisional Patent Application submitted on July 18th, 2005 number 60/700,699, the U.S. Provisional Patent Application of submitting on September 1st, 2005 number 60/714,042, title all is the right of priority of the recombinant microorganism of methionine(Met) " produce " (Methionine Producing RecombinantMicroorganism), and their full content all is incorporated herein by reference.

In addition, the U.S. Provisional Patent Application that the application relates on July 18th, 2005 and submits to number 60/700,698, the U.S. Provisional Patent Application of submitting on September 1st, 2005 number 60/713,907, title all is " purposes of dimethyl disulfide in the methionine(Met) of microorganism is produced " (Use of DimethylDisulfide for Methionine Production in Microrganisms), and their complete content all is incorporated herein by reference.

The U.S. Provisional Patent Application that the application also relates on July 18th, 2005 and submits to number 60/700,557, the U.S. Provisional Patent Application of submitting on September 1st, 2005 number 60/713,905, title all is " genus bacillus MetI gene methionine(Met) is produced in improving microorganism in purposes " (Use of aBacillus MetI Gene to Improve Methionine Production inMicroorganisms), and their complete content all is incorporated herein by reference.

With the complete content of each these patent application, include, but are not limited to specification sheets, claims and summary, with and any figure, table or accompanying drawing all clearly be incorporated herein by reference.

Background

Methionine(Met) is the amino acid that is used for many different industry, and described industry includes but not limited to, animal-feed, medicine, foodstuff additive, makeup and food additives.Can the scale operation methionine(Met) by many diverse ways.For example, by at first thiomethyl alcohol and acrolein reaction being produced intermediate 3-methyl mercapto propionic aldehyde (MMP), produce methionine(Met) by chemical process.Other processing comprise that it uses escharotic such as NaOH and Na then with MMP and prussic acid reaction formation 5-(2-methylmercaptoethyl) glycolylurea ₂CO ₃, NH ₃And CO ₂Hydrolysis.Subsequently, with DL-methionine(Met) sodium sulfuric acid and Na ₂CO ₃Neutralization is to produce DL-methionine(Met), Na ₂SO ₄And CO ₂This method is compared with the amount of methionine(Met) and is produced excessive obsolete compound, and it causes economic and ecological challenge.

In addition, the fermentation of microorganism can also potentially be used for the scale operation methionine(Met), as by using nutrition, include but not limited to sugared source, as sugar, as starch, the nitrogenous source of glucose, fructose or sucrose, hydrolysis, for example, ammonia and sulphur source, as vitriol and/or thiosulphate, and other necessary or replenish nutrient media components culturing micro-organisms.This method uses nontoxicity danger, nonflammable, stable harmless starting material to produce the L-methionine(Met) and as the biomass of by product.

Yet, use the titre of the methionine(Met) that existing method produces and output all too low and commercial infeasible.Therefore, need to find improved methionine(Met) production method, it avoids toxigenicity chemical and deleterious by product, and commercial be important.

Reported few to three kinds or still less expression of gene and/or their encoded protein matter, can obtain some amino acid whose high-level output by changing.For example, simply by changing E.C. 2.7.2.4., pyruvate carboxylase and homoserine dehydrogenase, can make up the bacterial strain (Ohnishi, J.et al., Appl.Microbiol.Biotechnol.58 (2): 217-223 (2002)) that produces 80g/l Methionin.

Gene below having reported in the independent change bacterium or cause methionine(Met) to produce: metF (See, WO/087386A2, WO 04/024931A2 and U.S.'s publication No. 2002049305) with the expression of the combination of other genes; MetH (See, WO 04/024933A2 and U.S.'s publication No. 2002/0048793); MetA (seeing WO/024932 A2); MetK (WO 03/100072 A2); SahH (seeing EP1507008); MetY (seeing U.S.'s publication No. 20050064551); MetR and/or metZ (seeing U.S.'s publication No. 2002/0102664); MetE (U.S.'s publication No. 20020110877); MetD (seeing U.S.'s publication No. 20050074802), cysQ (seeing WO 02/42466A2); CysD, cysN, cysK, cysE and cysH (seeing WO 02/0086373); With metZ, metC and rxa 00657 (seeing WO 01/66573).Also reported and produced similar resistant strain, can cause producing methionine(Met) (Kumar and Gomes, Biotechnology advances 23:41-61 (2005)) as the ethionine bacterial strain that produces the amino acid bacterium.

Yet, because the methionine(Met) biosynthesizing relates to mixing of reductive sulphur atom and is considered to more complicated than other amino acid whose biosynthesizing, so which kind of combination of the use of not clear gene that changes and/or resistant strain will be that the horizontal methionine(Met) of generation commercially attractive is required.

General introduction

The invention describes new the improving one's methods that is used to increase methionine(Met) output.Particularly, the present invention to small part based on following discovery: for example, change the output that some gene has increased methionine(Met) by genetically engineered in microorganism such as the Corynebacterium glutamicum and classical genetics.

The invention still further relates to recombinant microorganism, its methionine(Met) with respect to their the corresponding microorganisms of wild-type produces the methionine(Met) of improving the standard, and relates to the method that produces this type of method of microorganism and use this type of microorganisms methionine(Met).In some embodiments, some combination of the gene that is changed causes the methionine(Met) output that increases, any titre of report before it is higher than basically, for example, 15g/l at least, or 16g/l at least, or 17g/l or higher at least.

In some embodiments, recombinant microorganism described herein comprises and is selected from ask ^Fbr, hom ^FbrMetX, metY, metB, metH, metE, metF and zwf any two or more, or three kinds or more kinds of, or four kinds or more kinds of, or five kinds or more kinds of, or six kinds or more kinds of, or seven kinds or more kinds of, or the hereditary change in each of eight kinds or more kinds of genes, wherein this hereditary change causes cross expressing of described gene, thus cause the methionine(Met) output of described microorganism with respect to described two or more, or three kinds or more kinds of, or four kinds or more kinds of, or five kinds or more kinds of, or six kinds or more kinds of, or seven kinds or more kinds of, or do not exist the methionine(Met) output of hereditary change to increase in each of eight kinds or more kinds of genes.In some embodiments, recombinant microorganism is being selected from ask ^Fbr, hom ^Fbr, metX, metY, metB, metH, metE, metF and zwf each of at least five kinds of genes in have hereditary change, wherein said hereditary change causes cross expressing of described at least five kinds of genes, thereby the methionine(Met) output that causes the methionine(Met) output of this microorganism and produce when not having described hereditary change in each of described at least five kinds of genes increases.This paper has also described and has been selected from ask ^Fbr, hom ^Fbr, metX, metY, metB, metH, metE, metF and zwf any six kinds of genes each, each or any eight kinds of genes of any seven kinds of genes each in comprise the recombinant microorganism of hereditary change, wherein said hereditary change causes cross expressing of described gene, thereby the methionine(Met) output that causes the methionine(Met) output of this microorganism when not having described hereditary change in each of each or any eight kinds of genes of each of described any six kinds of genes, any seven kinds of genes increases.Recombinant microorganism also comprises all nine kinds of gene ask ^Fbr, hom ^Fbr, the hereditary change among metX, metY, metB, metH, metE, metF and the zwf, wherein said hereditary change causes cross expressing of these nine kinds of genes, thereby causes the methionine(Met) output of this microorganism to increase with respect to the methionine(Met) output when there is not described hereditary change in these nine kinds of genes in each.

As described herein, can cross expression by accomplished in many ways, described method includes but not limited to, increase gene transcription/translation, this can introduce promotor and/or enhancer sequence by for example upstream at this gene, with promotor with increasing described expression of gene or causing the allogeneic promoter of the constitutive expression of this gene to substitute, increase the copy number of this gene, use plasmid episomal, perhaps by the modifying factor sequence, with arbitrary combination of these class methods, make the enzyme of this genes encoding have the active of increase or one or more are suppressed the inhibition resistance of the increase of compound with respect to wild type counterparts.In addition, for example, wished the transcription factor of the expression of gene of expression, also can realize expression by passing through the common inhibition of disappearance or sudden change.

In some embodiments, recombinant microorganism as herein described comprises the hereditary change of any two kinds of genes in each that is selected from mcbR, hsk, metQ, metK and pepCK, wherein this hereditary change has reduced the activity of proteins (for example enzymatic activity) of any two kinds of expression of gene and/or described any two kinds of genes encodings, thereby the methionine(Met) output that causes the methionine(Met) output of microorganism when not having described hereditary change in each of described any two kinds of genes increases.In other embodiments, the recombinant microorganism that the present invention includes comprises the hereditary change in each of any three kinds of genes of being selected from mcbR, hsk, metQ, metK and pepCK or any four kinds of genes or all five kinds of genes, wherein said hereditary change has reduced the activity of proteins of described expression of gene and/or described genes encoding, thereby the methionine(Met) output that causes the methionine(Met) output of microorganism when not having described hereditary change in each of described any three kinds of genes or any four kinds of genes or all five kinds of genes increases.As used herein, can realize the reduction of genetic expression by many diverse ways, described method include but not limited to suddenly change this gene promotor, the promotor of gene is replaced with the allogeneic promoter that reduces this genetic expression, perhaps make the corresponding microorganism of its wild-type of its encoding ratio have more SA protein or enzyme by the modifying factor sequence.In some cases, make to produce the protein or the enzyme of lower level by disappearance or mutator gene sequence or do not produce protein or enzyme is realized the reduction expressed.In addition,, increase the expression of gene transcription repressor, can realize the reduction of genetic expression by for example.

In some embodiments, the recombinant microorganism that the present invention includes comprises and is selected from ask ^Fbr, hom ^FbrMetX, metY, metB, metH, metE, any two kinds of genes of metF and zwf, or any three kinds of genes, or any five kinds of genes, or any six kinds of genes, or any seven kinds of genes, or any eight kinds of genes, or the hereditary change in each of all nine kinds of genes, wherein said any two kinds of genes, or any three kinds of genes, or any five kinds of genes, or any six kinds of genes, or any seven kinds of genes, or any eight kinds of genes, or crossing of each of all nine kinds of genes expressed, with be selected from mcbR, hsk, metQ, any one gene of metK and pepCK, perhaps any two kinds of genes, perhaps any three kinds of genes, perhaps any four kinds of genes, the perhaps combination of the hereditary change in each of five kinds of genes, wherein said legacy changes and reduces described any one gene, perhaps any two kinds of genes, perhaps any three kinds of genes, perhaps any four kinds of genes, the methionine(Met) output that perhaps five kinds of expression of gene, wherein said combination cause the methionine(Met) output of this microorganism when not having this combination increases.In some embodiments, recombinant microorganism is being selected from ask ^Fbr, hom ^Fbr, metX, metY, metB, metH, metE, metF and zwf each of at least five kinds of genes in comprise hereditary change, wherein said hereditary change causes crossing of each of described at least five kinds of genes to be expressed, combination with hereditary change at least a gene that is selected from mcbR, hsk, metQ, metK and pepCK, thereby cause the expression of the reduction of described at least a gene, the methionine(Met) that wherein said microorganism produces when not having this combination produces the methionine(Met) of increase level.

For example, in some embodiments, recombinant microorganism described herein comprises and is selected from ask ^Fbr, hom ^Fbr, metH and ask ^Fbr, hom ^FbrHereditary change in every kind of gene of metE, thereby cause crossing of described every kind of gene to be expressed, each the combination of hereditary change with mcbR and hsk, thereby cause the expression of the reduction of mcbR and hsk, wherein the methionine level of microorganisms increases with respect to the methionine(Met) that does not exist described when combination to produce.In other embodiments as herein described, recombinant microorganism comprises and is selected from ask ^Fbr, hom ^Fbr, metX (being also referred to as metA), metY (being also referred to as metZ), metF, metH, metE and ask ^Fbr, hom ^Fb, metX, metY, metF and metE each of at least six kinds of genes in hereditary change, thereby cause crossing of described at least six kinds of genes to be expressed, combined with mcbR and the hsk hereditary change in each, wherein the methionine(Met) that produces when not having described combination of the methionine level of microorganisms increases.

Recombinant microorganism as herein described also comprises the hereditary change that causes one or more gene overexpressions in the halfcystine biosynthetic pathway.For example, in some embodiments, recombinant microorganism described herein comprises and is selected from ask ^Fbr, hom ^FbrMetX (being also referred to as metA), metY (being also referred to as metZ), metB, metK, metQ, metH, metE, metF, metC, zwf, frpA1, asd, cysE, cysK, cysN, cysD, cysH, cysI, cysC, cysX, cysM, cysA, cysQ, cysG, cysZ, cysJ, cysY, hsk, mcbR, pyc, two or more of pepCK and ilvA, or three kinds or more kinds of, or four kinds or more kinds of, or five kinds or more kinds of, or six kinds or more kinds of, or seven kinds or more kinds of, or eight kinds or more kinds of, or nine kinds or more kinds of, or ten kinds or more kinds of, or ten one or more kinds, or 12 kinds or more kinds of, or 13 kinds or more kinds of, or 14 kinds or more kinds of, or 15 kinds or more kinds of, or 16 kinds or more kinds of, or 17 kinds or more kinds of, or 18 kinds or more kinds of, or 19 kinds or more kinds of, or 20 kinds or more kinds of, or 20 one or more kinds, or 22 kinds or more kinds of, or 23 kinds or more kinds of, or 24 kinds or more kinds of, or 25 kinds or more kinds of, or 26 kinds or more kinds of, or 27 kinds or more kinds of, or 28 kinds or more kinds of, or 29 kinds or more kinds of, or 30 kinds or more kinds of, or hentriaconta-kind or more kinds of, or 32 kinds or more kinds of, or 33 kinds or more kinds of, or the hereditary change in 34 kinds of genes, thereby the methionine(Met) output that the output that causes methionine(Met) produces when not having described hereditary change is compared and has been increased.

In some embodiments, recombinant microorganism as herein described comprises and is selected from ask ^Fbr, hom ^FbrMetX (being also referred to as metA), metY (being also referred to as metZ), metB, metH, metE, metF, metC, zwf, frpA, asd, cysE, cysK, cysN, cysA, cysD, cysH, cysI, cysC, cysX, cysG, cysM, cysZ, cysJ and pyc at least two kinds, or at least three kinds, or at least four kinds, or at least five kinds, or at least six kinds, or at least seven kinds, or at least eight kinds, or at least nine kinds, or at least ten kinds, or at least ten one kinds, at least ten two kinds, or at least ten three kinds, or at least ten four kinds, or at least ten five kinds, or at least ten six kinds, or at least ten seven kinds, or at least ten eight kinds, or at least ten nine kinds, or at least two ten kinds, or at least two ten one kinds, at least two ten two kinds, or at least two ten three kinds, or at least two ten four kinds, or at least two ten five kinds, or each hereditary change of 26 kinds of genes, wherein said at least two kinds, or at least three kinds, or at least four kinds, or at least five kinds, or at least six kinds, or at least seven kinds, or at least eight kinds, or at least nine kinds, or at least ten kinds, or at least ten one kinds, at least ten two kinds, or at least ten three kinds, or at least ten four kinds, or at least ten five kinds, or at least ten six kinds, or at least ten seven kinds, or at least ten eight kinds, or at least ten nine kinds, or at least two ten kinds, or at least two ten one kinds, at least two ten two kinds, or at least two ten three kinds, or at least two ten four kinds, or at least two ten five kinds, or 26 kinds of gene overexpressions, thereby the methionine(Met) output of the output that causes methionine(Met) when not having described hereditary change increases.For example, in some embodiments, recombinant microorganism comprises and is selected from ask ^Fbr, hom ^Fbr, metX (being also referred to as metA), metY (being also referred to as metZ), metB, metH, metE, metF, metC, zwf, frpA, asd, cysE, cysK, cysN, cysA, cysD, cysH, cysI, cysC, cysX, cysG, cysM, cysZ, cysJ and pyc each of at least eight kinds of genes in hereditary change, wherein said hereditary change causes cross expressing of described at least eight kinds of genes, thereby the methionine(Met) of the generation of the output that causes methionine(Met) when not having described hereditary change increases.

In some embodiments, recombinant microorganism comprises and is selected from ask ^Fbr, hom ^FbrMetX, metY, metB, metH, metE, hereditary change in each of at least five kinds of genes of metF and zwf (wherein said hereditary change causes each cross to express of described at least five kinds of genes) be selected from cysE, cysK, cysN, cysA, cysD, cysH, cysI, cysC, cysX, cysG, cysM, hereditary change in each of at least six kinds of genes of cysZ and cysJ (wherein said hereditary change causes cross expressing of described at least six kinds of genes) is combined, thereby the output that this combination causes the methionine(Met) output of microorganism when not having described combination increases.

In other embodiments, recombinant microorganism comprises the hereditary change in each of at least two kinds of genes being selected from metK, metQ, cysQ, cysY, hsk, mcbR, pepCK and ilvA, wherein said at least two kinds of expression of gene reduce, thereby the methionine(Met) output of the output that causes methionine(Met) when not having described hereditary change increases.

In some embodiments; recombinant microorganism comprises and is selected from following at least two kinds; or at least three kinds; or at least four kinds; or at least five kinds; or at least six kinds; or at least seven kinds; or at least eight kinds; or at least nine kinds; or at least ten kinds; or at least ten one kinds; at least ten two kinds; or at least ten three kinds; or at least ten four kinds; or at least ten five kinds; or at least ten six kinds; or at least ten seven kinds; or at least ten eight kinds; or at least ten nine kinds; or at least two ten kinds; or at least two ten one kinds; at least two ten two kinds; or at least two ten three kinds; or at least two ten four kinds; or at least two ten five kinds of proteinic imbalances: E.C. 2.7.2.4.; homoserine dehydrogenase; homoserine acetyltransferase; homoserine succinyltransferase; cystathionine gamma-synthase; cystathionine beta-lyase; O-ethanoyl homoserine sulfhydrylase; O-succinyl homoserine sulfhydrylase; vitamin B12 dependency methionine synthases; vitamin B12 is methionine synthases independently; N5; 10-methylene radical-tetrahydrofolate reductase; sulfate adenylyl transferase subunit 1; sulfate adenylyl transferase subunit 2; the APS kinases; the APS reductase enzyme; adenosine phosphate phosphinylidyne sulfate reduction enzyme; the NADP-ferredoxin reductase; sulfite reductase subunit 1; sulfite reductase subunit 2; the vitriol translocator; Serine O-Transacetylase; O-ethanoyl Serine (thiol)-lyase A; the uroporphyrinogen III synthase; glucose-6-phosphate dehydrogenase (G6PD); pyruvate carboxylase and aspartate-semialdehyde dehydrogenase; wherein said imbalance comprises proteinic cross to express, thereby causes under appropriate condition giving birth to methionine(Met) with the volume production of 8g/l at least.In some embodiments, recombinant microorganism comprises at least five kinds of proteinic imbalances as herein described, thereby causes under appropriate condition giving birth to methionine(Met) with the volume production of 8g/l at least.In other embodiments, recombinant microorganism comprises at least eight kinds of proteinic imbalances as herein described, thereby causes under appropriate condition giving birth to methionine(Met) with the volume production of 16g/l at least.Appropriate condition as described herein is to cause recombinant microorganism as herein described to increase the condition that produces methionine(Met).

In embodiments more as herein described, recombinant microorganism under appropriate condition with 8g/l at least or at least 9g/l at least 10g/l or at least 11g/l at least 12g/l or 13g/l or at least 14g/l at least 15g/l or at least the volume production of 16g/l give birth to methionine(Met).In some embodiments, recombinant microorganism is given birth to methionine(Met) with the volume production of 8g/l at least.In other embodiments, recombinant microorganism as herein described is given birth to methionine(Met) with the volume production of 16g/l at least.

In some embodiments, recombinant microorganism comprises and is selected from ask ^Fbr, hom ^Fbr, metX, metY, metB, metH, metE, metF and zwf each of at least five kinds of genes in hereditary change (wherein this hereditary change causes crossing of each of described at least five kinds of genes to be expressed), combined with the hereditary change of at least a gene that is selected from metK, metQ, hsk, mcbR and pepCK, wherein this combination causes microorganism methionine(Met) output of 8g/l at least under conditions suitable as described herein.

In an exemplary, the recombinant microorganism that the present invention includes comprises the hereditary change in each of eight kinds of genes being selected from ask, hom, metX, metY, metE, metH, metF and mcbR, and wherein the titre of the methionine(Met) that produces under conditions suitable of this microorganism is 16g/l at least.

In some embodiments, the constitutive expression of expressing the polypeptide that comprises gene or this genes encoding excessively of gene.

In some embodiments, recombinant microorganism described herein is the ethionine resistance.Therefore, the present invention also comprises ethionine resistance recombinant microorganism, it comprises one of many combinations of hereditary change as described herein, and wherein the combination of ethionine resistance and the described hereditary change methionine(Met) that causes methionine(Met) output and produce when not having this combination increases.In some embodiments, the ethionine resistant microorganism of the combination that comprises hereditary change as described herein produces 8g/l or 9g/l or 10g/l or 11g/l or 12g/l or 13g/l or 14g/l or 15g/l or 16g/l or 17g/l or 18g/l or the 19g/l or the methionine(Met) of 20g/l amount at least at least at least at least at least at least at least at least at least at least at least at least at least during the fermentation.

In embodiments more as herein described, recombinant microorganism comprises: (1) is selected from ask ^Fbr, hom ^Fbr, metX (being also referred to as metA), metY (being also referred to as metZ), metH, metF and ask ^Fbr, hom ^Fbr, metX (being also referred to as metA), metY (being also referred to as metZ), metH, metF and metE each of at least six kinds of genes in hereditary change, thereby cause crossing of each of described at least six kinds of genes to be expressed; (2) each hereditary change of mcbR and hsk, thus the expression of the reduction of mcbR and hsk caused; (3) combination of ethionine resistant mutation; Wherein this microorganism produces the methionine(Met) of 16g/l at least under appropriate condition.

The invention still further relates to genetically engineered method of microorganism, described microorganism is to increase or enhanced level generation methionine(Met).In some embodiments, the invention provides carrier, it can import and be used to produce the included multiple hereditary change of the present invention in the microorganism.This type of hereditary change can increase expression of gene or reduce expression of gene.In some embodiments, carrier is used for the upstream with promotor and/or enhanser quiding gene, thereby increases this expression of gene.

Recombinant microorganism as herein described can be a Gram-positive or gram-negative.In some embodiments, recombinant microorganism belongs to the genus that is selected from genus bacillus (Bacillus), excellent bacillus (Cornyebacterium), Bacterium lacticum (Lactobacillus), galactococcus (Lactococci) and streptomycete (Streptomyces).In some embodiments, recombinant microorganism as herein described belongs to Corynebacterium, for example, and Corynebacterium glutamicum strain.

In some embodiments, the method that produces methionine(Met) comprises cultivates coryneform bacterial strains under certain conditions, thereby produce methionine(Met) and reclaim methionine(Met), described bacterial strain comprises and is selected from ask, hom, metX, metY, metB, metC, metH, metE, metF, metK, ilvA, metQ, fprA, asd, cysD, cysN, cysC, pyc, cysH, cysI, cysY, cysX, cysZ, cysE, cysK, cysG, zwf, hsk, mcbR and pepCK at least two kinds, or at least three kinds, or at least four kinds, or at least five kinds, or at least six kinds, or at least seven kinds, or the hereditary change in each of at least eight kinds of genes.In some embodiments, this type of coryneform bacterial strains comprises the hereditary change at least eight kinds of genes.

In some embodiments, the method for cultivation recombinant microorganism as herein described (for example, the reorganization Corynebacterium glutamicum) amount that causes the methionine(Met) that produces rises culture for 16g/ at least.

In some embodiments, carrier comprises the integration box, and it is used for nucleotide sequence is incorporated into desirable genomic gene seat specific in the microorganism.In some embodiments, integrate box and modify native gene by in the native gene sequence, inserting heterologous nucleic acid sequence.This type of heterologous nucleic acid sequence can comprise, for example, expresses the nucleotide sequence of the enzyme in the methionine(Met) biosynthetic pathway.Heterologous gene can be from different biological gene, modified native gene, the perhaps native genes of having removed from different chromosome positions.

The accompanying drawing summary

Fig. 1 is the synoptic diagram that is used for the methionine(Met) biosynthetic pathway of microorganism described herein.

Fig. 2 is the synoptic diagram of pH273 carrier.

Fig. 3 is the synoptic diagram of pH373 carrier.

Fig. 4 is the synoptic diagram of pH304 carrier.

Fig. 5 is the synoptic diagram of pH399 carrier.

Fig. 6 is the synoptic diagram of pH484 carrier.

Fig. 7 is the synoptic diagram of pH491 carrier.

Fig. 8 is the synoptic diagram of plasmid pOM62.

Fig. 9 is the synoptic diagram of pH357 carrier.

Figure 10 is the synoptic diagram of pH410 carrier.

Figure 11 is the synoptic diagram of pH295 carrier.

Figure 12 is the synoptic diagram of pH429 carrier.

Figure 13 is the synoptic diagram of pH170 carrier.

Figure 14 is the synoptic diagram of pH447 carrier.

Figure 15 is the synoptic diagram of pH449 carrier.

Figure 16 is the synoptic diagram of plasmid pOM423.

Describe in detail

The present invention is at least part of based on following discovery: some hereditary changes in the microorganism cause the methionine output of methionine to increase. On the other hand, the present invention is based on following discovery: the combination of hereditary change is especially favourable for methionine production in some genes.

Exist two kinds of alternative route to be used for sulphur atom is added to the synthetic middle substrate of methionine production in microorganisms, as described in Figure 1. For example, the bacteria Escherichia coli utilization turns the sulphur approach; Yet some other microorganisms utilize direct sulfhydrylation approach such as saccharomyces cerevisiae (Saccharomyces cerevisiae) and Corynebacterium glutamicum. As if yet many microorganisms utilize one or another kind of approach, Corynebacterium glutamicum utilizes two kinds of approach to be used for methionine production.

The present invention is at least part of to produce useful hereditary change based on having identified for methionine in excellent bacillus, the particularly Corynebacterium glutamicum. In order to maximize methionine production; useful is the feedback inhibition that reduces some key enzyme in this approach; described enzyme such as aspartokinase (ask gene code), homoserine dehydrogenase (hom gene code), O-acetyl group homoserine sulfhydrylase (metY gene code), homoserine acetyltransferase (metX gene code), N5,10-MTHFR (metF gene code) and methionine synthases (gene metH and metE coding). For example, reported the inhibition that aspartokinase (such as Ask) from multiple biology is subject to lysine and/or threonine. For example, 311 amino acids change over isoleucine (T311L) from threonine and have reduced the feedback inhibition of Ask the Corynebacterium glutamicum (see U.S. Patent number 6,893,848, it completely openly is incorporated herein by reference). Similarly, homoserine dehydrogenase (Hom) can be suppressed by threonine, methionine, lysine and isoleucine, such as Sritharan V.Journal of General Microbiology, 136:203-209 (1990); Chassagnole C.et al.Biochemical Journal 356:415-23 (2001); Eikmanns B.J.et al.Antonie van Leeuwenhoek 64:145-63 (1993-94); With Cremer J.et al.Journal of General Microbiology 134 (12): 3221-3229 (1988)), the complete of them openly is incorporated herein by reference. In addition, 393 amino acids are changed into phenylalanine (S393F) from serine and have been reduced the feedback inhibition of Hom (being also referred to as Hsdh) the Corynebacterium glutamicum, such as Sugimoto M et al.Bioscience, Biotechnology ﹠ Biochemistry, described in the 61:1760-1762 (1997), it completely openly is incorporated herein by reference. In addition; enzyme O-acetyl group homoserine sulfhydrylase (MetY) is suppressed (WO 2004/108894 A2) by methionine, and methionine synthases (MetH) also is (Chen et al. J.Biol.Chem.269:27193-27197 (1994)) like this.

It is to increase some gene (such as ask, hom (being also referred to as hsd), metX (being also referred to as metA), metY (being also referred to as metZ), metB, metH, metE, metF, metC) in the methionine biosynthesis pathway that the present invention illustrates useful, and/or in the cysteine biosynthesis pathway some gene (such as cysJ, cysE, cysK, cysN, cysD, cysH, cysA, cysI, cysG, cysZ, cysX and cysM) expression (for example, transcribe and/or translate) so that the methionine that increases in the microorganism produces.

In addition, also useful is the expression that reduces or reduce some gene, in order to increase methionine output, the product of described gene reduces methionine and produces under some conditions, such as mcbR (being also referred to as RXA00655), such as Rey D.A., Journal of Biotechnology 103:51-65 (2003); With Rey D.A.et al., described in the Molecular Microbiology 56:871-887 (2005), it completely openly is incorporated herein by reference hsk, cysQ, cysY, ilvA, pepCK, metK and metQ. For example, sudden change hsk gene (it causes having amino acid position 190 is changed into alanine (T190A) from threonine enzyme), and/or sudden change metK gene (cause having 94 amino acids and change into the S-adenosylmethionine synthase of alanine (C94A) from cysteine) methionine output is especially useful the Corynebacterium glutamicum for increasing.

The present invention has also described microorganism, and it contains and is selected from following gene: ask^fbr、hom ^fbr, the hereditary change of every kind of gene in any two kinds combination of metX (being also referred to as metA), metY (being also referred to as metZ), metB, metH, metE, metF and zwf or any three kinds combination or any four kinds combination or any five kinds combination or any six kinds combination or any seven kinds combination or any eight kinds combination, wherein said hereditary change causes described any two kinds or any three kinds or any four kinds or any five kinds or any six kinds or crossing of any seven kinds or any eight kinds of genes to be expressed, thereby the methionine that the output that causes methionine produces when not having described hereditary change increases. The present invention has also described the microorganism of hereditary change in each that contains nine kinds of genes listed above, and it strengthens the expression of all nine kinds of above-cited genes, thereby increases methionine output.

In some embodiments, recombinant microorganism described herein contains and is selected from following gene: ask^fbr、 hom ^fbr, the hereditary change in metX, metY, metB, metH, metE, metF and zwf any two kinds or any three kinds or any four kinds or any five kinds or any six kinds or any seven kinds or any eight kinds or any nine kinds every kind, with the combination of hereditary change at least a gene that is selected from following gene: mcbR, hsk, metQ, metK and pepCK, thereby increase methionine output. Be appreciated that to strengthen or increase to express and comprise activity or the level of transcribing/translate or increase the protein/enzyme of this gene code that increases gene. Similarly, reduce to express and to comprise and subtract oligogenic transcribing/translate or the reduce activity/level of the protein/enzyme of this gene code.

In order to be more readily understood the present invention, at first define in this article some terms.

Term " microorganism of generation methionine " refers to produce any microorganism of methionine as used herein, for example, and bacterium, yeast, fungi, archeobacteria etc. In some embodiments, the microorganism of generation methionine belongs to Corynebacterium. In other embodiments, the microorganism of generation methionine is Corynebacterium glutamicum. In other embodiments, the microorganism that produces methionine is selected from: belong to Corynebacterium microorganism, belong to the microorganism that enterobacteria belongs to, the microorganism that belongs to bacillus, and yeast. In some embodiments, the microorganism that belongs to Corynebacterium is Corynebacterium glutamicum; The microorganism that belongs to the enterobacteria genus is Escherichia coli. In other embodiments, the microorganism that belongs to bacillus is bacillus subtilis. In other embodiments, yeast is saccharomyces cerevisiae.

Term " the increase level of methionine output " refers to as described herein comprise two or more as used herein, or three kinds or more kinds of, or four kinds or more kinds of, or five kinds or more kinds of, or six kinds or more kinds of, or seven kinds or more kinds of, or eight kinds or more kinds of, or nine kinds or more kinds of, or ten kinds or more kinds of, or ten is one or more of, or 12 kinds or more kinds of, or 13 kinds or more kinds of, or 14 kinds or more kinds of, or 15 kinds or more kinds of, or 16 kinds or more kinds of, or 17 kinds or more kinds of, or 18 kinds or more kinds of, or 19 kinds or more kinds of, or 20 kinds or more kinds of, or 20 is one or more of, or 22 kinds or more kinds of, or 23 kinds or more kinds of, or 24 kinds or more kinds of, or 25 kinds or more kinds of, or 26 kinds or more kinds of, or 27 kinds or more kinds of, or 28 kinds or more kinds of, or 29 kinds or more kinds of, or 30 kinds or more kinds of, or 30 is one or more of, or 32 kinds or more kinds of, or 33 kinds or more kinds of, or the titer of the methionine of the microorganisms of the hereditary change in 34 kinds of genes (for example, under suitable fermentation condition in g/l), wherein this titer is greater than the amount that produces under similar fermentation condition of contrast microorganism, and described contrast microorganism normally lacks the microorganism of this type of hereditary change. Term " the increase level of methionine " also refers to comprise the titer of the methionine that the recombinant microorganism of the protein of at least two kinds of imbalances as herein described produces. Term " the increase level of methionine output " comprises the median of the value that the value of included methionine and scope and/or this paper provide. The level of the increase of methionine output also is intended to comprise the titer that is higher than foundation level that produces, and described foundation level is set up by the microorganism that does not have the genetically engineered insensitive biosynthetic enzyme of methionine with expressing heterologous. In some embodiments, the level of the increase of methionine refer to respect to genetically engineered (for example, modify or change) amount of the methionine of the bacterial strain generation before this genetically engineered bacterial strain just during the wild type of microorganism or parent's homologue or the strain construction in such as embodiment, discussed, the titer of the methionine of genetically engineered Institute of Micro-biology generation.

Term " biosynthesis pathway " and " biosynthetic process " refer in the body or external process as used herein, by this process, because one or more biochemical reactions produce molecules of interest or compound. Usually, begin with precursor molecule, the prototype biosynthetic process relates to one or more enzymes and in a step-wise fashion makes to produce molecules of interest or compound. Molecules of interest or compound for example comprise, little organic molecule, amino acid, peptide, cell co-factor, vitamin and similar chemical entities. Molecules of interest or compound especially comprise chemicals such as methionine, homocysteine, S-adenosylmethionine, glutathione, cysteine, biotin, thiamine, mycothiol, coacetylase, coenzyme M and lipoic acid. In some cases, one or more enzymes of performance function can be subject to the adjusting of the chemical product that produces in this process in biosynthesis pathway. In this type of situation, there is feedback loop, so that increase function or activity that the concentration of end-product or intermediate product is modified this approach endoenzyme. For example, the end product of biosynthesis pathway or intermediate can be reduced level or the activity of enzyme in the biosynthetic process, thereby reduce the speed that desirable end-product produces. For example, in the large scale fermentation method of using in the industry for the production of molecules of interest or compound, this situation is normally undesirable. At least part of commercial scale and the fermentation yield that increases the purpose compound that relate to of method as herein described and material. The typical example of feedback loop occurs in methionine as herein described is produced.

Term " methionine biosynthesis pathway " reference and be used for the formation of methionine or synthetic methionine biosynthetic enzyme (for example, the polypeptide of biosynthesis enzyme coding gene coding), the biosynthesis pathway of compound (for example, precursor, substrate, intermediate or product), co-factor etc. Term " methionine biosynthesis pathway " comprises the biosynthesis pathway that causes methionine production in microorganisms synthetic (for example, in the body) and causes the synthetic biosynthesis pathway of external methionine. Fig. 1 has described the diagram of methionine biosynthesis pathway.

Term " methionine biosynthetic enzyme " refers to be used to form any enzyme of the compound (for example, intermediate or product) of methionine biosynthesis pathway as used herein. " methionine biosynthetic enzyme " comprises and for example relates to " turning the sulphur approach " and " directly sulfhydrylation approach "---the enzyme in the synthetic alternative route of methionine. For example, as discussed above, Escherichia coli utilize and turn the sulphur approach, and other microorganisms utilize direct sulfhydrylation approach such as saccharomyces cerevisiae, Corynebacterium glutamicum and bacillus subtilis and these biological close relatives. Although many microorganisms utilize and turn sulphur approach or direct sulfhydrylation approach, rather than both, some microorganisms utilize two kinds of approach to carry out methionine such as Corynebacterium glutamicum and synthesize.

As describing among Fig. 1, undertaken by intermediate aspartic acid, aspartic acid (aspartoyl) phosphoric acid and aspartic acid semialdehyde from the synthetic methionine of oxaloacetic acid (OAA). The aspartic acid semialdehyde is transformed into homoserine by homoserine dehydrogenase (product of hom gene is also referred to as thrA, metL, hdh, hsd etc. in other biological). Later step during methionine is synthetic can by turn the sulphur approach and/or directly the sulfhydrylation approach carry out.

In turning the sulphur approach; homoserine is by the homoserine acetyltransferase (product of metX gene; be also referred to as metA) and extra substrate acetyl coenzyme A be transformed into O-acetyl group homoserine, perhaps by using extra substrate succinyl CoA and the product (homoserine succinyltransferase) of metA gene to be transformed into OSHS. Product cystathionine gamma-synthase by the metB gene is supplied with O-acetyl group homoserine or OSHS generation cystathionie with methylthio group from cysteine. Then the product cystathionine beta-lyase of metC gene (being also referred to as the aecD gene in some microorganisms) is transformed into homoserine with cystathionie.

In direct sulfhydrylation approach, the product O-acetyl group homoserine sulfhydrylase catalysis sulfide of metY gene (being also referred to as the metZ gene) directly is added to O-acetyl group homoserine and forms homocysteine. Product OSHS sulfhydrylase by the metZ gene directly is added to OSHS with sulfide group, also can form homocysteine in the modification of direct sulfhydrylation approach. As used herein, metY and metZ are used interchangeably, and metA and metX are used interchangeably.

From the beginning turn sulphur/sulfhydrylation enzyme unlike only existing only in to have in the synthetic biology of methionine, methionine synthases is present in many other biologicals the regeneration with the methyl of guaranteeing S-adenosylmethionine (SAM). In Escherichia coli, two types methionine synthases can be brought into play this function, and described enzyme is Cobastab₁₂-dependence methionine synthases (product of metH gene) and Cobastab₁₂-methionine synthases (product of metE gene) independently. (methyl-THF) supply with has or does not have the polyglutamic acid tail to the methyl of methionine, and its reduction by methylene-THF in the reaction of metF gene outcome catalysis forms by methyl-tetrahydrofolic acid. The S-adenosylmethionine synthase of metK gene code is responsible for forming SAM from methionine and ATP.

In addition, cysteine can be as turning the sulphur donor in the methionine biosynthesis in the sulphur approach. In bacterium, by from the sulfide of thiosulfate or mixing of sulphur atom, synthesize cysteine from serine. The gene outcome of cysK gene (O-acetyl group serine (thiol)-lyase A or CysK) is from O-acetyl group serine and the synthetic cysteine of sulfide, and the gene outcome of cysM gene (O-acetyl group serine (thiol)-lyase B or CysM) is utilized thiosulfate rather than sulfide in cysteine is synthetic.

When the final source of sulphur is sulfate, need a series of enzymes that the sulfate reduction sulphidisation is used for cysteine and methionine biosynthesis. Usually, under the help such as the transport protein of the gene code of cysZ (sulfate transporter) or cysP, Cell uptake sulfate. Sulfate is produced adewosine monophosphate-sulfuric acid (being also referred to as APS) by the activation of the product of cysD (sulfate adenylyl transferase subunit 2) and cysN (sulfate adenylyl transferase subunit 1) gene. Be reported in some biologies, adewosine monophosphate-sulfate is had the protein activation of adewosine monophosphate-sulfuric acid kinase activity to produce AMP-phosphoric acid-sulfuric acid (being also referred to as PAPS) in further, it is reduced by the PAPS reductase of cysH gene code subsequently. Alternatively, APS can be produced sulphite by APS reductase direct-reduction.

Because in Corynebacterium glutamicum, also there is not to identify the gene with adenylic acid sulfuric acid kinase activity, so not clear adewosine monophosphate sulfuric acid or the AMP-phosphoric acid-sulfuric acid substrate of the enzyme of cysH gene code whether. The product of reduction step is sulphite, and it is by the active further reduction of the sulfite reductase of gene cysI (sulfite reductase subunit 1) and cysJ (sulfate reduction enzyme subunit 2) coding.

Cysteine biosynthesis precursor is usually from serine, and its activity by serine-transacetylase (gene cysE coding) is converted into O-acetyl group serine. O-acetyl group serine and sulfide are as the substrate of O-acetyl group serine (thiol) the lyase A of cysK gene code. For the situation of thiosulfate as the sulphur source; comprising that Escherichia coli and Styphimurium (for example see; Neidhardt FC ed.ASM Press Washington (1996)) described the second cysteine synthase in some biologies, described biological utilisation O-acetyl group-serine and thiosulfate produce sulfocysteine. The gene of described the second cysteine synthase of encoding is also referred to as cysM (O-acetyl group serine (mercaptan) lyase A), and it is also found in Corynebacterium glutamicum.

Table 1a has listed plurality of enzymes and their gene of correspondence of coding in the methionine biosynthesis pathway. Table 1b has listed plurality of enzymes and their gene of correspondence of coding in the cysteine biosynthesis pathway. Table 1c has listed directly or the biosynthetic additional protein of remote-effects methionine and enzyme, and corresponding gene. For purpose easily, each distributes respectively alphanumeric codes to gene described herein. Be appreciated that in some microorganisms the title of the gene of the corresponding enzyme of encoding can be different from the listed title of this paper.

Table 1a: the enzyme in the methionine biosynthesis pathway and their gene of coding

Enzyme	Gene	Alphanumeric codes
Enzyme	Gene	Alphanumeric codes	E.C. 2.7.2.4.	ask	A(+)
Homoserine dehydrogenase	hom	D(+)	E.C. 2.7.2.4.	ask	A(+)
Homoserine dehydrogenase	hom	D(+)	Homoserine acetyltransferase	metX	X(+)
Homoserine succinyltransferase (for example, in intestinal bacteria)	metA	S(+)	Homoserine acetyltransferase	metX	X(+)

Cystathionine gamma-synthase	metB	B(+)
Cystathionine gamma-synthase	metB	B(+)	Cystathionine beta-lyase	metC	C(+)
O-ethanoyl homoserine sulfhydrylase	metY	Y(+)	Cystathionine beta-lyase	metC	C(+)
O-ethanoyl homoserine sulfhydrylase	metY	Y(+)	O-succinyl homoserine sulfhydrylase (for example, in rhizobium (Rhizobium))	metZ	Z(+)
Vitamin B12-dependency methionine synthases	metH	H(+)		metZ	Z(+)
Vitamin B12-dependency methionine synthases	metH	H(+)	The methionine synthases of vitamin B12-independently	metE	E(+)
N5,10-methylene radical-tetrahydrofolate reductase	metF	F(+)	The methionine synthases of vitamin B12-independently	metE	E(+)
N5,10-methylene radical-tetrahydrofolate reductase	metF	F(+)	The S-adenosylmethionine synthase	metK	K(-)
The D-methionine(Met) is in conjunction with the subunit of lipoprotein or methionine(Met) shooting system	metQ	Q(-)	The S-adenosylmethionine synthase	metK	K(-)

(+): refer to gene overexpression, it is that the output of increase of methionine(Met) is desirable

(-): refer to reduce or reduce and express or active gene, it is that the output of increase of methionine(Met) is desirable

Table 1b: enzyme in the halfcystine biosynthetic pathway and their gene of coding

Enzyme	Gene	Alphanumeric codes
Enzyme	Gene	Alphanumeric codes	Sulfate adenylyl transferase subunit 2	cysD	cD(+)
Gamma cystathionase	cysA	cA(+)	Sulfate adenylyl transferase subunit 2	cysD	cD(+)
Gamma cystathionase	cysA	cA(+)	Sulfate adenylyl transferase subunit 1	cysN	cN(+)
APS kinases (for example, in intestinal bacteria)	cysC	Cc(+)	Sulfate adenylyl transferase subunit 1	cysN	cN(+)
APS kinases (for example, in intestinal bacteria)	cysC	Cc(+)	APS reductase enzyme (for example, in Corynebacterium glutamicum), PAPS reductase enzyme (for example, in intestinal bacteria)	cysH	cH(+)
Sulfite reductase subunit 1	cysI	cI(+)		cysH	cH(+)
Sulfite reductase subunit 1	cysI	cI(+)	Sulfite reductase subunit 2 (in the intestinal bacteria)	cysJ	cJ(+)
CBS (oppositely approach)	cysY	cY(-)	Sulfite reductase subunit 2 (in the intestinal bacteria)	cysJ	cJ(+)
CBS (oppositely approach)	cysY	cY(-)	Attached effect sulphite reduction	cysX	cX(+)
The vitriol translocator	cysZ	cZ(+)	Attached effect sulphite reduction	cysX	cX(+)
The vitriol translocator	cysZ	cZ(+)	Serine O-Transacetylase	cysE	cE(+)
O-ethanoyl Serine (mercaptan)-lyase A	cysK	cK(+)	Serine O-Transacetylase	cysE	cE(+)
O-ethanoyl Serine (mercaptan)-lyase A	cysK	cK(+)	O-ethanoyl Serine (mercaptan)-lyase A (for example, exists	cysM	cM(+)

Intestinal bacteria are medium)
Intestinal bacteria are medium)			The uroporphyrinogen III synthase	cysG	cG(+)
APS Phosphoric acid esterase (for example, in intestinal bacteria)	cysQ	cQ(-)	The uroporphyrinogen III synthase	cysG	cG(+)

Table 1c: can change to increase the extra gene of methionine(Met) output

Enzyme/protein	Gene	Alphanumeric codes
Enzyme/protein	Gene	Alphanumeric codes	Glucose-6-phosphate dehydrogenase (G6PD)	zwf	W(+)
Homoserine kinase	hsk	V(-)	Glucose-6-phosphate dehydrogenase (G6PD)	zwf	W(+)
Homoserine kinase	hsk	V(-)	The metabolic TetR-type of sulphur transcriptional regulatory	mcbR	R(-)
Phosphoenolpyruvate carboxykinase	pepCK	P(-)		mcbR	R(-)
Phosphoenolpyruvate carboxykinase	pepCK	P(-)	Pyruvate carboxylase	pyc	Py(+)
The NADP-ferredoxin reductase	fprA	Fp(+)	Pyruvate carboxylase	pyc	Py(+)
The NADP-ferredoxin reductase	fprA	Fp(+)	Aspartate-semialdehyde dehydrogenase	asd	As(+)
Threonine dehydra(ta)se, biosynthetic	ilvA	Iv(-)	Aspartate-semialdehyde dehydrogenase	asd	As(+)
Threonine dehydra(ta)se, biosynthetic	ilvA	Iv(-)	Threonine dehydra(ta)se, catabolic	Cgl 0978， tdh	T(-)

Can change exemplary group with the gene that increases methionine(Met) output is combined in the Table II and describes.Yet, be appreciated that arbitrary combination that can change gene, as long as this combination causes enhanced methionine(Met) output.

Table II. the example combinations of the gene of change

A，D，X，Y，B

A，D，X，E，W

A，D，X，Y，H	A，X，Y，B，H
A，D，X，Y，H	A，X，Y，B，H	A，D，X，Y，E	A，X，Y，B，E
A，D，X，Y，F	A，X，Y，B，F	A，D，X，Y，E	A，X，Y，B，E
A，D，X，Y，F	A，X，Y，B，F	A，D，X，Y，W	A，X，Y，B，W
A，D，X，B，H	A，X，Y，H，E	A，D，X，Y，W	A，X，Y，B，W
A，D，X，B，H	A，X，Y，H，E	A，D，X，B，E	A，X，Y，H，F
A，D，X，B，F	A，X，Y，H，W	A，D，X，B，E	A，X，Y，H，F
A，D，X，B，F	A，X，Y，H，W	A，D，X，B，W	A，X，Y，E，F，
A，D，X，H，E	A，X，Y，E，W	A，D，X，B，W	A，X，Y，E，F，
A，D，X，H，E	A，X，Y，E，W	A，D，X，H，F	A，D，X，E，F
A，D，X，H，W		A，D，X，H，F	A，D，X，E，F

The recombinant microorganism that the present invention includes can be genetically engineered it causes the increase of methionine(Met) output to comprise the change of native gene, for example, change its increase or reduce some expression of gene by introducing in the gene.Alternatively, can import enzyme/protein that the native gene in this quasi-microorganism is encoded with expression by the genetic manipulation recombinant microorganism.In some embodiments, can recombinant microorganism is genetically engineered to change the expression of some enzyme/protein combination, wherein this type of combination causes the methionine(Met) output of the methionine(Met) output increase when not having this combination.For example, the expression by changing native gene and/or import heterologous gene in microorganism can realize the expression of suitable enzyme/proteinic combination.

Following Table III comprises from the Genbank searching number of the isolating several genes of Corynebacterium glutamicum and their encoded protein matter, wherein can change the multiple combination of gene, thereby cause enhanced methionine(Met) output.

Table III: the Genbank searching number that relates to the biosynthetic multiple Corynebacterium glutamicum gene of methionine(Met) and their encoded protein matter

The gene title	The gene searching number	The protein searching number
The gene title	The gene searching number	The protein searching number	MetK	Cgl1603	BAB98996.1
Hom	Cgl1183	BAB98576.1	MetK	Cgl1603	BAB98996.1
Hom	Cgl1183	BAB98576.1	hsk/thrA	Cgl1184	BAB98577.1
metY/Z	Cgl0653	BAB98046.1	hsk/thrA	Cgl1184	BAB98577.1
metY/Z	Cgl0653	BAB98046.1	metA/X	Cgl0652	BAB98045.1
MetH	Cgl1507	BAB98900.1	metA/X	Cgl0652	BAB98045.1
MetH	Cgl1507	BAB98900.1	MetE	Cgl1139	BAB98532.1
MetF	Cgl2171	BAB99564.1	MetE	Cgl1139	BAB98532.1
MetF	Cgl2171	BAB99564.1	MetC	Cgl2309	BAB99702.1
MetB	Cgl2446	BAB99839.1	MetC	Cgl2309	BAB99702.1
MetB	Cgl2446	BAB99839.1	ask/lysC	Cgl0251	BAB97644.1
Asd	Cgl0252	BAB97645.1	ask/lysC	Cgl0251	BAB97644.1
Asd	Cgl0252	BAB97645.1	Zwf	Cgl1576	BAB98969.1
PepCK	Cgl1585	BAB98978.1	Zwf	Cgl1576	BAB98969.1
PepCK	Cgl1585	BAB98978.1	CysE	Cgl2563	BAB99956.1
CysH (coding PAPS or APS reductase enzyme)	Cgl2816	BAC00210.1	CysE	Cgl2563	BAB99956.1
CysH (coding PAPS or APS reductase enzyme)	Cgl2816	BAC00210.1	The gene of coding sulfite reductase	Cgl2817	BAC00211.1
cysJ/fprA	Cgl2818	BAC00212.1	The gene of coding sulfite reductase	Cgl2817	BAC00211.1
cysJ/fprA	Cgl2818	BAC00212.1	CysN, coding vitriol adenylic acid (AMP) transferring enzyme subunit 1	Cgl2814	BAC00208.1
CysD, coding vitriol adenylic acid (AMP) transferring enzyme subunit 2	Cgl2815	BAC00209.1		Cgl2814	BAC00208.1
	Cgl2815	BAC00209.1	The gene of coding vitriol permease	Cgl1473	BAB98866.1
The gene of coding vitriol permease	Cgl1051	BAB98444.1	The gene of coding vitriol permease	Cgl1473	BAB98866.1
The gene of coding vitriol permease	Cgl1051	BAB98444.1	The proteinic gene of coding vitriol movement system permease	Cgl0870	BAB98263.1
The gene of coding vitriol permease	Cgl2812	BAC00206.1		Cgl0870	BAB98263.1
The gene of coding vitriol permease	Cgl2812	BAC00206.1	The gene of coding vitriol permease	Cgl2813	BAC00207.1

CysG	Cgl1998	BAB99391.1
CysG	Cgl1998	BAB99391.1	CysK	Cgl2562	BAB99955.1
CysM	Cgl2136	BAB99529.1	CysK	Cgl2562	BAB99955.1
CysM	Cgl2136	BAB99529.1	The gene of coding pyruvate carboxylase	Cgl0689	BAB98082.1

In some embodiments, the microorganism of the generation methionine(Met) that comprises of the present invention contains and is selected from ask ^FbrHom ^FbrMetX; MetY; MetB; MetH; MetE; MetF; With the hereditary change in each of any two kinds of genes of zwf or any three kinds of genes or any four kinds of genes or any five kinds of genes.The present invention has also described the microorganism that contains hereditary change, and described hereditary change comprises and is selected from ask ^Fbr, hom ^Fbr, metX, metY, metB, metH, metE, metF and zwf each of any six kinds of genes in hereditary change.In addition, the invention describes to contain and be selected from ask ^Fbr, hom ^Fbr, any seven kinds of genes of metX, metY, metB, metH, metE, metF and zwf or any eight kinds of genes or nine kinds of genes each in the microorganism of hereditary change.

The number of the possible combination of the several genes that can change can for example calculate based on following equation:

\frac{n!}{(n - r)! Xr!}

Wherein n is the gene number that can change, and r is the number gene that changes in microorganism.Therefore, be selected from ask ^Fbr, hom ^Fbr, metX, metY, metB, metH, metE, metF and zwf the number that may make up of any two kinds of genes that can change can followingly calculate:

\frac{9!}{(9 - 2)! X 2!} = 36 .

Similarly, be selected from ask ^Fbr, hom ^Fbr, metX, metY, metB, metH, metE, metF and zwf the number that may make up of any five kinds of genes that can change can followingly calculate:

\frac{9!}{(9 - 5)! X 5!} = 126 .

Therefore, the formula based on top is selected from ask ^Fbr, hom ^Fbr, metX, metY, metB, metH, metE, metF and any five kinds of genes that can change of zwf or the possible combined number of any six kinds of genes or any seven kinds of genes or any eight kinds of genes or any nine kinds of genes be respectively 126,84,36,9 and 1.

Similarly, any number that may make up that changes gene as described herein can easily be determined based on top formula.

Term " insensitive to methionine(Met) feedback " refers in the presence of methionine(Met) and can bring into play enzymatic functions and than living when not having methionine(Met) active at least 20% enzyme with conspicuous level as used herein.Methionine(Met) is fed back insensitive enzyme can in the presence of for example 1-10 μ M, 10-100 μ M or 100 μ M-1mM methionine(Met)s, bring into play function well.In some embodiments, the purpose endonuclease capable 1-10mM, 10-100mM methionine(Met) concentration or even greater concn under bring into play function.And in their native state, some methionine(Met) biosynthetic enzymes are to the feedback inhibition sensitivity of other amino acid such as Threonine and Methionin.The present invention describes methionine(Met), Methionin and/or Threonine to small part and feeds back insensitive enzyme, and they relate to methionine(Met) biosynthetic pathway or process, and described approach or process cause producing methionine(Met), and described enzyme is for example Ask ^FbrAnd Hom ^Fbr

In some embodiments, microorganism described herein belongs to Corynebacterium.In other embodiments, microorganism is a Corynebacterium glutamicum.In other embodiments, microorganism is selected from: gram negative bacterium (for example, intestinal bacteria or relevant enterobacteria), gram positive bacterium (for example, subtilis or relevant genus bacillus), yeast are (for example, yeast saccharomyces cerevisiae or relevant yeast strain), and archeobacteria.

In some embodiments, at least two of microorganism as herein described kinds or at least three kinds or at least four kinds or at least five kinds of methionine(Met) biosynthetic enzyme imbalances.In other embodiments, at least six of microorganism as herein described kinds of methionine(Met) biosynthetic enzyme imbalances.In some embodiments, at least seven of microorganism as herein described kinds or the imbalance of more methionine(Met) biosynthetic enzymes.Term used herein " imbalance " refers to respect to the level of the corresponding enzyme of the parent of biosynthetic enzyme or wild-type and/or than living, the level of described biosynthetic enzyme and/or actively raise or reduce or do not exist fully.In some embodiments, " imbalance " biosynthetic enzyme is by the genes encoding through changing as described herein.For example, " imbalance " biosynthetic enzyme can for example by changing the native gene of the described enzyme of coding, perhaps produce by importing heterologous gene to the microorganism that produces this enzyme.

In other embodiments, two or more or three kinds at least or more kinds of or four kinds or more kinds of or five kinds or more kinds of or six kinds or the imbalance of more kinds of enzyme of microorganism as herein described from the halfcystine biosynthetic pathway.In other embodiments, microorganism as herein described lacks of proper care from two or more enzymes of methionine(Met) biosynthetic pathway with from two or more enzymes of halfcystine biosynthetic pathway.For example, in some embodiments, recombinant microorganism comprises from the enzyme of five kinds of the methionine(Met) biosynthetic pathway or more kinds of imbalances with from six kinds or the enzyme of more kinds of imbalances of halfcystine biosynthetic pathway.In addition, the enzyme/protein that directly or indirectly influences methionine(Met) biosynthetic pathway and/or halfcystine biosynthetic pathway also can be lacked of proper care, for example, level and/or active the reduction, thus methionine(Met) output increased.For example, in some embodiments, recombinant microorganism comprises the hereditary change at least two kinds of genes, wherein this type of change causes at least two kinds of proteinic imbalances, described protein is selected from: the APS Phosphoric acid esterase of described genes encoding, CBS (oppositely approach), homoserine kinase, the metabolic TetR type of sulphur transcriptional regulatory, D-methionine(Met) are in conjunction with lipoprotein, phosphoenolpyruvate carboxykinase, S-adenosylmethionine synthase, and threonine dehydra(ta)se.

In some embodiments, the invention describes the new and improved method of the microorganisms methionine(Met) that uses hereditary change, in described microorganism, operated the methionine(Met) that the methionine(Met) biosynthetic pathway makes this microorganism to produce when not having described hereditary change and compared the methionine(Met) that produces elevated levels.

New and improved method as herein described comprise the method with the microorganisms methionine(Met), described microorganism comprises at least two kinds or at least three kinds or at least four kinds or at least five kinds or at least six kinds or at least seven kinds or at least eight kinds or more kinds of enzyme of being lacked of proper care of methionine(Met) biosynthetic pathway, thereby, produce methionine(Met) with the level that raises with respect to the microorganism that does not have this type of imbalance.For example; in some embodiments; microorganism as herein described comprises the hereditary change in five kinds or the more kinds of gene; it causes five kinds or the imbalance of more kinds of enzymes of described genes encoding; wherein said enzyme is selected from: E.C. 2.7.2.4.; homoserine dehydrogenase; homoserine acetyltransferase; cystathionine Gamma-synthetic enzyme; O-ethanoyl homoserine sulfhydrylase; O-succinyl homoserine sulfhydrylase; vitamin B12 dependency methionine synthases; N5; 10-methylene radical-tetrahydrofolate reductase; the S-adenosylmethionine synthase; cystathionine beta-lyase; homoserine succinyltransferase and vitamin B12 be methionine synthases independently.

The method of increase methionine(Met) output as herein described also comprises the method for microorganism that has hereditary change in the gene that is created in the halfcystine biosynthetic pathway, thereby with respect to not existing described hereditary change to produce methionine(Met) with the level that raises.

For example; in some embodiments; microorganism as herein described comprises two or more; or three kinds or more kinds of; or four kinds or more kinds of; or five kinds or more kinds of; or six kinds or more kinds of; or the hereditary change in seven kinds or the more kinds of gene; it causes the imbalance of the enzyme of described genes encoding, and wherein said enzyme is selected from: sulfate adenylyl transferase subunit 2; sulfate adenylyl transferase subunit 1; the cystathionine beta synthetic enzyme; the APS kinases; the APS reductase enzyme; the PAPS reductase enzyme; sulfite reductase subunit 1; sulfite reductase subunit 2; the sulphite reduction of booster action; the vitriol translocator; Serine O-Transacetylase; O-ethanoyl Serine (thiol) lyase A; the uroporphyrinogen III synthase; APS Phosphoric acid esterase and gamma cystathionase.In some embodiments, recombinant microorganism comprises the enzyme of six kinds of imbalances in the halfcystine biosynthetic pathway.

Method as herein described has been described as described herein and/or to cause increasing the mode cultured microorganism of methionine(Met) output, for example, and recombinant microorganism, and carrier and gene (for example, the gene of wild-type and/or sudden change).

Term " recombinant microorganism " refers to that microorganism (for example, bacterium, yeast cell, fungal cell or the like), it (has for example used genetic technique hereditary change in for example external DNA operative technique or the typical body, modification or through engineering approaches, genetically engineered), make and compare with the naturally occurring microorganism that it is originated, demonstrate change, modify or different genotype and/or phenotypes (for example, when this genetic modification influences the nucleic acid sequence encoding of microorganism).

" recombinant microorganism " as herein described can be selected from ask to comprise by genetically engineered, hom, metX, metB, metC, metY, metH, metE, metF, cysE, cysK, cysM, cysD, cysA, cysN, cysH, cysI, cysJ, cysX, cysZ, cysC, cysG, zwf, pyc, fprA and asd at least two kinds, or at least three kinds, or at least four kinds, or at least five kinds, or at least six kinds, or at least seven kinds, or at least eight kinds, or at least nine kinds, or at least ten kinds, or at least ten one kinds, at least ten two kinds, or at least ten three kinds, or at least ten four kinds, or at least ten five kinds, or at least ten six kinds, or at least ten seven kinds, or at least ten eight kinds, or at least ten nine kinds, or at least two ten kinds, or at least two ten one kinds, at least two ten two kinds, or at least two ten three kinds, or at least two ten four kinds, or at least two ten five kinds, or the hereditary change in whole 26 kinds of genes, wherein said hereditary change causes crossing of described gene to be expressed.In some embodiments, " recombinant microorganism " as herein described can be through genetically engineered and be selected from least two kinds of genes of metK, metQ, cysY, cysQ, hsk, mcbR, pepCK and ilvA or at least three kinds of genes or at least four kinds of genes or at least five kinds of genes or at least six kinds of genes or at least seven kinds of genes or at least eight kinds of genes and comprise hereditary change, and wherein said hereditary change causes the reduction of described genetic expression.In other embodiments, " recombinant microorganism " comprises the hereditary change in some genes, and it increases the hereditary change in those expression of gene and other genes, and it reduces this type of expression of gene, thus the methionine(Met) output that causes recombinant microorganism to increase.

The technician will understand the microorganism of expressing with respect to the increase that does not have gene with the microorganism of the horizontal expression gene that raises and produce the gained gene product with elevated levels and/or activity.Similarly, comprise that the microorganism of the reduction expression of gene produces the gene product of gained with lower level and/or activity with respect to the microorganism of the reduction expression that does not have described gene.

Term " recombinant microorganism " also refers to such microorganism as used herein, it by through engineering approaches (for example, genetically engineered) or modify and to make that at least two kinds of enzymes of methionine(Met) biosynthetic pathway of this microorganism and/or at least two kinds of enzymes of halfcystine biosynthetic pathway are lacked of proper care, make to produce methionine(Met) with the level that raises.In some embodiments, recombinant microorganism comprises that at least five kinds of enzymes of methionine(Met) biosynthetic pathway and at least six kinds of enzymes of halfcystine biosynthetic pathway are lacked of proper care, and makes to produce methionine(Met) with the level that raises.Can realize the modification or the through engineering approaches of this quasi-microorganism according to any method as herein described or known in the art, described method includes but not limited to, the change of the gene of encoding human route of synthesis enzyme.

Term " through what lack of proper care " or " through what operate " as reference enzyme or protein use are used interchangeably at this paper, and refer to enzyme or protein, its activity or level are changed or modify, make and to compare that the level or the speed of at least a upstream by enzyme or the flux of downstream precursor or intermediate, substrate or product are changed or modify with corresponding wild-type or naturally occurring enzyme or protein." through what operate " enzyme (for example, " through what operate " biosynthetic enzyme) comprises enzyme, its expression, output or activity are changed or modify, make and for example compare with corresponding wild-type or naturally occurring enzyme, at least a upstream or downstream precursor, substrate or the product of this enzyme is changed or modifies (for example, the level change of precursor, substrate and/or product or that modify, ratio etc.)." through operation " enzyme also comprises such enzyme, wherein to the inhibition of one or more products or intermediate, strengthens as the resistance of feedback inhibition.For example, can in the presence of methionine(Met) for example, bring into play the enzyme of enzymatic functions effectively.

Term " is expressed " excessively, " expressing excessively " refers to that gene product (for example, methionine(Met) biosynthetic enzyme or sulfate reduction path enzyme or halfcystine biosynthetic enzyme) expression, its expression level greater than the hereditary change of this microorganism before or the level that in also not having the suitable microorganism of hereditary change, exists.In some embodiments, can hereditary change (for example, genetically engineered) microorganism with the horizontal expression gene product of level to raise with respect to unaltered microorganism or the gene product that in the suitable microorganism that does not change, produces.Hereditary change comprises, but be not limited to, adjusting sequence that change or modification are relevant with concrete expression of gene or site are (for example, by adding strong promoter, inducible promoter or a plurality of promotor, perhaps make that by removing the adjusting sequence expressing is composing type), modify the chromosome position of concrete gene, change and the concrete adjacent nucleotide sequence of gene, as ribosome bind site or transcription terminator, increase the copy number of concrete gene, the protein of modification concrete gene transcription of participation and/or concrete gene product translation (for example, regulate protein, suppress son, enhanser, transcriptional activator or the like), perhaps any other conventional means of the conventional concrete genetic expression of imbalance (includes but not limited in this area, use antisense nucleic acid molecule, for example, the expression of blocking-up repressor) and/or use and to increase change allelotrope, for example, the bacterium allelotrope that strengthens hereditary variability and quicken for example adaptive evolution).

In some embodiments, can change microorganism with the expressing gene product on the physiology or on the environment, its level is with respect to unaltered microorganism or the also not expression level of the gene product of the suitable microorganism of change increase or lower.For example, can with known or suspect to increase the mass treatment microorganism of concrete genetic transcription and/or concrete gene translation or in the presence of described material culturing micro-organisms, make to strengthen or increase is transcribed and/or translated.Alternatively, can be under the temperature of the translation of selecting to be used for to increase concrete gene transcription and/or concrete gene product culturing micro-organisms, make to strengthen or increase and transcribe and/or translate.

Term " imbalance ", " imbalance " refer to the change or the modification of at least a gene in the microorganism, and wherein this change or modification cause increasing methionine(Met) output in the described microorganism with respect to the methionine(Met) output that does not have described change or modification.In some embodiments, the enzyme in the genes encoding biosynthetic pathway that is changed or modifies makes the level of biosynthetic enzyme described in this microorganism or activity be changed or modify.In some embodiments, at least a gene of enzyme is changed or modifies in the encoding human route of synthesis, makes the level of this enzyme or activity strengthen or increase with respect to the level under unaltered or the wild type gene existence.In other embodiments, at least two kinds of enzyme or at least three kinds or at least four kinds or at least five kinds of genes are changed or modify in the encoding human route of synthesis, make the level of enzyme of described genes encoding or active level under existing with respect to unaltered or wild type gene reduce or reduce.In some embodiments, described biosynthetic pathway is the methionine(Met) biosynthetic pathway.In other embodiments, described biosynthetic pathway is the halfcystine biosynthetic pathway.Imbalance also comprises the coding region that changes one or more genes to produce, for example, the feedback resistance or have higher or lower than enzyme alive.And imbalance comprises that also (for example, activator, the repressor hereditary change of) gene, described transcription factor is adjusted in expression of gene in methionine(Met) and/or the halfcystine biosynthetic pathway to the encoding transcription factor.

Term " approach of imbalance " refers to biosynthetic pathway, and wherein at least a gene of enzyme is changed or modifies and makes the level of described at least a biosynthetic enzyme or activity be changed or modify in the encoding human route of synthesis.Term " approach of imbalance " comprises biosynthetic pathway, and wherein more than one gene is changed or modifies, thereby changes the level and/or the activity of corresponding gene product/enzyme.In some cases, approach (for example in " imbalance " microorganism, with more than one gene in the given biosynthetic pathway of lacking of proper care simultaneously) cause by concrete phenomenon in the microorganism, in the described microorganism, more than one enzyme (for example, two or three biosynthetic enzyme) is by mutual adjacent genes encoding on the continuous fragment of the genetic material that is called " operon ".In other cases, for the imbalance approach, the several genes of in the through engineering approaches step of a series of orders, lacking of proper care.

Term " operon " refers to the co-ordination unit of genetic material, its contain promotor and possibly with one or more, the preferred regulatory element that combines of at least two kinds of structure genes (for example, codase is as the gene of biosynthetic enzyme).Adjustment structure expression of gene synergistically, for example, by adjusting albumen or anti-termination by transcribing in conjunction with regulatory element.Structure gene can be transcribed and the single mRNA of all structural protein that obtain encoding.Term " operon " comprises at least two adjacent genes or ORF, choose wantonly at least one gene or ORF 5 " or 3 ends overlapping on sequence.Term " operon " comprises the co-ordination unit of genetic expression, its contain promotor and possibly with one or more adjacent genes or the relevant regulatory element of ORF (for example, codase is as the gene of biosynthetic enzyme).The expression of regulatory gene synergistically, for example, by regulating in conjunction with the adjusting albumen of regulatory element or by the anti-termination of transcribing.The gene of operon (for example, structure gene) can be transcribed and the single mRNA of all proteins that obtains encoding.Because the collaborative adjusting of the gene that comprises in the operon, the change of single promotor and/or regulatory element or adjusting can cause the change or the modification of every kind of gene product of operon coding.The change or the modification of regulatory element comprise, but be not limited to, remove endogenesis promoter and/or regulatory element, add strong promoter, inducible promoter or a plurality of promotor or remove the adjusting sequence, make that the expression of gene product is modified, modify the chromosome position of operon, change the nucleotide sequence in or the operon adjacent with operon, for example, ribosome bind site, codon is selected, and increases the copy number of operon, modify relate to that operon is transcribed and/or the protein of operon gene product translation (for example, regulate albumen, suppress son, enhanser, transcription activating, or the like), perhaps any other conventional means of the imbalance genetic expression of this area routine (includes but not limited to, uses antisense nucleic acid molecule, for example, the expression of blocking-up repressor).

In some embodiments, recombinant microorganism as herein described is by genetically engineered gene or gene product with the expression bacterial origin.Term " bacterial origin " and " from bacterium " refer to the gene of natural discovery in bacterium or the gene product of encoding with bacterial gene.

In some embodiments, recombinant microorganism as herein described comprises and is selected from ask, hom, metX, metY, metB, metH, metE, metF, zwf, metC, fprA, cysE, cysK, cysM, cysD, cysH, cysA, cysN, cysI, cysJ, cysX, cysZ, cysC, cysG, the combination of any two kinds of genes of pyc and asd, the combination of perhaps any three kinds of genes, the combination of perhaps any four kinds of genes, the combination of perhaps any five kinds of genes, the combination of perhaps any six kinds of genes, the combination of perhaps any seven kinds of genes, the combination of perhaps any eight kinds of genes, the combination of perhaps any nine kinds of genes, the perhaps combination of any ten kinds of genes, the combination of perhaps any 11 kinds of genes, the combination of any 12 kinds of genes, the combination of perhaps any 13 kinds of genes, the combination of perhaps any 14 kinds of genes, the combination of perhaps any 15 kinds of genes, the combination of perhaps any 16 kinds of genes, the combination of perhaps any 17 kinds of genes, the combination of perhaps any 18 kinds of genes, the combination of perhaps any 19 kinds of genes, the combination of perhaps any 20 kinds of genes, the combination of perhaps any 21 kinds of genes, the combination of any 22 kinds of genes, the combination of perhaps any 23 kinds of genes, the combination of perhaps any 24 kinds of genes, the combination of perhaps any 25 kinds of genes, the hereditary change of every kind of gene in the combination of perhaps any 26 kinds of genes, wherein said hereditary change cause the expression excessively of gene in the described combination.In other embodiments, microorganism as herein described comprises and is selected from ask ^Fbr, hom ^Fbr, metX, metY, metB, metH, metE, metF and zwf the combination of any two kinds or any three kinds or any four kinds or any five kinds or any six kinds or any seven kinds or any eight kinds or all nine kinds of genes in hereditary change, wherein said hereditary change causes crossing of described gene to be expressed.For example, in some embodiments, microorganism as herein described comprises and is selected from ask ^Fbr, hom ^Fbr, metX, metY, metB, metH, metE, metF and zwf the combination of any five kinds of genes in hereditary change, wherein said hereditary change causes crossing of described any five kinds of genes to be expressed or constitutive expression.The microorganism that the present invention includes also is included in and is selected from ask ^Fbr, hom ^Fbr, metX, metY, metB, metH, metE, metF and any six kinds of genes of zwf or the microorganism of the hereditary change in any seven kinds of genes or any eight kinds of genes or any nine kinds of genes, wherein said hereditary change causes crossing of described any six kinds of genes or any seven kinds of genes or any eight kinds of genes or any nine kinds of genes to be expressed.Microorganism as herein described also is included in the hereditary change in two or more genes of being selected from mcbR, hsk, pepCK, metK and metQ or its combination, and wherein said hereditary change causes the reduction of genetic expression.The expression that reduces comprise reduce genes encoding gene product (for example, mRNA and/or protein) expression and/or the activity that reduces it (for example, the proteinic enzymatic activity of genes encoding) through changing, perhaps this gene of disappearance/sudden change, thus do not produce gene product.In some embodiments, microorganism includes and is beneficial to two or more genes (for example, ask that methionine(Met) is produced ^Fbr, hom ^Fbr, metX, metY, metB, metH, metE, metF and zwf) cross and express and the reduction of one or more genetic expressions, disappearance and/or reduce the expression that helps methionine(Met) production (for example, mcbR, hsk, pepCK, metK and metQ).

Term " gene " comprises that nucleic acid molecule (for example as used herein, dna molecular or its section), another gene in itself and the biology or other genes are by intergenic DNA (promptly, interleave or spacer DNA, it is natural be arranged in gene described in the biology chromosomal DNA flank and/or with described gene isolation) with another gene or other gene isolation.Alternatively, gene can be overlapping slightly with another gene (for example, with 3 ' end of first kind of gene of the terminal eclipsed of second kind of gene 5 '), and overlapping genes are by intergenic DNA and other gene isolation.Gene is synthetic enzyme or another kind of protein molecule (for example, it can comprise encoding sequence, for example, the successive open reading-frame (ORF) (ORF) of coded protein) or can be from function be arranged in biology directly.Gene in the biology can cluster in operon, and as defined herein, operon separates with other genes and/or operon by intergenic DNA." isolating gene " comprises such gene as used herein, its sequence that does not have natural this gene flank of karyomit(e) that is arranged in the biology that this gene originates basically (promptly, do not encode adjacent encoder sequence, adjacent structure sequence of second kind or different proteins or the like), and optional 5 ' and 3 ' the adjusting sequence that comprises, for example, promoter sequence and/or terminator sequence.In other embodiments, isolating gene mainly comprises the proteinic encoding sequence sequence of excellent thuringiensis protein (for example, encode).In other embodiments, isolating gene (for example comprises proteinic encoding sequence, the excellent thuringiensis protein of encoding) and the chromosomal DNA of this gene biology of originating adjacent 5 ' and/or 3 ' regulate sequence (for example, adjacent 5 ' and 3 ' the excellent bacillus adjusting sequence).In some embodiments, isolating gene contains and is less than about 10kb, 5kb, 2kb, 1kb, 0.5kb, 0.2kb, 0.1kb, 50bp, 25bp, 10bp or the nucleotide sequence of bp still less, and this sequence is the natural flank that is positioned at described gene in the chromosomal DNA of the biology that this gene is originated.

The term " gene of change ", " hereditary change ", " gene with change " and " mutator gene " that are used interchangeably as this paper refer to have and (for example comprise at least a modification, substitute, insert, disappearance) the gene of nucleotide sequence, thereby the polypeptide of modified genes encoding or protein demonstrate polypeptide or the different activity of protein with wild-type nucleic acid molecule or genes encoding.In some embodiments, gene or mutator gene coded polypeptide or protein with change, for example, when (for example under similar condition, measuring or measure, under the inhibition compound of uniform temp and/or same concentrations, measure in the cultured microorganism) time, described polypeptide or protein are compared the level with rising or the activity of increase with wild type gene encoded polypeptides or protein.In other embodiments, gene or mutator gene coded polypeptide or protein with change, when measuring under simulated condition or measuring, described polypeptide or protein are compared the activity with lower level or reduction with wild type gene encoded polypeptides or protein.In some embodiments, have the gene of change or mutator gene can not encode its wild type counterparts encoded protein matter or polypeptide.Term " gene of change ", " mutator gene ", " gene with change " and " hereditary change " comprise that also the modification or the adjusting sequence of the adjusting sequence of gene substitute with heterologous sequence, described heterologous sequence includes but not limited to promotor and/or enhanser, and it causes increase, minimizing or the disappearance of genetic expression.

Term " activity of increase " and " enzymatic activity of increase " refer to than the polypeptide of wild-type nucleic acid molecule or genes encoding or activity of proteins big 5% or 5-10% or 10-25% or 25-50% or 50-75% or the activity of 75-100% at least at least at least at least at least at least as used herein.Above-mentioned value intermediary scope also is intended to be included in herein as 75-85%, 85-90%, 90-95%." activity of increase " and " enzymatic activity of increase " also comprises the activity than wild type gene encoded polypeptides or activity of proteins big at least 1.25 times or at least 1.5 times or at least 2 times or at least 3 times or at least 4 times or at least 5 times or at least 10 times or at least 20 times or at least 50 times or at least 100 times as used herein.

Can be according to the arbitrary known assay method mensuration activity that is used to measure the specific purposes protein active.Can directly measure or measure activity, for example, by measuring cell crude extract or from the activity of proteins of cell or microorganism isolated or purified.Alternatively, can measure or measure activity in cell or microorganism or the extracellular substratum.For example,, for example, express the gene of sudden change or change in the thermally sensitive mutant microbial of for example described enzyme, and measure the ability of the enzymatic activity of complementary temperature sensitivity (Ts) mutant of this mutator gene, finish the mensuration of mutant by in microorganism.Coding " enzymatic activity of increase " the sudden change or the gene of change can be the more gene of effective supplement Ts mutant of ratio such as corresponding wild-type gene.Coding " reduce enzymatic activity " the sudden change or the gene of change be not as for example gene of corresponding wild-type effective supplement Ts mutant.

Do not wish to be bound by theory, the technician will understand single change in nucleic acid or the gene order (for example, the base of the amino acid change in the coding corresponding amino acid sequence substitutes), and comparing with corresponding wild-type polypeptide or protein can remarkably influenced encoded polypeptide and activity of proteins.The sudden change as defined herein or the gene of change are (for example, the polypeptide of encoding mutant or imbalance or protein) easily with the nucleic acid or the gene differentiation of coded protein, because sudden change or the genes encoding that changes have level or the active protein or the polypeptide of change, choose wantonly expressing mutator gene or produce in the microorganism (i.e. sudden change or recombinant microorganism) of mutein or polypeptide to compare and to be viewed as different or different phenotype with the corresponding microorganism of expressing wild type gene.Compare, mutator gene encoded protein matter can have identical or similar basically activity, when producing in microorganism, compares with the corresponding microorganism of expressing wild type gene, chooses wantonly to be difficult to distinguish on phenotype.Therefore, for example, not that only the sequence identity degree between nucleic acid molecule, gene, protein or the polypeptide is used to distinguish homologue and mutant, but the level of coded protein or polypeptide or active homologue and the mutant distinguished: have for example low (for example, 30-50% sequence identity) sequence identity and have the homologue of the functionally active that is equal to basically, for example, have 99% sequence identity and have the mutant of the functionally active of remarkable difference or change.

In some embodiments, gene or mutator gene coding with sudden change are compared with wild type gene encoded polypeptides or protein, has the active polypeptide or the protein that reduce or increase, as when under simulated condition, measuring, (for example, in the presence of the inhibitor of uniform temp or same concentrations, measuring in the cultured microorganism).Mutator gene is coded polypeptide or have the wild type peptide output of reduction level not.

Term " activity that reduces " and " enzymatic activity that reduces " refer to than the polypeptide of wild-type nucleic acid molecule or genes encoding or activity of proteins little 5% or 5-10% or 10-25% or 25-50% or 50-75% or the activity of 75-100% at least at least at least at least at least at least as used herein.Top fiducial value intermediary scope also is intended to be included in herein as 75-85%, 85-90%, 90-95%.As used herein, " activity that reduces " or " enzymatic activity that reduces " can also comprise and having lacked or the activity (for example, polypeptide or the activity of proteins than wild-type nucleic acid molecule or genes encoding is approximately little by 100%) of " knocking out ".

In some embodiments; recombinant microorganism described herein comprises and is selected from least two kinds of following protein; or at least three kinds of protein; or at least four kinds of protein; or at least five kinds of protein; or at least six kinds of protein; or at least seven kinds of protein; or at least eight kinds of protein; or at least nine kinds of protein; or at least ten kinds of protein; or at least ten one kinds of protein; at least ten two kinds of protein; or at least ten three kinds of protein; or at least ten four kinds of protein; or at least ten five kinds of protein; or at least ten six kinds of protein; or at least ten seven kinds of protein; or at least ten eight kinds of protein; or at least ten nine kinds of protein; or at least two ten kinds of protein; or at least two ten one kinds of protein; at least two ten two kinds of protein; or at least two ten three kinds of protein; or at least two ten four kinds of protein; or at least two ten five kinds of protein; or at least two ten five kinds of protein; or at least two ten six kinds of protein; or at least two ten seven kinds of protein; or at least two ten eight kinds of protein; or at least two ten nine kinds of protein; or at least three ten kinds of protein; or hentriaconta-kind protein at least; at least three ten two kinds of protein; or at least three ten three kinds of protein; or at least three ten four kinds of proteinic imbalances: E.C. 2.7.2.4.; homoserine dehydrogenase; homoserine acetyltransferase; O-succinyl homoserine sulfhydrylase; cystathionine gamma-synthase; cystathionine beta-lyase; O-ethanoyl homoserine sulfhydrylase; vitamin B12 dependency methionine synthases; vitamin B12 is methionine synthases independently; N5,10-methylene radical-tetrahydrofolate reductase; the S-adenosylmethionine synthase; methionine(Met) output albumen; the NADP-ferredoxin reductase; aspartate-semialdehyde dehydrogenase; the cystathionine beta synthetic enzyme; sulfite reductase (subunit 1 or 2 or both); serine acetyltransferase; O-ethanoyl Serine (thiol)-lyase A; sulfate adenylyl transferase (subunit 1 or 2 or both); adenosine phosphate phosphinylidyne sulfate reduction enzyme; gamma cystathionase; the APS kinases; the APS reductase enzyme; glucose-6-phosphate dehydrogenase (G6PD); pyruvate carboxylase; homoserine kinase; the uroporphyrinogen III synthase; the APS Phosphoric acid esterase; the sulfuric acid translocator; the reduction of booster action sulphite; threonate dehydrogenase; the metabolic TetR type of sulphur transcriptional regulatory and phosphoenolpyruvate carboxykinase.

In some embodiments; recombinant microorganism as herein described comprises two or more; or three kinds or more kinds of; or four kinds or more kinds of; or five kinds or more kinds of; or six kinds or more kinds of; or seven kinds or more kinds of; or eight kinds or more kinds of; or nine kinds or more kinds of; or ten kinds or more kinds of; or ten one or more kinds; or 12 kinds or more kinds of; or 13 kinds or more kinds of; or 14 kinds or more kinds of; or 15 kinds or more kinds of; or 16 kinds or more kinds of; or 17 kinds or more kinds of; or 18 kinds or more kinds of; or 19 kinds or more kinds of; or 20 kinds or more kinds of; or 20 one or more kinds; or 22 kinds or more kinds of; or 23 kinds or more kinds of; or 24 kinds or more kinds of; or 25 kinds or more kinds of; or 26 kinds or more kinds of; or the protein of 27 kinds or more kinds of imbalances; it is selected from E.C. 2.7.2.4.; homoserine dehydrogenase; homoserine acetyltransferase; O-succinyl homoserine sulfhydrylase; homoserine succinyltransferase; cystathionine gamma-synthase; cystathionine beta-lyase; O-ethanoyl homoserine sulfhydrylase; vitamin B12 dependency methionine synthases; vitamin B12 is methionine synthases independently; N5; 10-methylene radical-tetrahydrofolate reductase; the NADP-ferredoxin reductase; aspartate-semialdehyde dehydrogenase; sulfite reductase (subunit 1 or 2 or both); Serine O-Transacetylase; O-ethanoyl Serine (thiol)-lyase A; sulfate adenylyl transferase (subunit 1 or 2 or both); the APS kinases; the APS reductase enzyme; adenosine phosphate phosphinylidyne sulfate reduction enzyme; gamma cystathionase; glucose-6-phosphate dehydrogenase (G6PD); the uroporphyrinogen III synthase; the sulfuric acid translocator; the reduction of booster action sulphite; and pyruvate carboxylase; wherein Shi Tiao protein is with respect to comprising described proteinic wild type counterparts or not expressing expression or activity in the described proteinic microorganism, with higher level and/or bigger activity expression.

In some embodiments, recombinant microorganism as herein described comprises the protein of two or more imbalances, it is selected from methionine(Met) output albumen, S-adenosylmethionine synthase, cystathionine beta synthetic enzyme, APS Phosphoric acid esterase, homoserine kinase, the metabolic TetR type of sulphur transcriptional regulatory, phosphoenolpyruvate carboxykinase and threonine dehydra(ta)se, and the protein of wherein said two or more imbalances is with respect to the expression in the microorganism that comprises described proteinic wild type counterparts or active in lower level and/or the activity expression that reduces.

The protein that is appreciated that imbalance can have greater activity with respect to wild-type protein at the horizontal expression that is higher than the wild-type protein level and/or it.Alternatively, it can have level lower or that reduce at the horizontal expression that is lower than the wild-type level and/or with respect to wild-type protein.In some cases, the protein of imbalance be constitutive expression and in some cases, its enzymatic activity is not expressed or lost to the protein of imbalance.In some embodiments, the protein of being lacked of proper care is the enzyme in the methionine(Met) biosynthetic pathway.In other embodiments, the protein of being lacked of proper care is the enzyme in the halfcystine biosynthetic pathway.In other embodiments, the protein of being lacked of proper care is that gene transcription suppresses son or activates son in methionine(Met) biosynthetic pathway and/or the halfcystine biosynthetic pathway.In some cases, imbalance protein makes that it is to feed back resistance.The protein of imbalance is usually by genetic expression hereditary change in the microorganism or that modify.

Recombinant microorganism as herein described comprises the microorganism of genetic modification by this way or change, described mode makes them compare with the protein level that produces in the microorganism that does not have genetic modification or change, with higher or lower horizontal expression two or more, or three kinds or more kinds of, or four kinds or more kinds of, or five kinds or more kinds of, or six kinds or more kinds of, or seven kinds or more kinds of, or eight kinds or more kinds of, or nine kinds or more kinds of, or ten kinds or more kinds of, or ten one or more kinds, or 12 kinds or more kinds of, or 13 kinds or more kinds of, or 14 kinds or more kinds of, or 15 kinds or more kinds of, or 16 kinds or more kinds of, or 17 kinds or more kinds of, or 18 kinds or more kinds of, or 19 kinds or more kinds of, or 20 kinds or more kinds of, or 20 one or more kinds, or 22 kinds or more kinds of, or 23 kinds or more kinds of, or 24 kinds or more kinds of, or 25 kinds or more kinds of, or 26 kinds or more kinds of, or 27 kinds or more kinds of, or 28 kinds or more kinds of, or 29 kinds or more kinds of, or 30 kinds or more kinds of, or hentriaconta-kind or more kinds of, or 32 kinds or more kinds of, or 33 kinds or more kinds of, or 34 kinds or more kinds of protein.For example, in some embodiments, recombinant microorganism produces five kinds or more kinds of protein with activity (for example, enzymatic activity), and described activity is higher than or is lower than activity of proteins in the microorganism that does not also have genetic modification or change.

In some embodiments, recombinant microorganism as herein described comprises, for example, the combination of modified gene, wherein the summation of the methionine level that produces when having every kind of independent gene alteration in this combination of the level of the methionine(Met) that is produced (, the change of the assortment of genes to methionine(Met) production have add up or synergy).For example, the microorganism that the present invention includes comprises the microorganisms that comprise two or more genes through changing, wherein the methionine level sum that produces when having every kind of gene that changes separately of the level of the methionine(Met) that is produced.Therefore, for example, can change two or more or three kinds or more kinds of or five kinds or more kinds of or six kinds or more kinds of or seven kinds or more kinds of or eight kinds or more kinds of or nine kinds or more kinds of or ten kinds or more kinds of gene synergism for arbitrary multiple measurement of several genes as herein described.In some embodiments, the microorganisms methionine(Met) that comprises the assortment of genes through changing, its level are for example than having every kind of gene that changes separately or not having the methionine level sum that produces when changing 1-2% or 3-5% or 5-10% or 10-20% or 20-30% or 30-40% or 40-50% or 50-60% or 60-70% or 70-80% or 80-90% or 90-95% at least at least at least at least at least at least at least at least at least at least at least greatly at least.

In some embodiments, the level of methionine(Met) that comprises the microorganisms of the assortment of genes through changing is at least 2 times or at least 2.5 times or at least 3 times or at least 3.5 times or at least 4 times or at least 4.5 times or at least 5 times or at least 10 times or at least 15 times or at least 20 times or at least 25 times or at least 30 times or at least 35 times or at least 40 times or at least 45 times or at least 50 times or at least 100 times that have every kind of gene that changes separately or do not have the methionine level sum that produces when changing.

In other embodiments, the amount that comprises the methionine(Met) that the microorganism of the assortment of genes through changing produces under suitable fermentation condition is with respect to having every kind of gene that changes separately or not having every liter of the amount sum 5g greatly at least of methionine(Met) of the microorganisms of gene alteration, or 7g at least, or 8g at least, or 9g at least, or 10g at least, or 11g at least, or 12g at least, or 13g at least, or 14g at least, or 15g at least, or 16g at least, or 17g at least, or 18g at least, or 19g at least, or 20g at least, or 25g at least, or 30g at least, or 40g at least, or 50g at least.

The methionine level of microorganisms as herein described can use one or more assay methods as herein described easily to measure.

In some embodiments, " recombinant microorganism " that the present invention includes has the halfcystine biosynthetic pathway of imbalance.Term " microorganism with halfcystine biosynthetic pathway of imbalance " is included at least two kinds, or at least three kinds, or at least four kinds, or at least five kinds, or at least six kinds, or at least seven kinds, or at least eight kinds, or at least nine kinds, or at least ten kinds, or at least ten one kinds, at least ten two kinds, or have in the gene of at least ten three kinds of encoding aminothiopropionic acid biosynthetic pathway enzymes and change or modify or in the operon of the gene of the enzyme that comprises the encoding aminothiopropionic acid biosynthetic pathway, have the microorganism that changes or modify.In some embodiments, the microorganism of the halfcystine biosynthetic pathway with imbalance as herein described, is made that described gene is crossed to be expressed to have hereditary change in being selected from least two kinds of genes of cysJ, cysA, cysE, cysK, cysM, cysD, cysI, cysN, cysG, cysC, cysX, cysZ and cysH by genetically engineered.In some embodiments, the microorganism hereditary through engineering approaches of halfcystine biosynthetic pathway that will have imbalance to be comprising hereditary change in cysQ and/or cysY, thereby reduces one or both expression of gene.In other embodiments, the recombinant microorganism with halfcystine biosynthetic pathway of imbalance comprises the combination of hereditary change at least two kinds of being selected from cysJ, cysA, cysE, cysK, cysM, cysD, cysI, cysN, cysG, cysC, cysY, cysX, cysZ, cysH and cysQ or at least three kinds or at least four kinds or at least five kinds or at least six kinds of genes.

Other features of this paper are mutant microbials.As used herein, term " mutant microbial " comprises genetically engineered to express gene or proteinic recombinant microorganism sudden change or that change, and described gene or protein are common or natural in described microbial expression.For example, in some embodiments, gene or protein that mutant microbial is expressed sudden change make this microorganism demonstrate change, that modify or different phenotypes.In other embodiments, mutant microbial be changed or through engineering approaches make gene by the disappearance (that is, not producing the protein of this genes encoding).

In some embodiments, recombinant microorganism as herein described is Gram-positive biology (for example, owing to existing the gram-positive cell wall around microorganism to keep basic dyestuff, for example, the microorganism of Viola crystallina).In other embodiments, recombinant microorganism is to belong to the microorganism that is selected from bacillus, Corynebacterium, lactobacillus, lactococcus and streptococcus.In other embodiments, recombinant microorganism belongs to Corynebacterium, and in some embodiments, recombinant microorganism is selected from Corynebacterium glutamicum.

In some embodiments, recombinant microorganism is Gram-negative (exclusion basic dyestuff) biology.In other embodiments, recombinant microorganism is to belong to the microorganism that is selected from salmonella (Salmonella), Escherichia (Escherichia), Klebsiella (Klebsiella), Serratia (Serratia) and proteus (Proteus).In other embodiments, recombinant microorganism is a yeast, as be selected from yeast belong (Saccharomyces), genus kluyveromyces (Kluyveromyces), Pichia (Pichia), mycocandida (Candida), Schizosaccharomyces (Schizocaccharomyces), or the like the yeast or the archeobacteria of (for example, yeast saccharomyces cerevisiae).

An importance that the present invention includes is included in cultivates recombinant microorganism as herein described under the conditions suitable, make to produce methionine(Met).Term " cultivation " comprises the microorganism (for example, maintenance and/or grown culture or bacterial strain) of the maintenance and/or the work as herein described of growing.In some embodiments, culturing micro-organisms in the liquid medium within.In other embodiments, culturing micro-organisms in solid medium or semisolid medium.In other embodiments, at substratum (for example, aseptic liquid nutrient medium) culturing micro-organisms in, described substratum comprises keeping and/or (for example grow essential or useful nutrition for microorganism, carbon source or carbon substrate, for example complicated sugar is as soybean or cereal powder, starch, sugar, sugar alcohol, hydrocarbon, oil, fat, lipid acid, organic acid and alcohol; Nitrogenous source, for example, from vegetable-protein, peptone, peptide and the amino acid of cereal, beans and stem tuber, protein, peptide and amino acid, as meat, milk and animal byproducts, as peptone, meat extract and caseic hydrolysate from animal-origin; Inorganic nitrogen-sourced, as urea, ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate; The phosphorus source, for example, phosphoric acid, its sodium and sylvite; Trace element, for example, magnesium, iron, manganese, calcium, copper, zinc, boron, nickel, molybdenum and/or cobalt salt; And somatomedin, as amino acid, VITAMIN, growth stimulant or the like).

In some cases, under controlled pH, cultivate microorganism as herein described.Term " controlled pH " comprises any pH that causes producing methionine(Met).In some embodiments, culturing micro-organisms under about 7 pH.In other embodiments, culturing micro-organisms under 6.0 to 8.5 pH.Can keep desirable pH by any method known to those skilled in the art.

And, in some cases, under controlled ventilation, cultivate microorganism as herein described.Term " controlled ventilation " comprises the enough ventilations (for example, oxygen) that cause producing methionine(Met).In some embodiments, by regulating the oxygen level in the culture, for example, be dissolved in the amount control ventilation of oxygen in the substratum by adjusting.For example, control the ventilation of culture by the stir culture thing.By water screw or similar mechanical stirring equipment, by rotating or shaking growth container (for example, fermentor tank) or provide stirring by the moving equipment of multiple pump.Can also pass sterile air or oxygen control ventilation via substratum (for example, via fermenting mixture).And, there be not excess foam (for example, by adding defoamer) to have culturing micro-organisms down.

In addition, microorganism as herein described can be cultivated under controlled temperature.Term " controlled temperature " comprises any temperature that causes producing methionine(Met).In some embodiments, controlled temperature is set to specified temp, for example 15 ℃ to 95 ℃, 15 ℃ to 70 ℃, 20 ℃ to 55 ℃, 30 ℃ to 45 ℃ or 30 ℃ to 50 ℃ or 28 ℃ to 37 ℃.

Microorganism can (for example be cultivated in the liquid medium within, keep and/or growth), and continuously preferred or intermittently cultivation, cultivate as leaving standstill cultivation, test tube cultivation, wave and culture (for example, rotation wave and culture, shake-flask culture or the like), ventilation turn cultivation or fermentation by conventional cultural method.In some embodiments, culturing micro-organisms in shaking bottle.In other embodiments, culturing micro-organisms in fermentor tank (for example, fermentation process).Fermentation process includes, but not limited in batches, fed-batch and continuous ferment process.Phrase " batch processes " or " batch fermentation " refer to closed system, and wherein the composition of substratum, nutrition, supplemented by additives or the like is provided with when the fermentation beginning and is not during fermentation changed; Yet, can attempt to control the factor such as pH and oxygen concentration to prevent excessive substratum acidifying and/or microbial death.Phrase " fed-batch process " or " fed-batch " fermentation refer to batch fermentation, just along with the carrying out of fermentation, add one or more substrates or fill-in (for example, add or add continuously with increment).Phrase " successive processes " or " continuously fermenting " refer to such system, wherein the fermention medium of determining is joined continuously fermentor tank and equivalent is used or " condition " substratum remove simultaneously, for example be used to reclaim purpose product (for example, methionine(Met)).Multiple these class methods have been developed and they are well known in the art.

Microorganism as herein described can continuously or be distributed cultivation or be cultivated to produce methionine(Met) with fed-batch or repetition feed supplement apportioning method.Known cultural method summary can be seen Chmiel (Bioprozelitechnik 1.Einfiihrung in die Bioverfahrenstechnik (GustavFischer Verlag, Stuttgart, 1991)) or Storhas (Bioreaktoren und periphereEinrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994) textbook).The substratum that uses must be satisfied the needs of specific bacterial strain with suitable manner.The description of the substratum of multiple microorganism is included in U.S. bacteriology association " the Manual of Methods forGeneral Bacteriology " handbook (Washington D.C., USA, 1981).

Term " under the condition that produces purpose compound (for example methionine(Met)), cultivate " and " appropriate condition " refer to be fit to or enough obtain the generation of purpose compound or obtain particular compound that will generation purpose output condition (for example, temperature, pressure, pH, time length, etc.) keep down and/or the growth microorganism.For example, the time that culturing micro-organisms enough produces the methionine(Met) of desired amount under appropriate condition.In some embodiments, the culturing micro-organisms time enough is to reach the maximum production of methionine(Met) basically.In some embodiments, culturing micro-organisms is about 12 to 24 hours.In other embodiments, about 24 to 36 hours of culturing micro-organisms, about 36 to 48 hours, about 48 to 72 hours, about 72 to 96 hours, about 96 to 120 hours, about 120 to 144 hours, perhaps greater than time length of 144 hours.In other embodiments, cultivate the time of the output of the hope continue enough to reach methionine(Met), for example, culturing micro-organisms makes and produces at least about 7 to 10g/l, or at least 10 arrive 15g/l, or at least about 15 to 20g/l, or at least about 20 to 25g/l, or at least about 25 to 30g/l, or at least about 30 to 35g/l, or at least about 35 to 40g/l, or at least about 40 to the 50g/l methionine(Met).The amount of the methionine(Met) that the recombinant microorganism that the present invention includes in some embodiments, produces is 16g/l at least.In other embodiments, the amount of the methionine(Met) that produces under suitable fermentation condition of the recombinant microorganism of this paper is 17g/l at least.In other embodiments, under certain condition culturing micro-organisms make in about 24 hours, in about 36 hours, in about 48 hours, in about 72 hours or produce the preferred output of methionine(Met) in about 96 hours, for example, the output in the scope that provides above.

Method as herein described can also comprise the step that reclaims purpose compound (for example, methionine(Met)).Term " recovery " purpose compound (for example, methionine(Met)) refers to from substratum extraction, results, the described compound of isolated or purified.Can reclaim compound according to any conventional isolated or purified method known in the art, described method includes but not limited to, centrifugal, the evaporation, with conventional resin (for example, negatively charged ion or Zeo-karb, non-ionic adsorption resin or the like) handle, (for example use conventional sorbent material, gac, silicic acid, silica gel, Mierocrystalline cellulose, aluminum oxide or the like) handle, change pH, solvent extraction (for example, with conventional solvent as alcohol, ethyl acetate, hexane or the like), dialysis, filtration, concentrated, crystallization, recrystallization, pH regulator, freeze-drying or the like.For example, can reclaim methionine(Met) from substratum by at first reclaiming microorganism from culture.

In some embodiments, " extraction ", " separation " or " purifying " methionine(Met) make its essentially no other component (for example, not having nutrient media components and/or fermentation byproduct).Term " essentially no other components " refers to prepare the purpose compound, for example, methionine(Met), wherein methionine(Met) separates with the nutrient media components or the fermentation byproduct of the culture that produces it.In some embodiments, prepared product greater than about 80% (dry weight) methionine(Met) (for example has, less than about 20% other nutrient media componentses or fermentation byproduct), perhaps (for example greater than about 90% methionine(Met), less than about 10% other nutrient media componentses or fermentation byproduct), perhaps (for example greater than about 95% methionine(Met), less than about 5% other nutrient media componentses or fermentation byproduct), perhaps greater than about 98-99% methionine(Met) (for example, less than other nutrient media componentses of about 1-2% or fermentation byproduct).

In alternative embodiment, for example, when microorganism is biologically harmless (for example, safety), not from microorganism purifying methionine(Met).For example, whole culture (perhaps culture supernatant) can be as product source (for example, crude product).In one embodiment, culture (perhaps culture supernatant) need not be modified and directly use.In another embodiment, culture (perhaps culture supernatant) is concentrated.In a further embodiment, with culture (perhaps culture supernatant) drying or freeze-drying.

The present invention also comprises bioconversion method, it is characterized in that multiple recombinant microorganism as herein described.Term " bioconversion method " is also referred to as " biotransformation method " in this article and comprises the biological method that causes suitable substrate of generation (for example, conversion or transformation) and/or intermediate compound to become purpose product (for example, methionine(Met)).

The microorganism and/or the enzyme that are used for bioconversion reaction are to allow them to carry out the form of expectation function (for example, producing the purpose compound).This quasi-microorganism can be full cell, perhaps can only be those parts that must obtain desirable net result (for example, gene and/or enzyme) of cell.Can be (for example with these microbial suspensions, in suitable solution such as damping fluid or substratum), flushing (for example, the substratum of culturing micro-organisms is removed in flushing), acetone drying, (for example fix, with polyacrylamide gel or k-carrageenin or on synthetic support such as globule, matrix or the like), fixing, crosslinked or saturatingization is (for example, the film of having and/or wall, make compound can easily pass as substrate, intermediate or product as described in film or wall).

The present invention (for example also comprises recombinant nucleic acid molecules, recombinant DNA molecules), it (for example comprises gene as herein described, isolating gene), comprise excellent bacillus gene, as Corynebacterium glutamicum gene, more specifically, Corynebacterium glutamicum methionine(Met) biosynthesis gene and Corynebacterium glutamicum halfcystine biosynthesis gene.Term " recombinant nucleic acid molecules " through the nucleic acid molecule of change, modification or through engineering approaches (for example refers to, dna molecular), make it list with this recombinant nucleic acid molecules to originate at nucleotides sequence (for example, by add, disappearance or substitute one or more Nucleotide) original or natural acid molecule difference.In some embodiments, recombinant nucleic acid molecules (for example, recombinant DNA molecules) comprises the isolating gene of effective connection adjusting sequence.The nucleotide sequence of the gene of feeling the pulse with the finger-tip that phrase " effectively is connected to the adjusting sequence " is (for example to allow expression of gene, enhanced, increase, composing type, the basis, that weaken, that reduce or the expression prevented), for example, the expression of the gene product of this genes encoding (for example, when recombinant nucleic acid molecules is included in as defined herein recombinant vectors, and when importing in the microorganism) mode be connected to the adjusting sequence.

Term " heterologous nucleic acids " is used in reference to the nucleotide sequence that is not present in usually in the microorganism in this article.This type of nucleotide sequence also comprises and being present in the microorganism, but be not present in the hereditary locational nucleotide sequence that they are found usually in this microorganism.Similarly, term " heterologous gene " can comprise the gene that is not present in the wild-type microorganisms.Heterologous nucleic acids and heterologous gene comprise recombinant nucleic acid molecules usually.Heterologous nucleic acids or heterologous gene can comprise or not comprise modification (for example, by adding, lack or alternative one or more Nucleotide).

The present invention also comprises several genes as herein described and proteinic homologue." homologue " that relates to gene refers to the same basically nucleotide sequence to small part or its complementary strand or its part at this gene, and condition is the protein that this nucleotide sequence coded and protein of its homologous genes encoding are gone up identical activity/function substantially.The amino acid that can be by the homologue of inferring or the sequence of nucleotide sequence and described gene or their encoded protein matter are (for example, the nucleotide sequence of Corynebacterium glutamicum gene ask, hom, metX, metY, metB, metH, metE, metF, zwf, metC, metK, metQ, cysJ, cysE, cysK, cysM, cysD, cysH, cysA, mcbR, hsk and pepCK, perhaps their complementary strand) between per-cent identity identify the homologue of gene described herein.For example, by visual inspection, perhaps by using multiple computer program known in the art or as described herein to determine per-cent identity.For example, by using Devereux et al. (1984) Nucl.Acids.Res., described and the GAP computer program that can (UWGCG) obtain from wisconsin genetics computer group (Universityof Wisconsin Genetics Computer Group) of 12:387, by comparative sequences information, can determine the per-cent identity of two nucleotide sequences.By using Tatusova et al. (1999) FEMS Microbiol.Lett., described basic local comparison gopher (Basic Local Alignment Search the Tool) (BLAST of 174:247 ^TM) program, determine per-cent identity by comparing two nucleotide sequences.For example, for using BLAST ^TMProgram is carried out the nucleotide sequence comparison, and default setting is as follows: matching bonusing is 2, and the mispairing point penalty is-2, makes a breach and extend the breach point penalty to be respectively 5 and 2, and gap.times.dropoff is 50, and expected value is 10, and word length is 11, and filter is for closing (OFF).

Term " homology " and " homologous " are not limited to specify the protein with theoretic common hereditary ancestors as used herein, also comprise the protein that can have nothing to do in heredity and still still carry out identity function through evolution and/or have analog structure.The function homology of multiple proteins as herein described also comprises the protein of the corresponding protein active with its homologue.For protein has the function homology, do not need them in their aminoacid sequence, to have remarkable identity, but the protein with function homology define as enzymatic activity by having similar or identical activity.Similarly, the protein with structural homology is defined as having similar three grades (perhaps level Four) structures and needn't needs amino acid identity or their the nucleic acid identity of gene of encoding.In some cases, structural homology only can comprise in the activity of proteins site or binding site keeps the protein of structural homology.

Except the 26S Proteasome Structure and Function homology, the present invention also comprises the protein that has amino acid identity with multiple proteins as herein described and enzyme.In order to determine the per-cent identity of two aminoacid sequences, for the best compares purpose with sequence alignment (for example, can introduce breach to compare with another proteinic aminoacid sequence is best in a proteinic aminoacid sequence).Amino-acid residue on the relatively more corresponding then amino acid position.When same amino acid occupied the position in the sequence on the correspondence position in by another sequence, these two molecules were same on this position.Per-cent identity is the identity positional number purpose function (that is % identity=# same position/total number of positions * 100) that these sequences are enjoyed between the two sequences.

In some embodiments, the nucleic acid of molecule described herein and aminoacid sequence comprise with the hybridization of nucleic acid as herein described or aminoacid sequence or at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or the Nucleotide or the aminoacid sequence of higher identity.

This paper has also described and has been used for as herein describedly proteinicly genetically engineeredly having technology improved or the decorative features enzyme with generation.For example, known modifying protein in the instruction available in the art makes this protein have substrate binding affinity increase or that reduce.Also favourable and in the instruction of this area, be the protein that design has the enzyme speed that increases or reduce.Especially for multifunctional enzyme, usefully the proteinic various active of difference ground fine adjustments is to carry out best under specific environment.In addition, regulatory enzyme can participate in methionine(Met) or just may participate in or the amino acid of the combination of degenerative other cofactors or coordination is finished the ability of the susceptibility of feedback inhibition (for example, by methionine(Met)) by selectively changing.In addition, genetically engineered comprise with transcribe with translation skill on the relevant incident of adjusting expressed.For example, when operon completely or partially is used for the clone and expresses, adjusting sequence that can modifying factor, for example, promotor or enhancer sequence make their produce transcribing of level of hope.

" homologue " of any gene as herein described can also be by the activity identification of this homologue encoded protein matter.For example, this homologue can be complementary sudden change in its homologue gene.

Term " adjusting sequence " refers to the nucleotide sequence that influence (for example, regulation and control or adjusting) other nucleotide sequences (being gene) are expressed.In some embodiments, regulating sequence is included in the recombinant nucleic acid molecules, be in respect on specific purposes gene similar or the identical position and/or direction, as being to regulate sequence and the observed natural appearance of goal gene, as on natural place and/or direction.For example, can comprise goal gene in the recombinant nucleic acid molecules, it effectively is connected to the adjusting sequence, in natural biological, this adjusting sequence is followed or (for example, effectively is connected to " natural " and regulates sequence (for example, being connected to " natural " promotor) in abutting connection with goal gene.Alternatively, goal gene can be included in the recombinant nucleic acid molecules, and this recombinant nucleic acid molecules effectively is connected to be followed in natural biological or in abutting connection with the adjusting sequence of another (for example, different) gene.Alternatively, goal gene can be included in the recombinant nucleic acid molecules, and this recombinant nucleic acid molecules effectively is connected to the adjusting sequence from another biology.For example, the adjusting sequence (for example, other bacteriums are regulated sequence, phage is regulated sequence or the like) from other microorganisms can effectively be connected to the specific purposes gene.

In some embodiments, regulate sequence and be the sequence that non-natural or non-natural exist (for example, modified, the sequence suddenling change, substitute, derive, lack comprises the sequence of chemosynthesis).The example of regulating sequence (for example comprises promotor, enhanser, termination signal, antitermination signal and other expression controlling elementss, suppress son or elicitor bonded sequence and/or for example in the mRNA that transcribes, transcribe and/or translate and regulate the combination of proteins site).This type of regulates sequence description in for example Sambrook, J., Fritsh, E.F., and Maniatis, T.Molecular Cloning:ALaboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Patek, M.et al is among (2003) Journal of Biotechnology 104:311-323.Regulate sequence comprise instruct that nucleotides sequence is listed in constitutive expression in the microorganism those (for example, constitutive promoter and strong constitutive promoter), instruct that nucleotides sequence is listed in inducible expression in the microorganism those (for example, inducible promoter, for example, the wood sugar inducible promoter) and those (for example, attenuated signal or cis-acting repressor sequences) of weakening or preventing nucleotides sequence to be listed in to express in the microorganism.Also within the scope of the present invention be by removing or lack the adjusting sequence, regulate the expression of goal gene.For example, can remove the sequence that relates to the negative regulation of transcribing makes the expression of goal gene be enhanced.

In one embodiment, recombinant nucleic acid molecules described herein comprises that () nucleotide sequence or gene for example, the methionine(Met) biosynthetic enzyme, it effectively connects promotor or promoter sequence at least a bacterial gene product of coding.Promotor described herein includes but not limited to, excellent bacillus promotor and/or phage promoter (for example, infecting the phage of excellent bacillus or other bacteriums).For example, in some embodiments, promotor is excellent bacillus promotor, preferred strong excellent bacillus promotor (for example, with excellent bacillus in biological chemistry house-keeping gene bonded promotor).In other embodiments, promotor is a phage promoter.The extra promotor that is used for gram-positive microorganism includes but not limited to that superoxide-dismutase, groEL, groES, EF-T u, amy and SPO1 promotor are as P ₁₅And P ₂₆The example that is used for the promotor of gram-negative micro-organism includes, but not limited to cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacIQ, T7, T5, T3, gal, trc, ara, SP6, λ-PR and λ-PL.

In some embodiments, recombinant nucleic acid comprises one or more terminator sequences (for example, transcription termination sequence).Term " terminator sequence " comprises and is used to stop the adjusting sequence that mRNA transcribes.Terminator sequence (transcription terminator of perhaps connecting) can also be used for stable mRNA for example with opposing nuclease (for example, by adding structure to mRNA).

In some embodiments, recombinant nucleic acid molecules comprise allow to detect the carrier that contains described sequence sequence (for example, detectable and/or selective marker), for example, coding antibiotics resistance sequence or overcome the gene of auxotrophic mutation, for example, trpC, drug labelling, fluorescent mark, and/or colorimetric mark (for example, lacZ/ beta-galactosidase enzymes).In other embodiments, recombinant nucleic acid molecules comprises artificial ribosome bind site (RBS) or is transcribed into the sequence of artificial RBS.Term " artificial ribosome bind site (RBS) " (for example comprises the mRNA molecule, encode in the DNA) the internal ribosome combination is (for example, to start translation) the site, itself and natural RBS (for example, the RBS that finds in the naturally occurring gene) differ at least one Nucleotide.Preferred artificial RBS comprises about 5-6,7-8,9-10,11-12,13-14,15-16,17-18,19-20,21-22,23-24,25-26,27-28,29-30 or polynucleotide more, its about 1-2,3-4,5-6,7-8,9-10,11-12,13-15 or more polynucleotide and natural RBS (for example, the natural RBS of goal gene) difference.

The present invention also comprises the carrier (for example, recombinant plasmid and phagemid) that comprises nucleic acid molecule as described herein (for example, gene or comprise the recombinant nucleic acid molecules of described gene).Term " recombinant vectors " through the carrier of change, modification or through engineering approaches (for example comprises; the nucleic acid carrier of plasmid, phage, phagemid, virus, glutinous grain, the glutinous grain of F or other purifying), contain in the original or natural acid molecule that described change, modification or through engineering approaches make this carrier originate than this recombinant vectors more, still less or different nucleotide sequences.For example, recombinant vectors comprises the biosynthesizing enzyme coding gene or comprises the recombinant nucleic acid molecules of described gene, and it effectively is connected to the adjusting sequence, for example, and promoter sequence, terminator sequence and/or artificial ribosome bind site (RBS), as defined herein.In some embodiments, recombinant vectors comprises the sequence (for example, duplicating-enhancement sequences) of duplicating in the enhancing bacterium.In some embodiments, duplicate enhancement sequences and in intestinal bacteria or Corynebacterium glutamicum, bring into play function.In other embodiments, duplicate enhancement sequences, include but not limited to, pBR322, pACYC177, pACYC184 and pSC101 from plasmid.

In other embodiments, recombinant vectors of the present invention comprises the antibiotics resistance sequence.Term " antibiotics resistance sequence " comprises the sequence that promotes or give the antibiotics resistance of host living beings (for example, excellent bacillus).In some embodiments, the antibiotics resistance sequence is selected from cat (chlorampenicol resistant) sequence, tet (tetracyclin resistance) sequence, erm (erythromycin resistance) sequence, neo (neomycin resistance) sequence, kan (kalamycin resistance) sequence and spec (spectinomycin resistance) sequence.Recombinant vectors can also comprise homologous recombination sequence (for example, be designed for allow the recombinate chromosomal sequence of host living beings of goal gene).The design that it will be apparent to one skilled in the art that carrier can be according to such as expression level of the selection for the treatment of genetically engineered microorganism, desirable gene product or the like customization.

" Campbell advances " as used herein (Campbell in) refers to the transformant of initial host cell, wherein complete ring-type double chain DNA molecule (for example plasmid) is incorporated in the karyomit(e) by single homologous recombination incident ((cross in) incident is advanced in hybridization), and the linearizing form that effectively causes described ring-shaped DNA molecule is inserted in chromosomal first dna sequence dna, first dna sequence dna homology of this first dna sequence dna and described ring-shaped DNA molecule." advanced " the linearizing dna sequence dna in the karyomit(e) that (Campbelled in) refer to be incorporated into " Campbell advances " transformant by Campbell.The copy of first homologous DNA sequence is contained in " Campbell advances ", and its each copy comprises and center on the homologous recombination exchange spot of a copy.This title is from Alan Campbell professor, and he has proposed the reorganization of the type first.

" Campbell goes out " as used herein (Campbell out) refers to the progeny cell of " Campbell advances " transformant, wherein the DNA that the linearizing of " Campbell advances " DNA is inserted go up contained second dna sequence dna and and described linearizing insert between second dna sequence dna in segmental second dna sequence dna homologous karyomit(e) source homologous recombination incident (hybridizing (cross out) incident) for the second time taken place, this dna sequence dna that second time, recombination event caused disappearance (a casting) part to be integrated, but importantly, the DNA that is advanced by Campbell that also causes a part (this can be as small as single base) to be integrated is retained in the karyomit(e), thereby compare with initial host cell, " Campbell goes out " cell (for example contains one or more changes of having a mind in karyomit(e), single base is replaced, a plurality of bases are replaced, insert heterologous gene or dna sequence dna, insert the homologous gene of extra one or more copies or modified homologous gene, perhaps insert the dna sequence dna that comprises more than one these above-mentioned listed examples).

" Campbell goes out " cell or strain system usually but not necessary, by to " being advanced " dna sequence dna by Campbell, anti-selection of gene as the part (this part is abandoned in hope) of subtilis sacB gene obtains, and is lethal when expressing in the cell that described gene is being grown under having about 5% to 10% sucrose.Use or do not use anti-selection, by using the desirable cell of any phenotypic screen that screens, can obtain or identify " Campbell goes out " cell of hope, described phenotype be such as, but not limited to, colonial morphology, colony colour, existence or do not exist antibiotics resistance, (passing through the polymerase chain reaction) to exist or do not have given dna sequence dna, existence or do not have auxotroph, existence or do not have enzyme, bacterium colony nucleic acid hybridization, antibody screening, or the like.Term " Campbell advances " and " Campbell goes out " can also be used as verb and refer to aforesaid method in multiple tense.

Be appreciated that the homologous recombination incident that causes " Campbell advances " or " Campbell goes out " can take place in the DNA base scope in homologous DNA sequence, and because homologous sequence will for this scope be mutually identical to small part, where the exchange incident has taken place so can not point out with precision usually.In other words, from the DNA that inserts, which is at first from chromosomal DNA at first for which sequence that can not point out with precision.In addition, first homologous DNA sequence and second homologous DNA sequence separate by the non-homogeneous zone of part usually, and this non-homogeneous zone keeps being placed in the karyomit(e) of " Campbell goes out " cell.

For practicality, in Corynebacterium glutamicum, first is at least about 200 base pairs with second homologous DNA sequence length usually, and can reach several thousand base pairs, yet, can arrange step to use shorter or longer sequence.For example, the length of first and second homologous sequence can be about 500 to 2000 bases, and by first and second homologous sequence are arranged to about equal length, preferably differ and be less than 200 base pairs, what most preferably both were short is than at least 70% of elder's base pair length, can conveniently obtain " Campbell goes out " from " Campbell advances ".

Further illustrate the present invention by the following examples, these embodiment should be interpreted as restriction.The content of the patent application of all reference, patent and the announcement that the application is quoted in full is incorporated herein by reference.

Embodiment

The generation of embodiment 1:M2014 bacterial strain

Corynebacterium glutamicum strain ATCC 13032 usefulness DNA A (being also referred to as pH273) (SEQ IDNO:1) are transformed also " to be advanced by Campbell " to produce " Campbell advances " bacterial strain.Fig. 2 has shown the synoptic diagram of plasmid pH273.Then with " Campbell advances " bacterial strain " Campbell goes out " to produce " Campbell goes out " bacterial strain M440, it contains encoder feedback resistance homoserine dehydrogenase (hom ^Fbr) gene.The homoserine dehydrogenase protein of gained comprises amino acid change, and wherein S393 changes into F393 (being called HsdhS393F).

Subsequently bacterial strain M440 is transformed with DNA B (being also referred to as pH373) (SEQ ID NO:2) and obtain " Campbell advances " bacterial strain.Fig. 3 has described the synoptic diagram of plasmid pH373.Then with " Campbell advances " bacterial strain " Campbell goes out " to produce " Campbell goes out " bacterial strain M603, it contains encoder feedback resistance E.C. 2.7.2.4. (Ask ^Fbr) gene of (lysC coding).In the aspartokinase zymoprotein of gained, T311 changes into I311 (being called LysC T311I).

Find that bacterial strain M603 produces about 17.4mM Methionin, but and the ATCC13032 bacterial strain does not produce the Methionin of measuring vol.In addition, the M603 bacterial strain produces about 0.5mM homoserine, compares the ATCC13032 bacterial strain and does not produce measurable amount, as summarizing in the Table III.

The amount of homoserine, O-ethanoyl homoserine, methionine(Met) and Methionin that Table III: strains A TCC13032 and M603 produce

Bacterial strain	Homoserine (mM)	O-ethanoyl homoserine (mM)	Methionine(Met) (mM)	Methionin (mM)
Bacterial strain	Homoserine (mM)	O-ethanoyl homoserine (mM)	Methionine(Met) (mM)	Methionin (mM)	ATCC13032	0.0	0.4	0.0	0.0
M603	0.5	0.7	0.0	17.4	ATCC13032	0.0	0.4	0.0	0.0

Transform bacterial strain M603 with DNA C (be also referred to as pH304, its synoptic diagram is described at Fig. 4) (SEQ ID NO:3), obtain " Campbell advances " bacterial strain, it is obtained " Campbell goes out " bacterial strain M690 by " Campbell goes out " then.The M690 bacterial strain contains the metH gene and (is also referred to as P ₄₉₇MetH) the PgroES promotor of upstream.P ₄₉₇The sequence of promotor provides in SEQ ID NO:4.The M690 bacterial strain produces about 77.2mM Methionin and about 41.6mM homoserine, shown in following Table IV.

Table IV: the amount of homoserine, O-ethanoyl homoserine, methionine(Met) and Methionin that bacterial strain M603 and M690 produce

Bacterial strain	Homoserine (mM)	O-ethanoyl homoserine (mM)	Methionine(Met) (mM)	Methionin (mM)
Bacterial strain	Homoserine (mM)	O-ethanoyl homoserine (mM)	Methionine(Met) (mM)	Methionin (mM)	M603	0.5	0.7	0.0	17.4
M690	41.6	0.0	0.0	77.2	M603	0.5	0.7	0.0	17.4

Following subsequently mutagenesis M690 bacterial strain: the M603 overnight culture that will be grown in the BHI substratum (BECTONDICKINSON) is washed with 50mM citrate buffer solution pH 5.5, handles 20 minutes with N-methyl-N-nitrosoguanidine (10mg/ml among the 50mM citrate buffer solution pH 5.5) at 30 ℃.After the processing, cell wash once more with 50mM citrate buffer solution pH 5.5 and the substratum of plating composition below containing in: (all amounts of mentioning all are that the 500ml substratum is calculated) 10g (NH ₄) ₂SO ₄0.5g KH ₂PO ₄0.5g K ₂HPO ₄0.125g MgSO _4*7H ₂O; 21gMOPS; 50mg CaCl ₂The 15mg Protocatechuic Acid; 0.5mg vitamin H; 1mg VitB1; With 5g/l D, (SIGMA CHEMICALS CATALOG#E5139), is adjusted to pH 7.0 with KOH to the L-ethionine.In addition, substratum contains 0.5ml trace metal solution, and it consists of: 10g/lFeSO _4*7H ₂O; 1g/l MnSO ₄* H ₂O; 0.1g/l ZnSO ₄* 7H ₂O; 0.02g/l CuSO ₄With 0.002g/l NiCl ₂* 6H ₂O, all are dissolved among the 0.1M HCl.Add aseptic 50% glucose solution (40ml) and no bacterio-agar to 1.5% final concentration of 40ml to final substratum degerming and in substratum by filtration.Pour into the final substratum that contains agar in the agar plate and be labeled as minimum ethionine substratum.The bacterial strain of mutagenesis upward is coated with and cultivated 3-7 days at 30 ℃ at dull and stereotyped (minimum ethionine).Separate the clone be grown on the substratum and streak culture once more on identical minimum ethionine substratum.Selecting some clones to be used for methionine(Met) production analyzes.

Following analysis methionine(Met) is produced.Bacterial strain was grown two days under 30 ℃ on the CM nutrient agar, and this substratum contains: 10g/l D-glucose, 2.5g/l NaCl; 2g/l urea; 10g/l Bacto peptone (DIFCO); 5g/l yeast extract (DIFCO); 5g/l extractum carnis (DIFCO); 22g/l agar (DIFCO); It was about 121 ℃ of autoclavings 20 minutes.

Behind the strain growth, scrape off cell and be resuspended among the 0.15M NaCl.For main culture, initial OD and 0.5g solid and the autoclaved CaCO of the suspension of cell with 600nm will be scraped off ₃(RIEDEL DE HAEN) adds in 1.5 to the 10ml medium ii (seeing below) together, and shakes in the bottle at the 100ml that does not have baffle plate and to shake on the platform with about 200 rev/mins (rpm) 30 ℃ of following culturing cells at rail.Medium ii contains: 40g/l sucrose; 60g/l is from the total reducing sugar (according to sugared cubage) of molasses; 10g/l (NH ₄) ₂SO ₄0.4g/l MgSO ₄* 7H ₂O; 0.6g/lKH ₂PO ₄0.3mg/l VitB1 * HCl; The 1mg/l vitamin H; 2mg/l FeSO ₄And 2mg/lMnSO ₄With substratum NH ₄OH is adjusted to pH 7.8 and about 20 minutes of about 120 ℃ of autoclavings.After autoclaving and the cooling, add vitamins B from filtration sterilization stock solution (200 μ g/ml) ₁₂(cyanocobalamin) (SIGMA CHEMICALS) is to the final concentration of 100 μ g/l.

From the substratum sample thief and measure aminoacids content.Use the Agilent method of amino-acids to go up and measure the amino acid that produces, comprise methionine(Met) at the Agilent1100 series LC HPLC of system (Series LC System HPLC).With Phthalyldicarboxaldehyde the pre-column of sample is derived permission at the last quantitative amino acid that produces in back that separates of Hypersil AA-post (AGILENT).

Separate methionine(Met) titre and be among the M690 clone of twice at least.Be used for a kind of this type of clone called after M1179 of other experiments and be deposited in DSMZ bacterial strain preservation center on May 18th, 2005, the bacterial strain preserving number is DSM 17322.With the amino acid output of this bacterial strain and comparing of bacterial strain M690, as following Table V general introduction.

Table V: the amount of homoserine, O-ethanoyl homoserine, methionine(Met) and Methionin that bacterial strain M690 and M1197 produce

Bacterial strain	Homoserine (mM)	O-ethanoyl homoserine (mM)	Methionine(Met) (mM)	Methionin (mM)
Bacterial strain	Homoserine (mM)	O-ethanoyl homoserine (mM)	Methionine(Met) (mM)	Methionin (mM)	M690	41.6	0.0	0.0	77.2
M1179	26.4	1.9	0.7	79.2	M690	41.6	0.0	0.0	77.2

Bacterial strain M1179 is transformed generation " Campbell advances " bacterial strain with DNA F (be also referred to as pH399, its synoptic diagram is described at Fig. 5) (SEQ IDNO:5), and it is obtained bacterial strain M1494 by " Campbell goes out " subsequently.This bacterial strain contains sudden change in the gene of homoserine kinase, it causes in the homoserine kinase of gained amino acid to change into A190 (being called HskT190A) from T190.The amino acid output that bacterial strain M1494 is produced and the rate ratio of bacterial strain M1197 are summarized as following Table VI.

Table VI: the amount of homoserine, O-ethanoyl homoserine, methionine(Met) and Methionin that bacterial strain M1197 and M1494 produce

Bacterial strain	Homoserine (mM)	O-ethanoyl homoserine (mM)	Methionine(Met) (mM)	Methionin (mM)
Bacterial strain	Homoserine (mM)	O-ethanoyl homoserine (mM)	Methionine(Met) (mM)	Methionin (mM)	M1179	26.4	1.9	0.7	79.2
M1494	18.3	0.2	2.5	50.1	M1179	26.4	1.9	0.7	79.2

Bacterial strain M1494 is transformed generation " Campbell advances " bacterial strain with DNA D (be also referred to as pH484, its synoptic diagram is described at Fig. 6) (SEQ IDNO:6), and it is obtained the M1990 bacterial strain by " Campbell goes out " subsequently.The M1990 bacterial strain is crossed expression metY allelotrope, uses groES-promotor and EFTU (EF-T u)-promotor (to be called P ₄₉₇P ₁₂₈₄MetY).P ₄₉₇P ₁₂₈₄The sequence of promotor shows in SEQ ID NO:7.The amino acid output of bacterial strain M1494 is compared with the output of bacterial strain M1990, as summarizing in the following Table VII.

Table VII: the amount of homoserine, O-ethanoyl homoserine, methionine(Met) and Methionin that bacterial strain M1494 and M1990 produce

Bacterial strain	Homoserine (mM)	O-ethanoyl homoserine (mM)	Methionine(Met) (mM)	Methionin (mM)
Bacterial strain	Homoserine (mM)	O-ethanoyl homoserine (mM)	Methionine(Met) (mM)	Methionin (mM)	M1494	18.3	0.2	2.5	50.1

M1990

18.2

0.3

5.6

48.9

Bacterial strain M1990 is transformed generation " Campbell advances " bacterial strain with DNA E (be also referred to as pH 491, its synoptic diagram is described at Fig. 7) (SEQID NO:8), and it is obtained " Campbell goes out " bacterial strain M2014 by " Campbell goes out " subsequently.Use the superoxide-dismutase promotor (to be called P ₃₁₁₉MetA), the M2014 bacterial strain is crossed expression metA allelotrope.P ₃₁₁₉The sequence of promotor provides in SEQ ID NO:9.The amino acid output of bacterial strain M2014 is compared with the output of bacterial strain M2014, as summarizing in the following Table VIII.

Table VIII: the amount of homoserine, O-ethanoyl homoserine, methionine(Met) and Methionin that bacterial strain M1494 and M1990 produce

Bacterial strain	Homoserine (mM)	O-ethanoyl homoserine (mM)	Methionine(Met) (mM)	Methionin (mM)
Bacterial strain	Homoserine (mM)	O-ethanoyl homoserine (mM)	Methionine(Met) (mM)	Methionin (mM)	M1990	18.2	0.3	5.6	48.9
M2014	12.3	1.2	5.7	49.2	M1990	18.2	0.3	5.6	48.9

Embodiment 2: strengthen the expression of metF among the M2014

Methylene tetrahydrofolate reductase (MetF) catalysis 5, the 10-methylene tetrahydrofolate is reduced to 5-methyl tetrahydrofolate (5-MTF).5-MTF is the methyl donor that homocysteine methyl turns to methionine(Met).This methylates MetE or MetH catalysis.If the supply of 5-MTF is a suboptimum, this final step in the methionine(Met) biosynthesizing can be restricted so.Therefore, modify the metF gene and be used for constitutive expression.With the natural promoter of metF groES promotor (P ₄₉₇) (SEQ ID NO:4) replace and import among the Corynebacterium glutamicum strain M2014 on the bioAD locus.

Obtain Corynebacterium glutamicum metF gene and be connected between the XbaI and BamHI site of plasmid pOM35 by PCR, obtain pOM62 (SEQ ID NO:10).The synoptic diagram of pOM62 plasmid provides in Fig. 8.By at first selecting kalamycin resistance transformant (Campbell advances), use then that sacB is counter to select to separate the responsive derivative (Campbell goes out) of the kantlex that has lost the integrated plasmid skeleton, with P ₄₉₇The metF box imports among the M2014 on the bioAD chromogene seat.Bacterium colony by PCR screening gained finds to have P at the bioAD locus ₄₉₇The M2014 derivative of metF box.A kind of this type of Corynebacterium glutamicum isolate is called OM41.

In order to measure methionine(Met) and other amino acid whose generations, in standard molasses culture medium as described in example 3 above, with bacterial strain M2014 with duplicate and bacterial strain OM41 with quadruplicate growth shake-flask culture thing.As shown in Table IX, bacterial strain OM41 produces methionine(Met) with the level that is higher than the M2014 bacterial strain.

Table I X: in shake flat experiment, 48 hours the time, (contain P by Corynebacterium glutamicum M2014 and OM41 ₄₉₇The bacterial strain of metF box) amino acid of Chan Shenging ¹

¹Measure amino acid with g/L.The mean value of duplicate culturing bottle.

²Molasses culture medium.

³The glycine (Gly) and the homoserine (Hse) that in the amino acid analysis system that uses, move with identical retention time

Embodiment 3: shake flat experiment and HPLC assay method

With duplicate or quadruplicate, carry out shake flat experiment with bacterial strain with the standard molasses culture medium.Molasses culture medium contains in one liter of substratum: 40g glucose; The 60g molasses; 20g (NH ₄) ₂SO ₄0.4g MgSO ₄* 7H ₂O; 0.6g KH ₂PO ₄10g yeast extract (DIFCO); The 5ml400mM Threonine; 2mg FeSO ₄.7H ₂O; 2mg MnSO ₄.H ₂O; With 50g CaCO ₃(Riedel-de Haen) uses ddH ₂O supplies volume.Use 20%NH ₄OH regulates pH to 7.8, with substratum (so that the maintenance CaCO of 20ml continuously stirring ₃Suspending) Bellco that joins 250ml band baffle plate shakes in the bottle and will shake bottle autoclaving 20 minutes.Behind the autoclaving, every liter of basic medium (perhaps 80 μ l/ bottles) adds 4ml " 4B solution ".Every liter " 4B solution " contains: 0.25g vitamin (VITMAIN B1), 50mg cyanocobalamin (vitamin B12), 25mg vitamin H, 1.25g pyridoxine hydrochloride (vitamin B6) are also used 12.5mM KPO ₄, pH 7.0 bufferings are with the dissolving vitamin H, and filtration sterilization.Culture in cover with Bioshield paper with bungee fixed band baffle plate culturing bottle in shaking table at the bottom of 28 ℃ or 30 ℃ and the New Brunswick Scientific with 200 or 300rpm growth 48 hours.24 hours and/or 48 hours collected specimens.By the centrifugal cell of removing,, use Centricon 0.45 μ m column spinner membrane filtration solution then then with isopyknic 60% dilution in acetonitrile supernatant liquor.Methionine(Met), glycine add homoserine, O-ethanoyl homoserine, Threonine, Isoleucine, Methionin and other amino acid whose concentration of pointing out in the use HPLC mensuration filtrate.

For the HPLC assay method, with filtering supernatant liquor with the filtering 1mMNa of 0.45 μ m ₂EDTA is with dilution in 1: 100 and with 1 μ l solution borate buffer solution (80mM NaBO ₃, 2.5mM EDTA, pH 10.2) in OPA reagent (AGILENT) derive, and the Agilent 1100 serial HPLC that are expelled at equipment G1321A fluorimetric detector (AGILENT) go up on 200 * 4.1mm Hypersil, the 5 μ AA-ODS posts of operation.Excitation wavelength is that the 338nm and the emission wavelength of being monitored are 425nm.With amino acid standardized solution chromatography and be used for determining the retention time and the base peak area of multiple amino acids.The incidental software package Chem Station that Agilent provides is used for instrument control, data gathering and data manipulation.Hardware is HP Pentium 4 computers, and it supports the Microsoft Windows NT 4.0 with MicrosoftService Pack (SP6a) renewal.

Embodiment 4: MetA and MetZ activity have increased methionine(Met) output among enhancing M2014 and the OM41

Bacterial strain M2014 and OM41 are transformed with plasmid replication pH357, and this plasmid synoptic diagram shows (SEQ ID:11) in Fig. 9, contain P ₄₉₇MetZ, P ₃₁₁₉The metA box.The bacterial strain of gained is called M2014 (H357) and OM41 (H357), they and they parent strain is compared so that determine whether the extra expression of metZ and/or metA is useful for methionine(Met) production.In two kinds of bacterial strains, the existence of H357 plasmid has improved methionine(Met) output.As shown in Table X, in the standard molasses culture medium, the methionine(Met) titre of OM41 (H357) is high more about 75% than OM41's, shows that extra MetA and/or MetZ activity are useful (1.4g/l are to 0.8g/l) for increasing methionine(Met) titre.In addition, adding 1% yeast extract (YE) to substratum makes titre further increase 30-40%.

Table X:, be used for 30 ℃ of following 48 hours shake-flask culture of comparison OM41 and OM41 (H357) replenishing or not replenishing in the standard molasses culture medium of 1% yeast extract

Embodiment 5: the pepCK locus mixes P in M2014 ₄₉₇Hom ^FbrCause methionine(Met) output to increase

In the karyomit(e) of M2014, there is feedback resistance homoserine dehydrogenase gene (hom ^Fbr).Yet this gene uses its natural promoter to be used for expressing, and this promotor is subjected to the inhibition (Rey D.A.et al., J.Molecular Microbiology.56:871-887 (2005)) of methionine(Met) according to reports.In order to obtain containing the M2014 bacterial strain of the hom gene that is not subjected to the McbR adjusting, advance with Campbell to go out the P497hom that in the pepCK of M2014 locus, inserts from plasmid pH410 by Campbell ^FbrBox, its synoptic diagram show (SEQ ID NO:12) in Figure 10, and verify by PCR subsequently.The bacterial strain of gained is called OM224.

M2014 and OM224 bacterial strain carried out standard shake bottle research as mentioned previously.Shown in Table X I, OM224 compares with M2014 has increased the titre that glycine adds homoserine (Gly+Hse), O-ethanoyl homoserine (O-AcHse) and methionine(Met); Yet, to compare with M2014, Methionin titre reduces.Measure amino acid with g/l.

Bottle research is shaken in 48 hours of Table X I:M2014 derivative OM224

Bacterial strain	Box	Gly+ Hse	O-AcHse	Lys	Met
Bacterial strain	Box	Gly+ Hse	O-AcHse	Lys	Met	M2014	Do not have	2.0 2.1	1.3 1.4	4.9 5.1	0.5 0.6
OM224-1	P497hom ^fbr	3.2 2.9 3.1 3.6	3.0 2.1 2.6 2.7	2.9 2.6 3.4 3.9	0.7 0.7 0.8 0.9	M2014	Do not have	2.0 2.1	1.3 1.4	4.9 5.1	0.5 0.6

Use the plasmid pOM62 described in top embodiment 2, with P ₄₉₇The metF box is incorporated in the bioAD locus of OM224 bacterial strain, thereby obtains bacterial strain OM89.Further modify OM89 by the SAM synthase gene of integrating sudden change on MetK natural gene seat subsequently, described gene is a coding and the wild-type enzyme metK of the enzyme that significantly reduces of specific activity mutually ^*(C94A) (Reezkowski, R.S.and G.D.Markham, J.Biol.Chem., 270:18484-18490 (1995)).Expect that lower MetK activity will reduce the output of S-adenosylmethionine.The synoptic diagram of plasmid pH295 (SEQ ID NO:13) shows in Figure 11, and this plasmid Campbell is advanced with Campbell to go out OM89 to use metK ^*Replace the wild-type metK among the OM89, obtain bacterial strain OM99.Can identify metK ^*Allelotrope is because it introduces the PshAI restriction site in the chromosomal PCR product from OM99.Then with containing P ₄₉₇MetZ and P ₃₁₁₉The plasmid replication H357 of metA box transforms the OM99 bacterial strain, produces bacterial strain OM99 (H357).

OM89, OM99, OM99 (H357) and parent strain are carried out the standard shake flat experiment.As shown in Table X II, every kind of parent strain M2014 than them of OM41 and OM224 produces about 20% more methionine(Met).In this experiment, the behavior of OM89 is similar to M2014.MetK ^*As if gene is compared with parent strain to the integration of OM89 (bacterial strain OM99) has increased methionine(Met) titre.At last, OM99 (H357) causes the titre of 1.7g/l methionine(Met), increases about 70% than parent strain OM99.All amino acid are all measured with g/l.

Table X II: the shake flat experiment that carries out with multiple M2014 derivative

Bacterial strain	Box	Gly + Hse	O-Ac-Hse	Ile	Lys	Met
Bacterial strain	Box	Gly + Hse	O-Ac-Hse	Ile	Lys	Met	M2014		1.4 1.4	3.3 3.3	0.0 0.0	3.8 3.9	0.8 0.9
OM41	P ₄₉₇metF	0.8 0.9	4.8 5.9	0.0 0.0	4.0 4.6	1.0 1.1	M2014		1.4 1.4	3.3 3.3	0.0 0.0	3.8 3.9	0.8 0.9
OM41	P ₄₉₇metF	0.8 0.9	4.8 5.9	0.0 0.0	4.0 4.6	1.0 1.1	OM224	P ₄₉₇homf ^br	3.6 3.3	5.2 5.1	0.1 0.1	1.8 1.7	1.0 1.0
OM89	P ₄₉₇metFP ₄₉₇hom ^fbr	2.4 3.5	3.9 5.3	0.0 0.1	1.3 1.7	0.7 1.0	OM224	P ₄₉₇homf ^br	3.6 3.3	5.2 5.1	0.1 0.1	1.8 1.7	1.0 1.0
OM89	P ₄₉₇metFP ₄₉₇hom ^fbr	2.4 3.5	3.9 5.3	0.0 0.1	1.3 1.7	0.7 1.0	OM99	P ₄₉₇metFP ₄₉₇hom ^fbrmetK ^＊	2.7 3.0	3.4 3.0	0.1 0.2	1.6 1.5	1.0 1.0
OM99 (H357)	P ₄₉₇metFP ₄₉₇hom ^fbrmetK ^＊(H357)	1.7 1.7	1.7 1.8	0.2 0.1	0.5 0.5	1.7 1.7	OM99	P ₄₉₇metFP ₄₉₇hom ^fbrmetK ^＊	2.7 3.0	3.4 3.0	0.1 0.2	1.6 1.5	1.0 1.0

Also performance is good in the bench scale fermentation for OM99 (H357) bacterial strain, produces 8.5g/l methionine(Met) (seeing embodiment 11) after about 78 hours.

Embodiment 6: mcbR has increased methionine(Met) output from the M2014 disappearance

Plasmid pH429 contains RXA00655 disappearance (SEQ ID NO:14), and its synoptic diagram shows in Figure 12, and this plasmid is used for introducing mcbR disappearance (seeing WO 2004/050694 A1) by integrating and cutting out to Corynebacterium glutamicum.Select plasmid pH429 is transformed into (Campbell advances) in the M2014 bacterial strain with kalamycin resistance.Use that sacB is counter to be selected, separate the kantlex susceptibility derivative that transforms bacterial strain, it may lose the plasmid of integrating (Campbell goes out) by excision.The bacterial strain that is transformed produces the responsive derivative of kantlex, and this derivative produces little bacterium colony and bigger bacterium colony.Screen the bacterium colony of two kinds of sizes so that detect the existence of mcbR disappearance by PCR.Do not contain described disappearance than macrocolony, and 60-70% contain desired mcbR disappearance than small colonies.

When streak culture initial separation bacterium on the BHI flat board when separating single bacterium colony, the small colonies mixture appears.When small colonies was rule on BHI once more, the small colonies mixture appearred once more.When on BHI, small colonies being rule once more, the bacterium colony size normally little with homogeneous.Select two kinds little single bacterium colony isolate to be used for further research, described isolate is called OM403-4 and OM403-8.

Shake flat experiment (Table X III) shows at least twice of the amount of the methionine(Met) that OM403-8 produces for parent M2014.This bacterial strain also produces less than 1/5 of lysine amount that M2014 produces, and prompting carbon flux turns to homoserine from the aspartic acid semialdehyde.The Isoleucine accumulation that the third wonderful difference is OM403 increases by 10 times with respect to M2014.Grown culture is 48 hours in the standard molasses culture medium.

Table X III: the amino acid output of OM403 strains separation bacterium in the shake-flask culture of the cell of inoculating fresh growth

Also as shown in Table X IV, the accumulation of the O-ethanoyl homoserine of OM403 reduces more than 15 times with respect to M2014.This result's most probable explanation is that the most O-ethanoyl homoserine that accumulate in M2014 are converted to methionine(Met), homoserine and Isoleucine in OM403.Culture was grown 48 hours in the standard molasses culture medium.

Table X IV: the amino acid output of inoculating two isolates of OM403 in the shake-flask culture of cell of fresh growth

In order to improve in the OM403 background homocysteine to the conversion of methionine(Met), OM403-8 is transformed with plasmid replication, described plasmid causes that (being shown among Figure 13 of plasmid pH170 provides metH in the Corynebacterium glutamicum (pH170), sequence provides in SEQ ID NO:15) or cross the expressing of metE (pH447) (being shown among Figure 14 of plasmid pH447 provides, and sequence provides in SEQ ID NO:16) gene.New bacterial strain (being respectively OM418 and OM419) produces more methionine(Met) (Table X V) than OM403-8 in the medicine bottle experiment.

Table X V: with pH170 (P ₄₉₇MetH), pH447 (P ₄₉₇MetE) or pH448 (P ₁₂₈₄MetE) OM403-8's of Zhuan Huaing (M2014 Δ mcbR) shakes a bottle assay method

Culture is having or was not having in the standard molasses culture medium of 25 μ g/ml kantlex growth 48 hours.These bacterial strains of test in fermentor tank, wherein OM419 produces obviously more methionine(Met) than OM403-8.

Embodiment 7: increase the metF expression at OM419 and increased methionine(Met) output

For the metF that increases among the OM403-8 expresses, with original metF promotor intestinal bacteria lambda particles phage P _RPromotor is replaced.This uses standard Campbell to advance to go out technology with Campbell finishes with plasmid pOM427 (SEQ ID NO:17).The bacterial strain of gained is called OM428-2, and it is transformed with metE expression vector H447.Four isolates to the bacterial strain that is called OM448 of gained are measured methionine(Met) output with OM403-8 and OM428-2 in shaking bottle assay method.This result of experiment of describing in Table X VI shows that all four kinds of isolates of OM428-2 and OM448 produce obviously more methionine(Met) than OM403-8, but the only a kind of of four kinds of isolates of OM448 produces more methionine(Met) than OM428-2.

Table X VI:OM428-2 and OM448 shake a bottle assay method

Embodiment 8: the microorganism that produces the sulfate reduction approach that contains imbalance

Plasmid pOM423 (SEQ ID NO:18) is used to produce the bacterial strain of the sulfate reduction approach that contains imbalance.The synoptic diagram of plasmid pOM423 is described in Figure 16.Especially, with escherichia coli P _LAnd P _RDivergent promoter construct is used to replace the divergent promotor of natural sulfate reduction regulon.Bacterial strain OM41 is transformed and select kalamycin resistance (Campbell advances) with pOM423, after the anti-selection of sacB, separate the responsive derivative (Campbell goes out) of kantlex from transformant.Use pcr analysis to determine the promoter structure of sulfate reduction regulon subsequently them.Contain P _L-P _RThe isolate of divergent promotor is called OM429.Use DTNB test paper measurements determination OM429 four isolates sulfate reduction and in shaking bottle assay method test methionine(Met) output.In order to use the relative sulfide of DTNB test paper test evaluation to produce, be immersed in test strip in Ellman reagent (DTNB) solution and on the shake-flask culture thing of bacterial strain to be tested, hung 48 hours.The hydrogen sulfide reduction DTNB that the culture of growth produces produces yellow, itself and the H that produces ₂The amount of S is proportional basically.Thereby the intensity of the color of generation can be used to obtain the active thick step estimation of relative sulfate reduction of multiple bacterial strain.Result (Table X VII) shows that two kinds of four kinds of isolates demonstrate high-caliber relatively sulfate reduction.These two kinds of identical isolates also produce the methionine(Met) of highest level.Culture was grown 48 hours in the standard molasses culture medium.

The methionine(Met) output and the sulfate reduction of OM429 isolate in the Table X VII. shake-flask culture

Methionine(Met) output that embodiment 9. has reduced the metQ expression decreased

In order to reduce the output of methionine(Met) among the OM403-8, the promotor and the 5 ' part of disappearance metQ gene.The subunit of metQ genes encoding methionine(Met) output complex body, it is that this complex body function is required.This uses standard Campbell to advance to go out technology with Campbell finishes with plasmid pH449, and the synoptic diagram of this plasmid shows (SEQ ID NO:19) in Figure 15.The plasmid of gained is called OM456-2, and it is transformed with metE expression vector H447 or metF expression plasmid pOM436 (SEQ ID NO:20).Four isolates of every kind that are called OM464 and OM465 are measured methionine(Met) output with OM403-8 and OM456-2 in shaking bottle assay method.Result (Table X IVIII) shows that OM456-2 has produced the methionine(Met) of Duoing slightly than OM403-8, and all four isolates of OM464 and OM465 produce more methionine(Met) than OM403-8.Culture was grown 48 hours in the standard molasses culture medium.

Table X VIII.

The structure of embodiment 10.OM469 and OM508

Because the imbalance of the disappearance of metQ and metF has improved the output of methionine(Met) separately, so make up the bacterial strain that is called OM469, it contains two kinds of features.By using lambda particles phage P _RPromotor substitutes wild-type metF promotor, makes up OM469 from bacterial strain OM456-2.This uses standard Campbell to advance to go out technology with Campbell and uses plasmid pOM427 (SEQ ID NO:17) to finish.Four kinds of isolates to OM469 in the shake-flask culture assay method are measured methionine(Met) output, and wherein they all produce more methionine(Met) than OM456-2, as shown in Table X IX.

Table X IX.OM469 shakes a bottle assay method, and OM469 contains lambda particles phage P _RPromotor replaces the OM456-2 derivative of metF promotor

Culture was grown 48 hours in containing the standard molasses culture medium of 2mM Threonine.

In order to make up bacterial strain OM508, bacterial strain OM469-2 is transformed with plasmid replication pH357 (SEQ IDNO:11).In the shake-flask culture assay method, measure the methionine(Met) output of four isolates of OM508.Three of four isolates produce still less methionine(Met) than OM469, and isolate produces the methionine(Met) with the about same amount of OM469-2, as describing among the Table X X.All four kinds of isolate consumption rate OM469-2 glucose still less, the methionine(Met) of pointing out every mole of glucose higher yields.

Table X X. contains bottle assay method of shaking of metX metY expression cassette on replicating vector

^*Remaining glucose (g/l) when incubation finished in 48 hours.

Embodiment 11: fermentation in 110 jars of 7.5 liters of NBS BioFlo

In having 7 liters of New Brunswick Scientific (NBS) BioFlo jar of 5 liters of working volumes, carry out fed-batch fermentation.The aseptic batch culture base of operation M111 comprises: molasses 150g/l; Glucose 10g/l; Difco yeast extract 10g/l; (NH ₄) ₂SO ₄30g/l; MgSO ₄* 7H ₂O 1g/l; KH ₂PO ₄* 3H ₂O 5g/l; Mazu DF204C 1.5g/l (froth breaking reagent); The 25mM Threonine; The 25mg/l kantlex; 1X Met mineral substance; 1X Met VITAMIN; And dH ₂O to 2.0 liter.In this substratum, add 18 hours inoculum of growth in 28 ℃ of following BHI-10 (Becton Dickinson brain heart infusion agar adds 10g/l glucose).1X Met mineral substance has 10mg/lFeSO ₄* 7H ₂O, 10mg/l MnSO ₄* H ₂O, 1mg/l H ₃BO ₃* 4H ₂O, 2mg/lZnSO ₄* 7H ₂O, 0.25mg/l CuSO ₄, and 0.02mg/l Na ₂MoO ₄* 2H ₂The final concentration of O.1X Met VITAMIN has final concentration 6mg/l nicotinic acid, 9.2mg/l VitB1,0.8mg/l vitamin H, 0.4mg/l pyridoxal and 0.4mg/l cyanocobalamin (vitamins B ₁₂), from the storage liquid of 250X filtration sterilization, it contains the 12.5mM potassiumphosphate, and pH 7.0 is with the dissolving vitamin H.

Fermentation fed with constant rate of speed in 32 hours raise the Isoleucine that 400ml 12.5mM Threonine adds 12.5mM.Independent glucose charging contains dH ₂Glucose 750g/l among the O, MgSO ₄* 7H ₂O 2g/l, (NH ₄) ₂SO ₄20g/l and 10X Met VITAMIN.Independent the feeding of the fermentation of OM99 (H357) raised glucose and amino acid charging, and two kinds of chargings all begin when initial glucose level is reduced to 10g/l.

After inoculation, in preceding 16 to 24 hours, consume the initial carbohydrate of batch-wise in molasses and the glucose.Behind the initial glucose consumption of cell, raise the above-mentioned glucose solution that contains VITAMIN, sal epsom and ammonium sulfate, keep glucose concn between 10 to 15g/l by feeding.

Stir be arranged at first 200-300rpm (rev/min).When the dissolved oxygen concn was reduced to 25%, it was 20 ± 5%[pO to keep dissolved oxygen concentration that stir speed (S.S.) is regulated in computer control automatically ₂].By hard-wired maximum stir speed (S.S.) is 1200rpm.When 1200rpm is not enough to keep 20 ± 5%[pO ₂] dissolved oxygen levels the time, with the pure oxygen pulse in air supply.Fermentation remains on pH 7.0 ± 0.1 and 28 ° ± 0.5 ℃.Carry out computer control and data logging by New Brunswick Scientific Biocommand software.

Fermentation M111 produced the 8.5g/l methionine(Met) and produced the 11.5g/l methionine(Met) in 96 hour in 72 hours.At 96 hours, Methionin was that 16.5g/l and O-ethanoyl homoserine are 8.5g/l.Therefore, there is the precursor storehouse,, can additionally increases methionine(Met) output 20g/l so if it is converted into methionine(Met).

The fed-batch fermentation of embodiment 12:OM448-1, fermentation M190

Fermentation OM448-1 as described in example 11 above, but begin with following initial batch substratum for operation M190: molasses 150g/l, glucose 10g/l, Difco yeast extract 20g/l, (NH ₄) ₂SO ₄30g/l, MgSO ₄* 7H ₂O 1g/l, KH ₂PO ₄* 3H ₂O 12g/l, HySoyT 20g/l, Mazu DF204C 1.5g/l, 25mM Threonine, 25mg/l kantlex, 1X Met mineral substance, 10X Met VITAMIN, and dH ₂O to 1.5 liter.In this substratum, add in BHySoy-10 (Becton Dickinson brain heart infusion agar adds 10g/l glucose and 10g/l HySoy) 24 hours 500ml OM448-2 inoculum of 30 ℃ of growths to produce 2 liters initial volume.

Feed the 30mM Threonine of raising 400ml to fermentation with 12.5ml/ hour speed.Independent glucose charging contains glucose 750g/l, MgSO ₄* 7H ₂O 2g/l, (NH ₄) ₂SO ₄30g/l, 1X Met mineral substance and 25X Met VITAMIN.

The fermentation of OM448-2 produced the 16.6g/l methionine(Met) and produced the 17.1g/l methionine(Met) in 76 hour in the above-mentioned substratum in 72 hours.

The fed-batch fermentation of embodiment 13:OM508-4, fermentation operation M322

Fermentation OM508-4 as described in example 11 above, but begin with following initial batch substratum for operation M322: molasses 150g/l, Difco yeast extract 20g/l, (NH ₄) ₂SO ₄30g/l, MgSO ₄* 7H ₂O 1g/l, KH ₂PO ₄* 3H ₂O 20g/l, HySoyT 20g/l, Mazu DF204C1.5g/l, Threonine 6g/l, Serine 10g/l, 25mg/l kantlex, 1X Met mineral substance, batch VITAMIN, and dH ₂O to 1.5 liter.In initial batch substratum, add VITAMIN and obtain final concentration 15mg/l nicotinic acid, 23mg/l VitB1,2mg/l vitamin H, 1mg/l pyridoxal and 1mg/l cyanocobalamin.In this substratum of 1.5L, add in BHySoy-15 (Becton Dickinson brain heart infusion agar adds 15g/l glucose and 10g/l HySoy) 30 ℃ down 24 hours 500ml OM508-4 inoculum of growth to produce 2 liters initial volume.

Charging contains glucose 750g/l, MgSO ₄* 7H ₂O 2g/l, (NH ₄) ₂SO ₄40g/l, Serine 10g/l, Threonine 3.6g/l, 1X Met mineral substance and charging VITAMIN.

VITAMIN joined obtain final concentration 75mg/l nicotinic acid, 115mg/l VitB1,10mg/l vitamin H, 5mg/l pyridoxal and 5mg/l cyanocobalamin in the feedstock solution in the glucose charging.The fermentation of OM508-4 produced the 25.8g/l methionine(Met) in 56 hours in above-mentioned substratum.

The instruction of the reference that this specification sheets is quoted in is to specifications done the most completely and is understood, and described reference is incorporated herein by reference.What the embodiment in the specification sheets provided the embodiment that the present invention includes illustrates and should not be construed the restriction scope of the invention.Easy clear many other embodiments that the present invention includes of technician.All publications of being quoted and patent and the sequence of complete identified by searching number as herein described or database reference number are incorporated herein by reference.If the reference that is incorporated herein by reference contradicts with this specification sheets or be inconsistent, present disclosure will have precedence over any this type of material.Quoting of any reference of this paper is not to recognize that this type of reference is a prior art of the present invention.

Unless otherwise noted, the expression specification sheets, all numerals that comprise the amount of the composition that uses in claims, cell culture, treatment condition or the like all are construed as in all situations modifies with term " about ".Therefore, opposite unless otherwise noted, digital parameters and approximation can be sought the desirable properties that obtains and be become according to the present invention.Unless otherwise noted, the term " at least " before a series of compositions will be understood that every kind of composition in the series.Those skilled in the art will recognize that or only use normal experiment just can determine many equivalents of particular of the present invention described herein.This type of equivalent is intended to be comprised by following claims.

Sequence table

＜110〉BASF Aktiengesellchaft

＜120〉recombinant microorganism of generation methionine(Met)

<130>BGI-180PC

<140>

<141>

<150>60/714,042

<151>2005-09-01

<150>60/700,699

<151>2005-07-18

<160>20

＜170〉PatentIn version 3 .3

<210>1

<211>7070

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Nucleotide sequence

<400>1

tcgagaggcc tgacgtcggg cccggtacca cgcgtcatat gactagttgg agaatcatga 60

cctcagcatc tgccccaagc tttaaccccg gcaagggtcc cggctcagca gtcggaattg 120

cccttttagg attcggaaca gtcggcactg aggtgatgcg tctgatgacc gagtacggtg 180

atgaacttgc gcaccgcatt ggtggcccac tggaggttcg tggcattgct gtttctgata 240

tctcaaagcc acgtgaaggc gttgcacctg agctgctcac tgaggacgct tttgcactca 300

tcgagcgcga ggatgttgac atcgtcgttg aggttatcgg cggcattgag tacccacgtg 360

aggtagttct cgcagctctg aaggccggca agtctgttgt taccgccaat aaggctcttg 420

ttgcagctca ctctgctgag cttgctgatg cagcggaagc cgcaaacgtt gacctgtact 480

tcgaggctgc tgttgcaggc gcaattccag tggttggccc actgcgtcgc tccctggctg 540

gcgatcagat ccagtctgtg atgggcatcg ttaacggcac caccaacttc atcttggacg 600

ccatggattc caccggcgct gactatgcag attctttggc tgaggcaact cgtttgggtt 660

acgccgaagc tgatccaact gcagacgtcg aaggccatga cgccgcatcc aaggctgcaa 720

ttttggcatc catcgctttc cacacccgtg ttaccgcgga tgatgtgtac tgcgaaggta 780

tcagcaacat cagcgctgcc gacattgagg cagcacagca ggcaggccac accatcaagt 840

tgttggccat ctgtgagaag ttcaccaaca aggaaggaaa gtcggctatt tctgctcgcg 900

tgcacccgac tctattacct gtgtcccacc cactggcgtc ggtaaacaag tcctttaatg 960

caatctttgt tgaagcagaa gcagctggtc gcctgatgtt ctacggaaac ggtgcaggtg 1020

gcgcgccaac cgcgtctgct gtgcttggcg acgtcgttgg tgccgcacga aacaaggtgc 1080

acggtggccg tgctccaggt gagtccacct acgctaacct gccgatcgct gatttcggtg 1140

agaccaccac tcgttaccac ctcgacatgg atgtggaaga tcgcgtgggg gttttggctg 1200

aattggctag cctgttctct gagcaaggaa tcttcctgcg tacaatccga caggaagagc 1260

gcgatgatga tgcacgtctg atcgtggtca cccactctgc gctggaatct gatctttccc 1320

gcaccgttga actgctgaag gctaagcctg ttgttaaggc aatcaacagt gtgatccgcc 1380

tcgaaaggga ctaattttac tgacatggca attgaactga acgtcggtcg taaggttacc 1440

gtcacggtac ctggatcttc tgcaaacctc ggacctggct ttgacacttt aggtttggca 1500

ctgtcggtat acgacactgt cgaagtggaa attattccat ctggcttgga agtggaagtt 1560

tttggcgaag gccaaggcga agtccctctt gatggctccc acctggtggt taaagctatt 1620

cgtgctggcc tgaaggcagc tgacgctgaa gttcctggat tgcgagtggt gtgccacaac 1680

aacattccgc agtctcgtgg tcttggctcc tctgctgcag cggcggttgc tggtgttgct 1740

gcagctaatg gtttggcgga tttcccgctg actcaagagc agattgttca gttgtcctct 1800

gcctttgaag gccacccaga taatgctgcg gcttctgtgc tgggtggagc agtggtgtcg 1860

tggacaaatc tgtctatcga cggcaagagc cagccacagt atgctgctgt accacttgag 1920

gtgcaggaca atattcgtgc gactgcgctg gttcctaatt tccacgcatc caccgaagct 1980

gtgcgccgag tccttcccac tgaagtcact cacatcgatg cgcgatttaa cgtgtcccgc 2040

gttgcagtga tgatcgttgc gttgcagcag cgtcctgatt tgctgtggga gggtactcgt 2100

gaccgtctgc accagcctta tcgtgcagaa gtgttgccta ttacctctga gtgggtaaac 2160

cgcctgcgca accgtggcta cgcggcatac ctttccggtg ccggcccaac cgccatggtg 2220

ctgtccactg agccaattcc agacaaggtt ttggaagatg ctcgtgagtc tggcattaag 2280

gtgcttgagc ttgaggttgc gggaccagtc aaggttgaag ttaaccaacc ttaggcccaa 2340

caaggaaggc ccccttcgaa tcaagaaggg ggccttatta gtgcagcaat tattcgctga 2400

acacgtgaac cttacaggtg cccggcgcgt tgagtggttt gagttccagc tggatgcggt 2460

tgttttcacc gaggctttct tggatgaatc cggcgtggat ggcgcagacg aaggctgatg 2520

ggcgtttgtc gttgaccaca aatgggcagc tgtgtagagc gagggagttt gcttcttcgg 2580

tttcggtggg gtcaaagccc atttcgcgga ggcggttaat gagcggggag agggcttcgt 2640

cgagttcttc ggcttcggcg tggttaatgc ccatgacgtg tgcccactgg gttccgatgg 2700

aaagtgcttt ggcgcggagg tcggggttgt gcattgcgtc atcgtcgaca tcgccgagca 2760

tgttggccat gagttcgatc agggtgatgt attctttggc gacagcgcgg ttgtcgggga 2820

cgcgtgtttg gaagatgagg gaggggcggg atcctctaga cccgggattt aaatcgctag 2880

cgggctgcta aaggaagcgg aacacgtaga aagccagtcc gcagaaacgg tgctgacccc 2940

ggatgaatgt cagctactgg gctatctgga caagggaaaa cgcaagcgca aagagaaagc 3000

aggtagcttg cagtgggctt acatggcgat agctagactg ggcggtttta tggacagcaa 3060

gcgaaccgga attgccagct ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa 3120

actggatggc tttcttgccg ccaaggatct gatggcgcag gggatcaaga tctgatcaag 3180

agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg 3240

ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg 3300

atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc 3360

tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga 3420

cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc 3480

tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag 3540

tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat 3600

tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg 3660

tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca 3720

ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct 3780

tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg 3840

gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg 3900

gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc 3960

gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat 4020

gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta 4080

tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg 4140

ggatctcatg ctggagttct tcgcccacgc tagcggcgcg ccggccggcc cggtgtgaaa 4200

taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca 4260

ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 4320

taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 4380

agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 4440

cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 4500

tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 4560

tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 4620

gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 4680

acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 4740

acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 4800

cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 4860

gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 4920

gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 4980

agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 5040

ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 5100

ggatcttcac ctagatcctt ttaaaggccg gccgcggccg ccatcggcat tttcttttgc 5160

gtttttattt gttaactgtt aattgtcctt gttcaaggat gctgtctttg acaacagatg 5220

ttttcttgcc tttgatgttc agcaggaagc tcggcgcaaa cgttgattgt ttgtctgcgt 5280

agaatcctct gtttgtcata tagcttgtaa tcacgacatt gtttcctttc gcttgaggta 5340

cagcgaagtg tgagtaagta aaggttacat cgttaggatc aagatccatt tttaacacaa 5400

ggccagtttt gttcagcggc ttgtatgggc cagttaaaga attagaaaca taaccaagca 5460

tgtaaatatc gttagacgta atgccgtcaa tcgtcatttt tgatccgcgg gagtcagtga 5520

acaggtacca tttgccgttc attttaaaga cgttcgcgcg ttcaatttca tctgttactg 5580

tgttagatgc aatcagcggt ttcatcactt ttttcagtgt gtaatcatcg tttagctcaa 5640

tcataccgag agcgccgttt gctaactcag ccgtgcgttt tttatcgctt tgcagaagtt 5700

tttgactttc ttgacggaag aatgatgtgc ttttgccata gtatgctttg ttaaataaag 5760

attcttcgcc ttggtagcca tcttcagttc cagtgtttgc ttcaaatact aagtatttgt 5820

ggcctttatc ttctacgtag tgaggatctc tcagcgtatg gttgtcgcct gagctgtagt 5880

tgccttcatc gatgaactgc tgtacatttt gatacgtttt tccgtcaccg tcaaagattg 5940

atttataatc ctctacaccg ttgatgttca aagagctgtc tgatgctgat acgttaactt 6000

gtgcagttgt cagtgtttgt ttgccgtaat gtttaccgga gaaatcagtg tagaataaac 6060

ggatttttcc gtcagatgta aatgtggctg aacctgacca ttcttgtgtt tggtctttta 6120

ggatagaatc atttgcatcg aatttgtcgc tgtctttaaa gacgcggcca gcgtttttcc 6180

agctgtcaat agaagtttcg ccgacttttt gatagaacat gtaaatcgat gtgtcatccg 6240

catttttagg atctccggct aatgcaaaga cgatgtggta gccgtgatag tttgcgacag 6300

tgccgtcagc gttttgtaat ggccagctgt cccaaacgtc caggcctttt gcagaagaga 6360

tatttttaat tgtggacgaa tcaaattcag aaacttgata tttttcattt ttttgctgtt 6420

cagggatttg cagcatatca tggcgtgtaa tatgggaaat gccgtatgtt tccttatatg 6480

gcttttggtt cgtttctttc gcaaacgctt gagttgcgcc tcctgccagc agtgcggtag 6540

taaaggttaa tactgttgct tgttttgcaa actttttgat gttcatcgtt catgtctcct 6600

tttttatgta ctgtgttagc ggtctgcttc ttccagccct cctgtttgaa gatggcaagt 6660

tagttacgca caataaaaaa agacctaaaa tatgtaaggg gtgacgccaa agtatacact 6720

ttgcccttta cacattttag gtcttgcctg ctttatcagt aacaaacccg cgcgatttac 6780

ttttcgacct cattctatta gactctcgtt tggattgcaa ctggtctatt ttcctctttt 6840

gtttgataga aaatcataaa aggatttgca gactacgggc ctaaagaact aaaaaatcta 6900

tctgtttctt ttcattctct gtatttttta tagtttctgt tgcatgggca taaagttgcc 6960

tttttaatca caattcagaa aatatcataa tatctcattt cactaaataa tagtgaacgg 7020

caggtatatg tgatgggtta aaaaggatcg gcggccgctc gatttaaatc 7070

<210>2

<211>7070

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Nucleotide sequence

<400>2

tcgagaggcc tgacgtcggg cccggtacca cgcgtcatat gactagttgg agaatcatga 60

cctcagcatc tgccccaagc tttaaccccg gcaagggtcc cggctcagca gtcggaattg 120

cccttttagg attcggaaca gtcggcactg aggtgatgcg tctgatgacc gagtacggtg 180

atgaacttgc gcaccgcatt ggtggcccac tggaggttcg tggcattgct gtttctgata 240

tctcaaagcc acgtgaaggc gttgcacctg agctgctcac tgaggacgct tttgcactca 300

tcgagcgcga ggatgttgac atcgtcgttg aggttatcgg cggcattgag tacccacgtg 360

aggtagttct cgcagctctg aaggccggca agtctgttgt taccgccaat aaggctcttg 420

ttgcagctca ctctgctgag cttgctgatg cagcggaagc cgcaaacgtt gacctgtact 480

tcgaggctgc tgttgcaggc gcaattccag tggttggccc actgcgtcgc tccctggctg 540

gcgatcagat ccagtctgtg atgggcatcg ttaacggcac caccaacttc atcttggacg 600

ccatggattc caccggcgct gactatgcag attctttggc tgaggcaact cgtttgggtt 660

acgccgaagc tgatccaact gcagacgtcg aaggccatga cgccgcatcc aaggctgcaa 720

ttttggcatc catcgctttc cacacccgtg ttaccgcgga tgatgtgtac tgcgaaggta 780

tcagcaacat cagcgctgcc gacattgagg cagcacagca ggcaggccac accatcaagt 840

tgttggccat ctgtgagaag ttcaccaaca aggaaggaaa gtcggctatt tctgctcgcg 900

tgcacccgac tctattacct gtgtcccacc cactggcgtc ggtaaacaag tcctttaatg 960

caatctttgt tgaagcagaa gcagctggtc gcctgatgtt ctacggaaac ggtgcaggtg 1020

gcgcgccaac cgcgtctgct gtgcttggcg acgtcgttgg tgccgcacga aacaaggtgc 1080

acggtggccg tgctccaggt gagtccacct acgctaacct gccgatcgct gatttcggtg 1140

agaccaccac tcgttaccac ctcgacatgg atgtggaaga tcgcgtgggg gttttggctg 1200

aattggctag cctgttctct gagcaaggaa tcttcctgcg tacaatccga caggaagagc 1260

gcgatgatga tgcacgtctg atcgtggtca cccactctgc gctggaatct gatctttccc 1320

gcaccgttga actgctgaag gctaagcctg ttgttaaggc aatcaacagt gtgatccgcc 1380

tcgaaaggga ctaattttac tgacatggca attgaactga acgtcggtcg taaggttacc 1440

gtcacggtac ctggatcttc tgcaaacctc ggacctggct ttgacacttt aggtttggca 1500

ctgtcggtat acgacactgt cgaagtggaa attattccat ctggcttgga agtggaagtt 1560

tttggcgaag gccaaggcga agtccctctt gatggctccc acctggtggt taaagctatt 1620

cgtgctggcc tgaaggcagc tgacgctgaa gttcctggat tgcgagtggt gtgccacaac 1680

aacattccgc agtctcgtgg tcttggctcc tctgctgcag cggcggttgc tggtgttgct 1740

gcagctaatg gtttggcgga tttcccgctg actcaagagc agattgttca gttgtcctct 1800

gcctttgaag gccacccaga taatgctgcg gcttctgtgc tgggtggagc agtggtgtcg 1860

tggacaaatc tgtctatcga cggcaagagc cagccacagt atgctgctgt accacttgag 1920

gtgcaggaca atattcgtgc gactgcgctg gttcctaatt tccacgcatc caccgaagct 1980

gtgcgccgag tccttcccac tgaagtcact cacatcgatg cgcgatttaa cgtgtcccgc 2040

gttgcagtga tgatcgttgc gttgcagcag cgtcctgatt tgctgtggga gggtactcgt 2100

gaccgtctgc accagcctta tcgtgcagaa gtgttgccta ttacctctga gtgggtaaac 2160

cgcctgcgca accgtggcta cgcggcatac ctttccggtg ccggcccaac cgccatggtg 2220

ctgtccactg agccaattcc agacaaggtt ttggaagatg ctcgtgagtc tggcattaag 2280

gtgcttgagc ttgaggttgc gggaccagtc aaggttgaag ttaaccaacc ttaggcccaa 2340

caaggaaggc ccccttcgaa tcaagaaggg ggccttatta gtgcagcaat tattcgctga 2400

acacgtgaac cttacaggtg cccggcgcgt tgagtggttt gagttccagc tggatgcggt 2460

tgttttcacc gaggctttct tggatgaatc cggcgtggat ggcgcagacg aaggctgatg 2520

ggcgtttgtc gttgaccaca aatgggcagc tgtgtagagc gagggagttt gcttcttcgg 2580

tttcggtggg gtcaaagccc atttcgcgga ggcggttaat gagcggggag agggcttcgt 2640

cgagttcttc ggcttcggcg tggttaatgc ccatgacgtg tgcccactgg gttccgatgg 2700

aaagtgcttt ggcgcggagg tcggggttgt gcattgcgtc atcgtcgaca tcgccgagca 2760

tgttggccat gagttcgatc agggtgatgt attctttggc gacagcgcgg ttgtcgggga 2820

cgcgtgtttg gaagatgagg gaggggcggg atcctctaga cccgggattt aaatcgctag 2880

cgggctgcta aaggaagcgg aacacgtaga aagccagtcc gcagaaacgg tgctgacccc 2940

ggatgaatgt cagctactgg gctatctgga caagggaaaa cgcaagcgca aagagaaagc 3000

aggtagcttg cagtgggctt acatggcgat agctagactg ggcggtttta tggacagcaa 3060

gcgaaccgga attgccagct ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa 3120

actggatggc tttcttgccg ccaaggatct gatggcgcag gggatcaaga tctgatcaag 3180

agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg 3240

ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg 3300

atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc 3360

tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga 3420

cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc 3480

tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag 3540

tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat 3600

tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg 3660

tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca 3720

ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct 3780

tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg 3840

gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg 3900

gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc 3960

gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat 4020

gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta 4080

tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg 4140

ggatctcatg ctggagttct tcgcccacgc tagcggcgcg ccggccggcc cggtgtgaaa 4200

taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca 4260

ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 4320

taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 4380

agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 4440

cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 4500

tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 4560

tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 4620

gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 4680

acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 4740

acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 4800

cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 4860

gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 4920

gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 4980

agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 5040

ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 5100

ggatcttcac ctagatcctt ttaaaggccg gccgcggccg ccatcggcat tttcttttgc 5160

gtttttattt gttaactgtt aattgtcctt gttcaaggat gctgtctttg acaacagatg 5220

ttttcttgcc tttgatgttc agcaggaagc tcggcgcaaa cgttgattgt ttgtctgcgt 5280

agaatcctct gtttgtcata tagcttgtaa tcacgacatt gtttcctttc gcttgaggta 5340

cagcgaagtg tgagtaagta aaggttacat cgttaggatc aagatccatt tttaacacaa 5400

ggccagtttt gttcagcggc ttgtatgggc cagttaaaga attagaaaca taaccaagca 5460

tgtaaatatc gttagacgta atgccgtcaa tcgtcatttt tgatccgcgg gagtcagtga 5520

acaggtacca tttgccgttc attttaaaga cgttcgcgcg ttcaatttca tctgttactg 5580

tgttagatgc aatcagcggt ttcatcactt ttttcagtgt gtaatcatcg tttagctcaa 5640

tcataccgag agcgccgttt gctaactcag ccgtgcgttt tttatcgctt tgcagaagtt 5700

tttgactttc ttgacggaag aatgatgtgc ttttgccata gtatgctttg ttaaataaag 5760

attcttcgcc ttggtagcca tcttcagttc cagtgtttgc ttcaaatact aagtatttgt 5820

ggcctttatc ttctacgtag tgaggatctc tcagcgtatg gttgtcgcct gagctgtagt 5880

tgccttcatc gatgaactgc tgtacatttt gatacgtttt tccgtcaccg tcaaagattg 5940

atttataatc ctctacaccg ttgatgttca aagagctgtc tgatgctgat acgttaactt 6000

gtgcagttgt cagtgtttgt ttgccgtaat gtttaccgga gaaatcagtg tagaataaac 6060

ggatttttcc gtcagatgta aatgtggctg aacctgacca ttcttgtgtt tggtctttta 6120

ggatagaatc atttgcatcg aatttgtcgc tgtctttaaa gacgcggcca gcgtttttcc 6180

agctgtcaat agaagtttcg ccgacttttt gatagaacat gtaaatcgat gtgtcatccg 6240

catttttagg atctccggct aatgcaaaga cgatgtggta gccgtgatag tttgcgacag 6300

tgccgtcagc gttttgtaat ggccagctgt cccaaacgtc caggcctttt gcagaagaga 6360

tatttttaat tgtggacgaa tcaaattcag aaacttgata tttttcattt ttttgctgtt 6420

cagggatttg cagcatatca tggcgtgtaa tatgggaaat gccgtatgtt tccttatatg 6480

gcttttggtt cgtttctttc gcaaacgctt gagttgcgcc tcctgccagc agtgcggtag 6540

taaaggttaa tactgttgct tgttttgcaa actttttgat gttcatcgtt catgtctcct 6600

tttttatgta ctgtgttagc ggtctgcttc ttccagccct cctgtttgaa gatggcaagt 6660

tagttacgca caataaaaaa agacctaaaa tatgtaaggg gtgacgccaa agtatacact 6720

ttgcccttta cacattttag gtcttgcctg ctttatcagt aacaaacccg cgcgatttac 6780

ttttcgacct cattctatta gactctcgtt tggattgcaa ctggtctatt ttcctctttt 6840

gtttgataga aaatcataaa aggatttgca gactacgggc ctaaagaact aaaaaatcta 6900

tctgtttctt ttcattctct gtatttttta tagtttctgt tgcatgggca taaagttgcc 6960

tttttaatca caattcagaa aatatcataa tatctcattt cactaaataa tagtgaacgg 7020

caggtatatg tgatgggtta aaaaggatcg gcggccgctc gatttaaatc 7070

<210>3

<211>8766

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Nucleotide sequence

<400>3

tcgagaggcc tgacgtcggg cccggtacca cgcgtcatat gactagttcg gacctaggga 60

tatcgtcgac atcgatgctc ttctgcgtta attaacaatt gggatctctc aactaatgca 120

gcgatgcgtt ctttccagaa tgctttcatg acagggatgc tgtcttgatc aggcaggcgt 180

ctgtgctgga tgccgaagct ggatttattg tcgcctttgg aggtgaagtt gacgctcact 240

cgagaatcat cggccaacca tttggcattg aatgttctag gttcggaggc ggaggttttc 300

tcaattagtg cgggatcgag ccactgcgcc cgcaggtcat cgtctccgaa gagcttccac 360

actttttcga ccggcaggtt aagggttttg gaggcattgg ccgcgaaccc atcgctggtc 420

atcccgggtt tgcgcatgcc acgttcgtat tcataaccaa tcgcgatgcc ttgagcccac 480

cagccactga catcaaagtt gtccacgatg tgctttgcga tgtgggtgtg agtccaagag 540

gtggctttta cgtcgtcaag caattttagc cactcttccc acggctttcc ggtgccgttg 600

aggatagctt caggggacat gcctggtgtt gagccttgcg gagtggagtc agtcatgcga 660

ccgagactag tggcgctttg ggtaccgggc cccccctcga ggtcgagcgg cttaaagttt 720

ggctgccatg tgaattttta gcaccctcaa cagttgagtg ctggcactct cgggggtaga 780

gtgccaaata ggttgtttga cacacagttg ttcacccgcg acgacggctg tgctggaaac 840

ccacaaccgg cacacacaaa atttttctca tggagggatt catcatgtcg acttcagtta 900

cttcaccagc ccacaacaac gcacattcct ccgaattttt ggatgcgttg gcaaaccatg 960

tgttgatcgg cgacggcgcc atgggcaccc agctccaagg ctttgacctg gacgtggaaa 1020

aggatttcct tgatctggag gggtgtaatg agattctcaa cgacacccgc cctgatgtgt 1080

tgaggcagat tcaccgcgcc tactttgagg cgggagctga cttggttgag accaatactt 1140

ttggttgcaa cctgccgaac ttggcggatt atgacatcgc tgatcgttgc cgtgagcttg 1200

cctacaaggg cactgcagtg gctagggaag tggctgatga gatggggccg ggccgaaacg 1260

gcatgcggcg tttcgtggtt ggttccctgg gacctggaac gaagcttcca tcgctgggcc 1320

atgcaccgta tgcagatttg cgtgggcact acaaggaagc agcgcttggc atcatcgacg 1380

gtggtggcga tgcctttttg attgagactg ctcaggactt gcttcaggtc aaggctgcgg 1440

ttcacggcgt tcaagatgcc atggctgaac ttgatacatt cttgcccatt atttgccacg 1500

tcaccgtaga gaccaccggc accatgctca tgggttctga gatcggtgcc gcgttgacag 1560

cgctgcagcc actgggtatc gacatgattg gtctgaactg cgccaccggc ccagatgaga 1620

tgagcgagca cctgcgttac ctgtccaagc acgccgatat tcctgtgtcg gtgatgccta 1680

acgcaggtct tcctgtcctg ggtaaaaacg gtgcagaata cccacttgag gctgaggatt 1740

tggcgcaggc gctggctgga ttcgtctccg aatatggcct gtccatggtg ggtggttgtt 1800

gtggcaccac acctgagcac atccgtgcgg tccgcgatgc ggtggttggt gttccagagc 1860

aggaaacctc cacactgacc aagatccctg caggccctgt tgagcaggcc tcccgcgagg 1920

tggagaaaga ggactccgtc gcgtcgctgt acacctcggt gccattgtcc caggaaaccg 1980

gcatttccat gatcggtgag cgcaccaact ccaacggttc caaggcattc cgtgaggcaa 2040

tgctgtctgg cgattgggaa aagtgtgtgg atattgccaa gcagcaaacc cgcgatggtg 2100

cacacatgct ggatctttgt gtggattacg tgggacgaga cggcaccgcc gatatggcga 2160

ccttggcagc acttcttgct accagctcca ctttgccaat catgattgac tccaccgagc 2220

cagaggttat tcgcacaggc cttgagcact tgggtggacg aagcatcgtt aactccgtca 2280

actttgaaga cggcgatggc cctgagtccc gctaccagcg catcatgaaa ctggtaaagc 2340

agcacggtgc ggccgtggtt gcgctgacca ttgatgagga aggccaggca cgtaccgctg 2400

agcacaaggt gcgcattgct aaacgactga ttgacgatat caccggcagc tacggcctgg 2460

atatcaaaga catcgttgtg gactgcctga ccttcccgat ctctactggc caggaagaaa 2520

ccaggcgaga tggcattgaa accatcgaag ccatccgcga gctgaagaag ctctacccag 2580

aaatccacac caccctgggt ctgtccaata tttccttcgg cctgaaccct gctgcacgcc 2640

aggttcttaa ctctgtgttc ctcaatgagt gcattgaggc tggtctggac tctgcgattg 2700

cgcacagctc caagattttg ccgatgaacc gcattgatga tcgccagcgc gaagtggcgt 2760

tggatatggt ctatgatcgc cgcaccgagg attacgatcc gctgcaggaa ttcatgcagc 2820

tgtttgaggg cgtttctgct gccgatgcca aggatgctcg cgctgaacag ctggccgcta 2880

tgcctttgtt tgagcgtttg gcacagcgca tcatcgacgg cgataagaat ggccttgagg 2940

atgatctgga agcaggcatg aaggagaagt ctcctattgc gatcatcaac gaggaccttc 3000

tcaacggcat gaagaccgtg ggtgagctgt ttggttccgg acagatgcag ctgccattcg 3060

tgctgcaatc ggcagaaacc atgaaaactg cggtggccta tttggaaccg ttcatggaag 3120

aggaagcaga agctaccgga tctgcgcagg cagagggcaa gggcaaaatc gtcgtggcca 3180

ccgtcaaggg tgacgtgcac gatatcggca agaacttggt ggacatcatt ttgtccaaca 3240

acggttacga cgtggtgaac ttgggcatca agcagccact gtccgccatg ttggaagcag 3300

cggaagaaca caaagcagac gtcatcggca tgtcgggact tcttgtgaag tccaccgtgg 3360

tgatgaagga aaaccttgag gagatgaaca acgccggcgc atccaattac ccagtcattt 3420

tgggtggcgc tgcgctgacg cgtacctacg tggaaaacga tctcaacgag gtgtacaccg 3480

gtgaggtgta ctacgcccgt gatgctttcg agggcctgcg cctgatggat gaggtgatgg 3540

cagaaaagcg tggtgaagga cttgatccca actcaccaga agctattgag caggcgaaga 3600

agaaggcgga acgtaaggct cgtaatgagc gttcccgcaa gattgccgcg gagcgtaaag 3660

ctaatgcggc tcccgtgatt gttccggagc gttctgatgt ctccaccgat actccaaccg 3720

cggcaccacc gttctgggga acccgcattg tcaagggtct gcccttggcg gagttcttgg 3780

gcaaccttga tgagcgcgcc ttgttcatgg ggcagtgggg tctgaaatcc acccgcggca 3840

acgagggtcc aagctatgag gatttggtgg aaactgaagg ccgaccacgc ctgcgctact 3900

ggctggatcg cctgaagtct gagggcattt tggaccacgt ggccttggtg tatggctact 3960

tcccagcggt cgcggaaggc gatgacgtgg tgatcttgga atccccggat ccacacgcag 4020

ccgaacgcat gcgctttagc ttcccacgcc agcagcgcgg caggttcttg tgcatcgcgg 4080

atttcattcg cccacgcgag caagctgtca aggacggcca agtggacgtc atgccattcc 4140

agctggtcac catgggtaat cctattgctg atttcgccaa cgagttgttc gcagccaatg 4200

aataccgcga gtacttggaa gttcacggca tcggcgtgca gctcaccgaa gcattggccg 4260

agtactggca ctcccgagtg cgcagcgaac tcaagctgaa cgacggtgga tctgtcgctg 4320

attttgatcc agaagacaag accaagttct tcgacctgga ttaccgcggc gcccgcttct 4380

cctttggtta cggttcttgc cctgatctgg aagaccgcgc aaagctggtg gaattgctcg 4440

agccaggccg tatcggcgtg gagttgtccg aggaactcca gctgcaccca gagcagtcca 4500

cagacgcgtt tgtgctctac cacccagagg caaagtactt taacgtctaa tctagacccg 4560

ggatttaaat cgctagcggg ctgctaaagg aagcggaaca cgtagaaagc cagtccgcag 4620

aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca 4680

agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg 4740

gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg 4800

aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga 4860

tcaagatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg 4920

cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag 4980

acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt 5040

tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta 5100

tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg 5160

ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt 5220

gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat 5280

ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg 5340

atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca 5400

gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc 5460

catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc 5520

gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat 5580

attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc 5640

gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga 5700

ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt 5760

ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga 5820

tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccacgctagc ggcgcgccgg 5880

ccggcccggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct 5940

tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 6000

gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 6060

atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 6120

ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 6180

cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 6240

tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 6300

gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 6360

aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 6420

tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 6480

aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 6540

aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 6600

ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 6660

ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 6720

atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 6780

atgagattat caaaaaggat cttcacctag atccttttaa aggccggccg cggccgccat 6840

cggcattttc ttttgcgttt ttatttgtta actgttaatt gtccttgttc aaggatgctg 6900

tctttgacaa cagatgtttt cttgcctttg atgttcagca ggaagctcgg cgcaaacgtt 6960

gattgtttgt ctgcgtagaa tcctctgttt gtcatatagc ttgtaatcac gacattgttt 7020

cctttcgctt gaggtacagc gaagtgtgag taagtaaagg ttacatcgtt aggatcaaga 7080

tccattttta acacaaggcc agttttgttc agcggcttgt atgggccagt taaagaatta 7140

gaaacataac caagcatgta aatatcgtta gacgtaatgc cgtcaatcgt catttttgat 7200

ccgcgggagt cagtgaacag gtaccatttg ccgttcattt taaagacgtt cgcgcgttca 7260

atttcatctg ttactgtgtt agatgcaatc agcggtttca tcactttttt cagtgtgtaa 7320

tcatcgttta gctcaatcat accgagagcg ccgtttgcta actcagccgt gcgtttttta 7380

tcgctttgca gaagtttttg actttcttga cggaagaatg atgtgctttt gccatagtat 7440

gctttgttaa ataaagattc ttcgccttgg tagccatctt cagttccagt gtttgcttca 7500

aatactaagt atttgtggcc tttatcttct acgtagtgag gatctctcag cgtatggttg 7560

tcgcctgagc tgtagttgcc ttcatcgatg aactgctgta cattttgata cgtttttccg 7620

tcaccgtcaa agattgattt ataatcctct acaccgttga tgttcaaaga gctgtctgat 7680

gctgatacgt taacttgtgc agttgtcagt gtttgtttgc cgtaatgttt accggagaaa 7740

tcagtgtaga ataaacggat ttttccgtca gatgtaaatg tggctgaacc tgaccattct 7800

tgtgtttggt cttttaggat agaatcattt gcatcgaatt tgtcgctgtc tttaaagacg 7860

cggccagcgt ttttccagct gtcaatagaa gtttcgccga ctttttgata gaacatgtaa 7920

atcgatgtgt catccgcatt tttaggatct ccggctaatg caaagacgat gtggtagccg 7980

tgatagtttg cgacagtgcc gtcagcgttt tgtaatggcc agctgtccca aacgtccagg 8040

ccttttgcag aagagatatt tttaattgtg gacgaatcaa attcagaaac ttgatatttt 8100

tcattttttt gctgttcagg gatttgcagc atatcatggc gtgtaatatg ggaaatgccg 8160

tatgtttcct tatatggctt ttggttcgtt tctttcgcaa acgcttgagt tgcgcctcct 8220

gccagcagtg cggtagtaaa ggttaatact gttgcttgtt ttgcaaactt tttgatgttc 8280

atcgttcatg tctccttttt tatgtactgt gttagcggtc tgcttcttcc agccctcctg 8340

tttgaagatg gcaagttagt tacgcacaat aaaaaaagac ctaaaatatg taaggggtga 8400

cgccaaagta tacactttgc cctttacaca ttttaggtct tgcctgcttt atcagtaaca 8460

aacccgcgcg atttactttt cgacctcatt ctattagact ctcgtttgga ttgcaactgg 8520

tctattttcc tcttttgttt gatagaaaat cataaaagga tttgcagact acgggcctaa 8580

agaactaaaa aatctatctg tttcttttca ttctctgtat tttttatagt ttctgttgca 8640

tgggcataaa gttgcctttt taatcacaat tcagaaaata tcataatatc tcatttcact 8700

aaataatagt gaacggcagg tatatgtgat gggttaaaaa ggatcggcgg ccgctcgatt 8760

taaatc 8766

<210>4

<211>184

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Promoter sequence

<400>4

ggtcgagcgg cttaaagttt ggctgccatg tgaattttta gcaccctcaa cagttgagtg 60

ctggcactct cgggggtaga gtgccaaata ggttgtttga cacacagttg ttcacccgcg 120

acgacggctg tgctggaaac ccacaaccgg cacacacaaa atttttctca tggagggatt 180

catc 184

<210>5

<211>7070

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Nucleotide sequence

<400>5

tcgagaggcc tgacgtcggg cccggtacca cgcgtcatat gactagttgg agaatcatga 60

cctcagcatc tgccccaagc tttaaccccg gcaagggtcc cggctcagca gtcggaattg 120

cccttttagg attcggaaca gtcggcactg aggtgatgcg tctgatgacc gagtacggtg 180

atgaacttgc gcaccgcatt ggtggcccac tggaggttcg tggcattgct gtttctgata 240

tctcaaagcc acgtgaaggc gttgcacctg agctgctcac tgaggacgct tttgcactca 300

tcgagcgcga ggatgttgac atcgtcgttg aggttatcgg cggcattgag tacccacgtg 360

aggtagttct cgcagctctg aaggccggca agtctgttgt taccgccaat aaggctcttg 420

ttgcagctca ctctgctgag cttgctgatg cagcggaagc cgcaaacgtt gacctgtact 480

tcgaggctgc tgttgcaggc gcaattccag tggttggccc actgcgtcgc tccctggctg 540

gcgatcagat ccagtctgtg atgggcatcg ttaacggcac caccaacttc atcttggacg 600

ccatggattc caccggcgct gactatgcag attctttggc tgaggcaact cgtttgggtt 660

acgccgaagc tgatccaact gcagacgtcg aaggccatga cgccgcatcc aaggctgcaa 720

ttttggcatc catcgctttc cacacccgtg ttaccgcgga tgatgtgtac tgcgaaggta 780

tcagcaacat cagcgctgcc gacattgagg cagcacagca ggcaggccac accatcaagt 840

tgttggccat ctgtgagaag ttcaccaaca aggaaggaaa gtcggctatt tctgctcgcg 900

tgcacccgac tctattacct gtgtcccacc cactggcgtc ggtaaacaag tcctttaatg 960

caatctttgt tgaagcagaa gcagctggtc gcctgatgtt ctacggaaac ggtgcaggtg 1020

gcgcgccaac cgcgtctgct gtgcttggcg acgtcgttgg tgccgcacga aacaaggtgc 1080

acggtggccg tgctccaggt gagtccacct acgctaacct gccgatcgct gatttcggtg 1140

agaccaccac tcgttaccac ctcgacatgg atgtggaaga tcgcgtgggg gttttggctg 1200

aattggctag cctgttctct gagcaaggaa tcttcctgcg tacaatccga caggaagagc 1260

gcgatgatga tgcacgtctg atcgtggtca cccactctgc gctggaatct gatctttccc 1320

gcaccgttga actgctgaag gctaagcctg ttgttaaggc aatcaacagt gtgatccgcc 1380

tcgaaaggga ctaattttac tgacatggca attgaactga acgtcggtcg taaggttacc 1440

gtcacggtac ctggatcttc tgcaaacctc ggacctggct ttgacacttt aggtttggca 1500

ctgtcggtat acgacactgt cgaagtggaa attattccat ctggcttgga agtggaagtt 1560

tttggcgaag gccaaggcga agtccctctt gatggctccc acctggtggt taaagctatt 1620

cgtgctggcc tgaaggcagc tgacgctgaa gttcctggat tgcgagtggt gtgccacaac 1680

aacattccgc agtctcgtgg tcttggctcc gctgctgcag cggcggttgc tggtgttgct 1740

gcagctaatg gtttggcgga tttcccgctg actcaagagc agattgttca gttgtcctct 1800

gcctttgaag gccacccaga taatgctgcg gcttctgtgc tgggtggagc agtggtgtcg 1860

tggacaaatc tgtctatcga cggcaagagc cagccacagt atgctgctgt accacttgag 1920

gtgcaggaca atattcgtgc gactgcgctg gttcctaatt tccacgcatc caccgaagct 1980

gtgcgccgag tccttcccac tgaagtcact cacatcgatg cgcgatttaa cgtgtcccgc 2040

gttgcagtga tgatcgttgc gttgcagcag cgtcctgatt tgctgtggga gggtactcgt 2100

gaccgtctgc accagcctta tcgtgcagaa gtgttgccta ttacctctga gtgggtaaac 2160

cgcctgcgca accgtggcta cgcggcatac ctttccggtg ccggcccaac cgccatggtg 2220

ctgtccactg agccaattcc agacaaggtt ttggaagatg ctcgtgagtc tggcattaag 2280

gtgcttgagc ttgaggttgc gggaccagtc aaggttgaag ttaaccaacc ttaggcccaa 2340

caaggaaggc ccccttcgaa tcaagaaggg ggccttatta gtgcagcaat tattcgctga 2400

acacgtgaac cttacaggtg cccggcgcgt tgagtggttt gagttccagc tggatgcggt 2460

tgttttcacc gaggctttct tggatgaatc cggcgtggat ggcgcagacg aaggctgatg 2520

ggcgtttgtc gttgaccaca aatgggcagc tgtgtagagc gagggagttt gcttcttcgg 2580

tttcggtggg gtcaaagccc atttcgcgga ggcggttaat gagcggggag agggcttcgt 2640

cgagttcttc ggcttcggcg tggttaatgc ccatgacgtg tgcccactgg gttccgatgg 2700

aaagtgcttt ggcgcggagg tcggggttgt gcattgcgtc atcgtcgaca tcgccgagca 2760

tgttggccat gagttcgatc agggtgatgt attctttggc gacagcgcgg ttgtcgggga 2820

cgcgtgtttg gaagatgagg gaggggcggg atcctctaga cccgggattt aaatcgctag 2880

cgggctgcta aaggaagcgg aacacgtaga aagccagtcc gcagaaacgg tgctgacccc 2940

ggatgaatgt cagctactgg gctatctgga caagggaaaa cgcaagcgca aagagaaagc 3000

aggtagcttg cagtgggctt acatggcgat agctagactg ggcggtttta tggacagcaa 3060

gcgaaccgga attgccagct ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa 3120

actggatggc tttcttgccg ccaaggatct gatggcgcag gggatcaaga tctgatcaag 3180

agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg 3240

ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg 3300

atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc 3360

tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga 3420

cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc 3480

tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag 3540

tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat 3600

tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg 3660

tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca 3720

ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct 3780

tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg 3840

gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg 3900

gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc 3960

gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat 4020

gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta 4080

tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg 4140

ggatctcatg ctggagttct tcgcccacgc tagcggcgcg ccggccggcc cggtgtgaaa 4200

taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca 4260

ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 4320

taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 4380

agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 4440

cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 4500

tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 4560

tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 4620

gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 4680

acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 4740

acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 4800

cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 4860

gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 4920

gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 4980

agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 5040

ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 5100

ggatcttcac ctagatcctt ttaaaggccg gccgcggccg ccatcggcat tttcttttgc 5160

gtttttattt gttaactgtt aattgtcctt gttcaaggat gctgtctttg acaacagatg 5220

ttttcttgcc tttgatgttc agcaggaagc tcggcgcaaa cgttgattgt ttgtctgcgt 5280

agaatcctct gtttgtcata tagcttgtaa tcacgacatt gtttcctttc gcttgaggta 5340

cagcgaagtg tgagtaagta aaggttacat cgttaggatc aagatccatt tttaacacaa 5400

ggccagtttt gttcagcggc ttgtatgggc cagttaaaga attagaaaca taaccaagca 5460

tgtaaatatc gttagacgta atgccgtcaa tcgtcatttt tgatccgcgg gagtcagtga 5520

acaggtacca tttgccgttc attttaaaga cgttcgcgcg ttcaatttca tctgttactg 5580

tgttagatgc aatcagcggt ttcatcactt ttttcagtgt gtaatcatcg tttagctcaa 5640

tcataccgag agcgccgttt gctaactcag ccgtgcgttt tttatcgctt tgcagaagtt 5700

tttgactttc ttgacggaag aatgatgtgc ttttgccata gtatgctttg ttaaataaag 5760

attcttcgcc ttggtagcca tcttcagttc cagtgtttgc ttcaaatact aagtatttgt 5820

ggcctttatc ttctacgtag tgaggatctc tcagcgtatg gttgtcgcct gagctgtagt 5880

tgccttcatc gatgaactgc tgtacatttt gatacgtttt tccgtcaccg tcaaagattg 5940

atttataatc ctctacaccg ttgatgttca aagagctgtc tgatgctgat acgttaactt 6000

gtgcagttgt cagtgtttgt ttgccgtaat gtttaccgga gaaatcagtg tagaataaac 6060

ggatttttcc gtcagatgta aatgtggctg aacctgacca ttcttgtgtt tggtctttta 6120

ggatagaatc atttgcatcg aatttgtcgc tgtctttaaa gacgcggcca gcgtttttcc 6180

agctgtcaat agaagtttcg ccgacttttt gatagaacat gtaaatcgat gtgtcatccg 6240

catttttagg atctccggct aatgcaaaga cgatgtggta gccgtgatag tttgcgacag 6300

tgccgtcagc gttttgtaat ggccagctgt cccaaacgtc caggcctttt gcagaagaga 6360

tatttttaat tgtggacgaa tcaaattcag aaacttgata tttttcattt ttttgctgtt 6420

cagggatttg cagcatatca tggcgtgtaa tatgggaaat gccgtatgtt tccttatatg 6480

gcttttggtt cgtttctttc gcaaacgctt gagttgcgcc tcctgccagc agtgcggtag 6540

taaaggttaa tactgttgct tgttttgcaa actttttgat gttcatcgtt catgtctcct 6600

tttttatgta ctgtgttagc ggtctgcttc ttccagccct cctgtttgaa gatggcaagt 6660

tagttacgca caataaaaaa agacctaaaa tatgtaaggg gtgacgccaa agtatacact 6720

ttgcccttta cacattttag gtcttgcctg ctttatcagt aacaaacccg cgcgatttac 6780

ttttcgacct cattctatta gactctcgtt tggattgcaa ctggtctatt ttcctctttt 6840

gtttgataga aaatcataaa aggatttgca gactacgggc ctaaagaact aaaaaatcta 6900

tctgtttctt ttcattctct gtatttttta tagtttctgt tgcatgggca taaagttgcc 6960

tttttaatca caattcagaa aatatcataa tatctcattt cactaaataa tagtgaacgg 7020

caggtatatg tgatgggtta aaaaggatcg gcggccgctc gatttaaatc 7070

<210>6

<211>6625

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Nucleotide sequence

<400>6

tcgagaggcc tgacgtcggg cccggtaccg ttgctcgctg atctttcggc ttaacaactt 60

tgtattcaat cagtcgggca tagaaagaaa acgcaatgat ataggaacca actgccgcca 120

aaaccagcca cacagagttg attgtttcgc cacgggagaa agcgattgct ccccaaccca 180

ccgccgcgat aaccccaaag acaaggagac caacgcgggc ggtcggtgac attttagggg 240

acttcttcac gcctactgga aggtcagtag cgttgctgta caccaaatca tcgtcattga 300

tgttgtcagt ctgttttatg gtcacgatct ttactgtttt ctcttcgggt cgtttcaaag 360

ccactatgcg tagaaacagc gggcagaaac agcgggcaga aactgtgtgc agaaatgcat 420

gcagaaaaag gaaagttcgg ccagatgggt gtttctgtat gccgatgatc ggatctttga 480

cagctgggta tgcgacaaat caccgagagt tgttaattct taacaatgga aaagtaacat 540

tgagagatga tttataccat cctgcaccat ttagagtggg gctagtcata cccccataac 600

cctagctgta cgcaatcgat ttcaaatcag ttggaaaaag tcaagaaaat tacccgagac 660

atatgcggct taaagtttgg ctgccatgtg aatttttagc accctcaaca gttgagtgct 720

ggcactctcg agggtagagt gccaaatagg ttgtttgaca cacagttgtt cacccgcgac 780

gacggctgtg ctggaaaccc acaaccggca cacacaaaat ttttctcatg gccgttaccc 840

tgcgaatgtc cacagggtag ctggtagttt gaaaatcaac gccgttgccc ttaggattca 900

gtaactggca cattttgtaa tgcgctagat ctgtgtgctc agtcttccag gctgcttatc 960

acagtgaaag caaaaccaat tcgtggctgc gaaagtcgta gccaccacga agtccaggag 1020

gacatacaat gccaaagtac gacaattcca atgctgacca gtggggcttt gaaacccgct 1080

ccattcacgc aggccagtca gtagacgcac agaccagcgc acgaaacctt ccgatctacc 1140

aatccaccgc tttcgtgttc gactccgctg agcacgccaa gcagcgtttc gcacttgagg 1200

atctaggccc tgtttactcc cgcctcacca acccaaccgt tgaggctttg gaaaaccgca 1260

tcgcttccct cgaaggtggc gtccacgctg tagcgttctc ctccggacag gccgcaacca 1320

ccaacgccat tttgaacctg gcaggagcgg gcgaccacat cgtcacctcc ccacgcctct 1380

acggtggcac cgagactcta ttccttatca ctcttaaccg cctgggtatc gatgtttcct 1440

tcgtggaaaa ccccgacgac cctgagtcct ggcaggcagc cgttcagcca aacaccaaag 1500

cattcttcgg cgagactttc gccaacccac aggcagacgt cctggatatt cctgcggtgg 1560

ctgaagttgc gcaccgcaac agcgttccac tgatcatcga caacaccatc gctaccgcag 1620

cgctcgtgcg cccgctcgag ctcggcgcag acgttgtcgt cgcttccctc accaagttct 1680

acaccggcaa cggctccgga ctgggcggcg tgcttatcga cggcggaaag ttcgattgga 1740

ctgtcgaaaa ggatggaaag ccagtattcc cctacttcgt cactccagat gctgcttacc 1800

acggattgaa gtacgcagac cttggtgcac cagccttcgg cctcaaggtt cgcgttggcc 1860

ttctacgcga caccggctcc accctctccg cattcaacgc atgggctgca gtccagggca 1920

tcgacaccct ttccctgcgc ctggagcgcc acaacgaaaa cgccatcaag gttgcagaat 1980

tcctcaacaa ccacgagaag gtggaaaagg ttaacttcgc aggcctgaag gattcccctt 2040

ggtacgcaac caaggaaaag cttggcctga agtacaccgg ctccgttctc accttcgaga 2100

tcaagggcgg caaggatgag gcttgggcat ttatcgacgc cctgaagcta cactccaacc 2160

ttgcaaacat cggcgatgtt cgctccctcg ttgttcaccc agcaaccacc acccattcac 2220

agtccgacga agctggcctg gcacgcgcgg gcgttaccca gtccaccgtc cgcctgtccg 2280

ttggcatcga gaccattgat gatatcatcg ctgacctcga aggcggcttt gctgcaatct 2340

agcactagtt cggacctagg gatatcgtcg acatcgatgc tcttctgcgt taattaacaa 2400

ttgggatcct ctagacccgg gatttaaatc gctagcgggc tgctaaagga agcggaacac 2460

gtagaaagcc agtccgcaga aacggtgctg accccggatg aatgtcagct actgggctat 2520

ctggacaagg gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg 2580

gcgatagcta gactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc 2640

gccctctggt aaggttggga agccctgcaa agtaaactgg atggctttct tgccgccaag 2700

gatctgatgg cgcaggggat caagatctga tcaagagaca ggatgaggat cgtttcgcat 2760

gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 2820

ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 2880

gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 2940

ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 3000

cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 3060

tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 3120

gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 3180

cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 3240

gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg 3300

cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 3360

ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 3420

agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 3480

cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 3540

cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg 3600

ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt 3660

ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc 3720

cacgctagcg gcgcgccggc cggcccggtg tgaaataccg cacagatgcg taaggagaaa 3780

ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 3840

gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 3900

ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 3960

ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4020

acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4080

tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4140

ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4200

ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4260

ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4320

actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4380

gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4440

tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4500

caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4560

atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4620

acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 4680

ggccggccgc ggccgccatc ggcattttct tttgcgtttt tatttgttaa ctgttaattg 4740

tccttgttca aggatgctgt ctttgacaac agatgttttc ttgcctttga tgttcagcag 4800

gaagctcggc gcaaacgttg attgtttgtc tgcgtagaat cctctgtttg tcatatagct 4860

tgtaatcacg acattgtttc ctttcgcttg aggtacagcg aagtgtgagt aagtaaaggt 4920

tacatcgtta ggatcaagat ccatttttaa cacaaggcca gttttgttca gcggcttgta 4980

tgggccagtt aaagaattag aaacataacc aagcatgtaa atatcgttag acgtaatgcc 5040

gtcaatcgtc atttttgatc cgcgggagtc agtgaacagg taccatttgc cgttcatttt 5100

aaagacgttc gcgcgttcaa tttcatctgt tactgtgtta gatgcaatca gcggtttcat 5160

cacttttttc agtgtgtaat catcgtttag ctcaatcata ccgagagcgc cgtttgctaa 5220

ctcagccgtg cgttttttat cgctttgcag aagtttttga ctttcttgac ggaagaatga 5280

tgtgcttttg ccatagtatg ctttgttaaa taaagattct tcgccttggt agccatcttc 5340

agttccagtg tttgcttcaa atactaagta tttgtggcct ttatcttcta cgtagtgagg 5400

atctctcagc gtatggttgt cgcctgagct gtagttgcct tcatcgatga actgctgtac 5460

attttgatac gtttttccgt caccgtcaaa gattgattta taatcctcta caccgttgat 5520

gttcaaagag ctgtctgatg ctgatacgtt aacttgtgca gttgtcagtg tttgtttgcc 5580

gtaatgttta ccggagaaat cagtgtagaa taaacggatt tttccgtcag atgtaaatgt 5640

ggctgaacct gaccattctt gtgtttggtc ttttaggata gaatcatttg catcgaattt 5700

gtcgctgtct ttaaagacgc ggccagcgtt tttccagctg tcaatagaag tttcgccgac 5760

tttttgatag aacatgtaaa tcgatgtgtc atccgcattt ttaggatctc cggctaatgc 5820

aaagacgatg tggtagccgt gatagtttgc gacagtgccg tcagcgtttt gtaatggcca 5880

gctgtcccaa acgtccaggc cttttgcaga agagatattt ttaattgtgg acgaatcaaa 5940

ttcagaaact tgatattttt catttttttg ctgttcaggg atttgcagca tatcatggcg 6000

tgtaatatgg gaaatgccgt atgtttcctt atatggcttt tggttcgttt ctttcgcaaa 6060

cgcttgagtt gcgcctcctg ccagcagtgc ggtagtaaag gttaatactg ttgcttgttt 6120

tgcaaacttt ttgatgttca tcgttcatgt ctcctttttt atgtactgtg ttagcggtct 6180

gcttcttcca gccctcctgt ttgaagatgg caagttagtt acgcacaata aaaaaagacc 6240

taaaatatgt aaggggtgac gccaaagtat acactttgcc ctttacacat tttaggtctt 6300

gcctgcttta tcagtaacaa acccgcgcga tttacttttc gacctcattc tattagactc 6360

tcgtttggat tgcaactggt ctattttcct cttttgtttg atagaaaatc ataaaaggat 6420

ttgcagacta cgggcctaaa gaactaaaaa atctatctgt ttcttttcat tctctgtatt 6480

ttttatagtt tctgttgcat gggcataaag ttgccttttt aatcacaatt cagaaaatat 6540

cataatatct catttcacta aataatagtg aacggcaggt atatgtgatg ggttaaaaag 6600

gatcggcggc cgctcgattt aaatc 6625

<210>7

<211>363

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Promoter sequence

<400>7

cggcttaaag tttggctgcc atgtgaattt ttagcaccct caacagttga gtgctggcac 60

tctcgagggt agagtgccaa ataggttgtt tgacacacag ttgttcaccc gcgacgacgg 120

ctgtgctgga aacccacaac cggcacacac aaaatttttc tcatggccgt taccctgcga 180

atgtccacag ggtagctggt agtttgaaaa tcaacgccgt tgcccttagg attcagtaac 240

tggcacattt tgtaatgcgc tagatctgtg tgctcagtct tccaggctgc ttatcacagt 300

gaaagcaaaa ccaattcgtg gctgcgaaag tcgtagccac cacgaagtcc aggaggacat 360

aca 363

<210>8

<211>6350

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Nucleotide sequence

<400>8

tcgagctcgg cgcagacgtt gtcgtcgctt ccctcaccaa gttctacacc ggcaacggct 60

ccggactggg cggcgtgctt atcgacggcg gaaagttcga ttggactgtc gaaaaggatg 120

gaaagccagt attcccctac ttcgtcactc cagatgctgc ttaccacgga ttgaagtacg 180

cagaccttgg tgcaccagcc ttcggcctca aggttcgcgt tggccttcta cgcgacaccg 240

gctccaccct ctccgcattc aacgcatggg ctgcagtcca gggcatcgac accctttccc 300

tgcgcctgga gcgccacaac gaaaacgcca tcaaggttgc agaattcctc aacaaccacg 360

agaaggtgga aaaggttaac ttcgcaggcc tgaaggattc cccttggtac gcaaccaagg 420

aaaagcttgg cctgaagtac accggctccg ttctcacctt cgagatcaag ggcggcaagg 480

atgaggcttg ggcatttatc gacgccctga agctacactc caaccttgca aacatcggcg 540

atgttcgctc cctcgttgtt cacccagcaa ccaccaccca ttcacagtcc gacgaagctg 600

gcctggcacg cgcgggcgtt acccagtcca ccgtccgcct gtccgttggc atcgagacca 660

ttgatgatat catcgctgac ctcgaaggcg gctttgctgc aatctagcac tagttcggac 720

ctagggatat cgtcgagagc tgccaattat tccgggcttg tgacccgcta cccgataaat 780

aggtcggctg aaaaatttcg ttgcaatatc aacaaaaagg cctatcattg ggaggtgtcg 840

caccaagtac ttttgcgaag cgccatctga cggattttca aaagatgtat atgctcggtg 900

cggaaaccta cgaaaggatt ttttacccat gcccaccctc gcgccttcag gtcaacttga 960

aatccaagcg atcggtgatg tctccaccga agccggagca atcattacaa acgctgaaat 1020

cgcctatcac cgctggggtg aataccgcgt agataaagaa ggacgcagca atgtcgttct 1080

catcgaacac gccctcactg gagattccaa cgcagccgat tggtgggctg acttgctcgg 1140

tcccggcaaa gccatcaaca ctgatattta ctgcgtgatc tgtaccaacg tcatcggtgg 1200

ttgcaacggt tccaccggac ctggctccat gcatccagat ggaaatttct ggggtaatcg 1260

cttccccgcc acgtccattc gtgatcaggt aaacgccgaa aaacaattcc tcgacgcact 1320

cggcatcacc acggtcgccg cagtacttgg tggttccatg ggtggtgccc gcaccctaga 1380

gtgggccgca atgtacccag aaactgttgg cgcagctgct gttcttgcag tttctgcacg 1440

cgccagcgcc tggcaaatcg gcattcaatc cgcccaaatt aaggcgattg aaaacgacca 1500

ccactggcac gaaggcaact actacgaatc cggctgcaac ccagccaccg gactcggcgc 1560

cgcccgacgc atcgcccacc tcacctaccg tggcgaacta gaaatcgacg aacgcttcgg 1620

caccaaagcc caaaagaacg aaaacccact cggtccctac cgcaagcccg accagcgctt 1680

cgccgtggaa tcctacttgg actaccaagc agacaagcta gtacagcgtt tcgacgccgg 1740

ctcctacgtc ttgctcaccg acgccctcaa ccgccacgac attggtcgcg accgcggagg 1800

cctcaacaag gcactcgaat ccatcaaagt tccagtcctt gtcgcaggcg tagataccga 1860

tattttgtac ccctaccacc agcaagaaca cctctccaga aacctgggaa atctactggc 1920

aatggcaaaa atcgtatccc ctgtcggcca cgatgctttc ctcaccgaaa gccgccaaat 1980

ggatcgcatc gtgaggaact tcttcagcct catctcccca gacgaagaca acccttcgac 2040

ctacatcgag ttctacatct aacatatgac tagttcggac ctagggatat cgtcgacatc 2100

gatgctcttc tgcgttaatt aacaattggg atcctctaga cccgggattt aaatcgctag 2160

cgggctgcta aaggaagcgg aacacgtaga aagccagtcc gcagaaacgg tgctgacccc 2220

ggatgaatgt cagctactgg gctatctgga caagggaaaa cgcaagcgca aagagaaagc 2280

aggtagcttg cagtgggctt acatggcgat agctagactg ggcggtttta tggacagcaa 2340

gcgaaccgga attgccagct ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa 2400

actggatggc tttcttgccg ccaaggatct gatggcgcag gggatcaaga tctgatcaag 2460

agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg 2520

ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg 2580

atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc 2640

tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga 2700

cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc 2760

tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag 2820

tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat 2880

tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg 2940

tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca 3000

ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct 3060

tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg 3120

gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg 3180

gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc 3240

gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat 3300

gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta 3360

tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg 3420

ggatctcatg ctggagttct tcgcccacgc tagcggcgcg ccggccggcc cggtgtgaaa 3480

taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca 3540

ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 3600

taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 3660

agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 3720

cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 3780

tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 3840

tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 3900

gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 3960

acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 4020

acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 4080

cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 4140

gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 4200

gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 4260

agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 4320

ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 4380

ggatcttcac ctagatcctt ttaaaggccg gccgcggccg ccatcggcat tttcttttgc 4440

gtttttattt gttaactgtt aattgtcctt gttcaaggat gctgtctttg acaacagatg 4500

ttttcttgcc tttgatgttc agcaggaagc tcggcgcaaa cgttgattgt ttgtctgcgt 4560

agaatcctct gtttgtcata tagcttgtaa tcacgacatt gtttcctttc gcttgaggta 4620

cagcgaagtg tgagtaagta aaggttacat cgttaggatc aagatccatt tttaacacaa 4680

ggccagtttt gttcagcggc ttgtatgggc cagttaaaga attagaaaca taaccaagca 4740

tgtaaatatc gttagacgta atgccgtcaa tcgtcatttt tgatccgcgg gagtcagtga 4800

acaggtacca tttgccgttc attttaaaga cgttcgcgcg ttcaatttca tctgttactg 4860

tgttagatgc aatcagcggt ttcatcactt ttttcagtgt gtaatcatcg tttagctcaa 4920

tcataccgag agcgccgttt gctaactcag ccgtgcgttt tttatcgctt tgcagaagtt 4980

tttgactttc ttgacggaag aatgatgtgc ttttgccata gtatgctttg ttaaataaag 5040

attcttcgcc ttggtagcca tcttcagttc cagtgtttgc ttcaaatact aagtatttgt 5100

ggcctttatc ttctacgtag tgaggatctc tcagcgtatg gttgtcgcct gagctgtagt 5160

tgccttcatc gatgaactgc tgtacatttt gatacgtttt tccgtcaccg tcaaagattg 5220

atttataatc ctctacaccg ttgatgttca aagagctgtc tgatgctgat acgttaactt 5280

gtgcagttgt cagtgtttgt ttgccgtaat gtttaccgga gaaatcagtg tagaataaac 5340

ggatttttcc gtcagatgta aatgtggctg aacctgacca ttcttgtgtt tggtctttta 5400

ggatagaatc atttgcatcg aatttgtcgc tgtctttaaa gacgcggcca gcgtttttcc 5460

agctgtcaat agaagtttcg ccgacttttt gatagaacat gtaaatcgat gtgtcatccg 5520

catttttagg atctccggct aatgcaaaga cgatgtggta gccgtgatag tttgcgacag 5580

tgccgtcagc gttttgtaat ggccagctgt cccaaacgtc caggcctttt gcagaagaga 5640

tatttttaat tgtggacgaa tcaaattcag aaacttgata tttttcattt ttttgctgtt 5700

cagggatttg cagcatatca tggcgtgtaa tatgggaaat gccgtatgtt tccttatatg 5760

gcttttggtt cgtttctttc gcaaacgctt gagttgcgcc tcctgccagc agtgcggtag 5820

taaaggttaa tactgttgct tgttttgcaa actttttgat gttcatcgtt catgtctcct 5880

tttttatgta ctgtgttagc ggtctgcttc ttccagccct cctgtttgaa gatggcaagt 5940

tagttacgca caataaaaaa agacctaaaa tatgtaaggg gtgacgccaa agtatacact 6000

ttgcccttta cacattttag gtcttgcctg ctttatcagt aacaaacccg cgcgatttac 6060

ttttcgacct cattctatta gactctcgtt tggattgcaa ctggtctatt ttcctctttt 6120

gtttgataga aaatcataaa aggatttgca gactacgggc ctaaagaact aaaaaatcta 6180

tctgtttctt ttcattctct gtatttttta tagtttctgt tgcatgggca taaagttgcc 6240

tttttaatca caattcagaa aatatcataa tatctcattt cactaaataa tagtgaacgg 6300

caggtatatg tgatgggtta aaaaggatcg gcggccgctc gatttaaatc 6350

<210>9

<211>192

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Promoter sequence

<400>9

gagctgccaa ttattccggg cttgtgaccc gctacccgat aaataggtcg gctgaaaaat 60

ttcgttgcaa tatcaacaaa aaggcctatc attgggaggt gtcgcaccaa gtacttttgc 120

gaagcgccat ctgacggatt ttcaaaagat gtatatgctc ggtgcggaaa cctacgaaag 180

gattttttac cc 192

<210>10

<211>7080

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Nucleotide sequence

<400>10

cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc 60

cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa 120

ttgggcccga cgtcgcatgc tcccggccgg gccatgggcc gggcgtgcgc aatagactcg 180

tcaccaaaac cgatggagtg tttttgacgc tggaagatgg cagcaccgtg attgacgcga 240

tgagctcctg gtggtcggca attcatggac acggacaccc ccgactgaaa gctgccgccc 300

aaaaacaaat cgacaccatg agtcacgtca tgtttggcgg actaacccac gagcccgcca 360

ttaagctcac ccacaaactc ctcaatctca ctggaaattc ctttgaccac gtcttttatt 420

ccgattcggg ctcggtctca gtggaggtcg ccatcaaaat ggcactgcag gcctccaaag 480

gacaaggcca cccggaacga acaaaactcc tcacctggcg gtccggctac cacggagaca 540

cattcaccgc gatgagcgtg tgcgacccag aaaatggcat gcatagcctc tggaaaggca 600

cactccccga gcagattttc gcccccgccc caccagttcg ggggtcatcg ccgcaggcga 660

tttccgagta cctgcgcagc atggaattgc ttatcgacga gaccgtctcc gcaatcatca 720

tcgaaccgat cgtccaaggc gctggaggca tgcgcgcggc cgcacagcga tcccagagga 780

aatatcctct ggggtcgctg tgtcgacctt aaagtttggc tgccatgtga atttttagca 840

ccctcaacag ttgagtgctg gcactctcgg gggtagagtg ccaaataggt tgtttgacac 900

acagttgttc acccgcgacg acggctgtgc tggaaaccca caaccggcac acacaaaatt 960

tttctagtag gagcacaaac acatgtccct aacgaacatc ccagcctcat ctcaatgggc 1020

aattagcgac gttttgaagc gtccttcacc cggccgagta cctttttctg tcgagtttat 1080

gccaccccgc gacgatgcag ctgaagagcg tctttaccgc gcagcagagg tcttccatga 1140

cctcggtgca tcgtttgtct ccgtgactta tggtgctggc ggatcaaccc gtgagagaac 1200

ctcacgtatt gctcgacgat tagcgaaaca accgttgacc actctggtgc acctgaccct 1260

ggttaaccac actcgcgaag agatgaaggc aattcttcgg gaatacctag agctgggatt 1320

aacaaacctg ttggcgcttc gaggagatcc gcctggagac ccattaggcg attgggtgag 1380

caccgatgga ggactgaact atgcctctga gctcatcgat cttattaagt ccactcctga 1440

gttccgggaa ttcgacctcg gtatcgcctc cttccccgaa gggcatttcc gggcgaaaac 1500

tctagaagaa gacaccaaat acactctggc gaagctgcgt ggaggggcag agtactccat 1560

cacgcagatg ttctttgatg tggaagacta cctgcgactt cgtgatcgcc ttgtcgctgc 1620

agaccccatt catggtgcga agccaatcat tcctggcatc atgcccatta cgagcctgcg 1680

gtctgtgcgt cgacaggtcg aactctctgg tgctcaattg ccgagccaac tagaagaatc 1740

acttgttcga gctgcaaacg gcaatgaaga agcgaacaaa gacgagatcc gcaaggtggg 1800

cattgaatat tccaccaata tggcagagcg actcattgcc gaaggtgcgg aagatctgca 1860

cttcatgacg cttaacttca cccgtgcaac ccaagaagtg ttgtacaacc ttggcatggc 1920

gcctgcttgg ggagcagagc acggccaaga cgcggtgcgt taagggatcc gccctcccgc 1980

acgctttgcg ggagggcggt accggaactg gggttgggaa aaccttctcc acagccgttt 2040

tggttcgata cttagccgat caaggacacg atgttctgcc cgtaaagcta gtccaaaccg 2100

gtgaacttcc aggcgaggga gacatcttta acattgaacg cttgactgga attgctggag 2160

aggaatttgc tcgtttcaaa gaccctcttg cgccaaatct ggcagcccga cgagaggggg 2220

tcgagccaat acagtttgat cagattatct cgtggcttcg tggttttgac gacccagatc 2280

gcatcattgt ggtggagggc gctggtggcc tgctggtcag attaggggaa gatttcaccc 2340

tggcagatgt tgcctccgct ttgaatgcac ccttagtgat tgtgacaagc accggattgg 2400

gaagcctcaa cgctgctgaa ttaagcgttg aggcagcaaa ccgccgagga ctcacagtgt 2460

tgggagtcct cggcggttcg atccctcaaa atcctgatct agctacgatg cttaatctcg 2520

aagaatttga gagagtcacc ggcgtgccct tttggggagc tttgccggaa gggttgtcac 2580

gggtggaggg gttcgtcgaa aagcaatctt ttccggccct tgatgccttt aagaaaccgc 2640

cggcaaggct cccaacgcgt tggatgcata gcttgagtat tctatagtgt cacctaaata 2700

gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 2760

cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 2820

aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 2880

agctgaaatc gctagcgggc tgctaaagga agcggaacac gtagaaagcc agtccgcaga 2940

aacggtgctg accccggatg aatgtcagct actgggctat ctggacaagg gaaaacgcaa 3000

gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg gcgatagcta gactgggcgg 3060

ttttatggac agcaagcgaa ccggaattgc cagctggggc gccctctggt aaggttggga 3120

agccctgcaa agtaaactgg atggctttct tgccgccaag gatctgatgg cgcaggggat 3180

caagatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc 3240

acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga 3300

caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt 3360

ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat 3420

cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg 3480

gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg 3540

ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc 3600

cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga 3660

tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag 3720

ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc 3780

atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg 3840

actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata 3900

ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg 3960

ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac 4020

tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc 4080

caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat 4140

gatcctccag cgcggggatc tcatgctgga gttcttcgcc cacgctagcg gcgcgccggc 4200

cggcccggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt 4260

ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 4320

ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 4380

tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 4440

tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 4500

gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 4560

ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 4620

tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 4680

agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 4740

atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 4800

acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 4860

actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 4920

tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 4980

tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 5040

tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 5100

tgagattatc aaaaaggatc ttcacctaga tccttttaaa ggccggccgc ggccgccatc 5160

ggcattttct tttgcgtttt tatttgttaa ctgttaattg tccttgttca aggatgctgt 5220

ctttgacaac agatgttttc ttgcctttga tgttcagcag gaagctcggc gcaaacgttg 5280

attgtttgtc tgcgtagaat cctctgtttg tcatatagct tgtaatcacg acattgtttc 5340

ctttcgcttg aggtacagcg aagtgtgagt aagtaaaggt tacatcgtta ggatcaagat 5400

ccatttttaa cacaaggcca gttttgttca gcggcttgta tgggccagtt aaagaattag 5460

aaacataacc aagcatgtaa atatcgttag acgtaatgcc gtcaatcgtc atttttgatc 5520

cgcgggagtc agtgaacagg taccatttgc cgttcatttt aaagacgttc gcgcgttcaa 5580

tttcatctgt tactgtgtta gatgcaatca gcggtttcat cacttttttc agtgtgtaat 5640

catcgtttag ctcaatcata ccgagagcgc cgtttgctaa ctcagccgtg cgttttttat 5700

cgctttgcag aagtttttga ctttcttgac ggaagaatga tgtgcttttg ccatagtatg 5760

ctttgttaaa taaagattct tcgccttggt agccatcttc agttccagtg tttgcttcaa 5820

atactaagta tttgtggcct ttatcttcta cgtagtgagg atctctcagc gtatggttgt 5880

cgcctgagct gtagttgcct tcatcgatga actgctgtac attttgatac gtttttccgt 5940

caccgtcaaa gattgattta taatcctcta caccgttgat gttcaaagag ctgtctgatg 6000

ctgatacgtt aacttgtgca gttgtcagtg tttgtttgcc gtaatgttta ccggagaaat 6060

cagtgtagaa taaacggatt tttccgtcag atgtaaatgt ggctgaacct gaccattctt 6120

gtgtttggtc ttttaggata gaatcatttg catcgaattt gtcgctgtct ttaaagacgc 6180

ggccagcgtt tttccagctg tcaatagaag tttcgccgac tttttgatag aacatgtaaa 6240

tcgatgtgtc atccgcattt ttaggatctc cggctaatgc aaagacgatg tggtagccgt 6300

gatagtttgc gacagtgccg tcagcgtttt gtaatggcca gctgtcccaa acgtccaggc 6360

cttttgcaga agagatattt ttaattgtgg acgaatcaaa ttcagaaact tgatattttt 6420

catttttttg ctgttcaggg atttgcagca tatcatggcg tgtaatatgg gaaatgccgt 6480

atgtttcctt atatggcttt tggttcgttt ctttcgcaaa cgcttgagtt gcgcctcctg 6540

ccagcagtgc ggtagtaaag gttaatactg ttgcttgttt tgcaaacttt ttgatgttca 6600

tcgttcatgt ctcctttttt atgtactgtg ttagcggtct gcttcttcca gccctcctgt 6660

ttgaagatgg caagttagtt acgcacaata aaaaaagacc taaaatatgt aaggggtgac 6720

gccaaagtat acactttgcc ctttacacat tttaggtctt gcctgcttta tcagtaacaa 6780

acccgcgcga tttacttttc gacctcattc tattagactc tcgtttggat tgcaactggt 6840

ctattttcct cttttgtttg atagaaaatc ataaaaggat ttgcagacta cgggcctaaa 6900

gaactaaaaa atctatctgt ttcttttcat tctctgtatt ttttatagtt tctgttgcat 6960

gggcataaag ttgccttttt aatcacaatt cagaaaatat cataatatct catttcacta 7020

aataatagtg aacggcaggt atatgtgatg ggttaaaaag gatcggcggc cgctcgattt 7080

<210>11

<211>8554

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Nucleotide sequence

<400>11

tcgagaggcc tgacgtcggg cccggtaccg ttgctcgctg atctttcggc ttaacaactt 60

tgtattcaat cagtcgggca tagaaagaaa acgcaatgat ataggaacca actgccgcca 120

aaaccagcca cacagagttg attgtttcgc cacgggagaa agcgattgct ccccaaccca 180

ccgccgcgat aaccccaaag acaaggagac caacgcgggc ggtcggtgac attttagggg 240

acttcttcac gcctactgga aggtcagtag cgttgctgta caccaaatca tcgtcattga 300

tgttgtcagt ctgttttatg gtcacgatct ttactgtttt ctcttcgggt cgtttcaaag 360

ccactatgcg tagaaacagc gggcagaaac tgtgtgcaga aatgcatgca gaaaaaggaa 420

agttcggcca gatgggtgtt tctgtatgcc gatgatcgga tctttgacag ctgggtatgc 480

gacaaatcac cgagagttgt taattcttaa caatggaaaa gtaacattga gagatgattt 540

ataccatcct gcaccattta gagtggggct agtcataccc ccataaccct agctgtacgc 600

aatcgatttc aaatcagttg gaaaaagtca agaaaattac ccgagacata tgcggcttaa 660

agtttggctg ccatgtgaat ttttagcacc ctcaacagtt gagtgctggc actctcgagg 720

gtagagtgcc aaataggttg tttgacacac agttgttcac ccgcgacgac ggctgtgctg 780

gaaacccaca accggcacac acaaaatttt tctcatggag ggattcatca tgccaaagta 840

cgacaattcc aatgctgacc agtggggctt tgaaacccgc tccattcacg caggccagtc 900

agtagacgca cagaccagcg cacgaaacct tccgatctac caatccaccg ctttcgtgtt 960

cgactccgct gagcacgcca agcagcgttt cgcacttgag gatctaggcc ctgtttactc 1020

ccgcctcacc aacccaaccg ttgaggcttt ggaaaaccgc atcgcttccc tcgaaggtgg 1080

cgtccacgct gtagcgttct cctccggaca ggccgcaacc accaacgcca ttttgaacct 1140

ggcaggagcg ggcgaccaca tcgtcacctc cccacgcctc tacggtggca ccgagactct 1200

attccttatc actcttaacc gcctgggtat cgatgtttcc ttcgtggaaa accccgacga 1260

ccctgagtcc tggcaggcag ccgttcagcc aaacaccaaa gcattcttcg gcgagacttt 1320

cgccaaccca caggcagacg tcctggatat tcctgcggtg gctgaagttg cgcaccgcaa 1380

cagcgttcca ctgatcatcg acaacaccat cgctaccgca gcgctcgtgc gcccgctcga 1440

gctcggcgca gacgttgtcg tcgcttccct caccaagttc tacaccggca acggctccgg 1500

actgggcggc gtgcttatcg acggcggaaa gttcgattgg actgtcgaaa aggatggaaa 1560

gccagtattc ccctacttcg tcactccaga tgctgcttac cacggattga agtacgcaga 1620

ccttggtgca ccagccttcg gcctcaaggt tcgcgttggc cttctacgcg acaccggctc 1680

caccctctcc gcattcaacg catgggctgc agtccagggc atcgacaccc tttccctgcg 1740

cctggagcgc cacaacgaaa acgccatcaa ggttgcagaa ttcctcaaca accacgagaa 1800

ggtggaaaag gttaacttcg caggcctgaa ggattcccct tggtacgcaa ccaaggaaaa 1860

gcttggcctg aagtacaccg gctccgttct caccttcgag atcaagggcg gcaaggatga 1920

ggcttgggca tttatcgacg ccctgaagct acactccaac cttgcaaaca tcggcgatgt 1980

tcgctccctc gttgttcacc cagcaaccac cacccattca cagtccgacg aagctggcct 2040

ggcacgcgcg ggcgttaccc agtccaccgt ccgcctgtcc gttggcatcg agaccattga 2100

tgatatcatc gctgacctcg aaggcggctt tgctgcaatc tagcactagt tcggacctag 2160

ggatatcgtc gagagctgcc aattattccg ggcttgtgac ccgctacccg ataaataggt 2220

cggctgaaaa atttcgttgc aatatcaaca aaaaggccta tcattgggag gtgtcgcacc 2280

aagtactttt gcgaagcgcc atctgacgga ttttcaaaag atgtatatgc tcggtgcgga 2340

aacctacgaa aggatttttt acccatgccc accctcgcgc cttcaggtca acttgaaatc 2400

caagcgatcg gtgatgtctc caccgaagcc ggagcaatca ttacaaacgc tgaaatcgcc 2460

tatcaccgct ggggtgaata ccgcgtagat aaagaaggac gcagcaatgt cgttctcatc 2520

gaacacgccc tcactggaga ttccaacgca gccgattggt gggctgactt gctcggtccc 2580

ggcaaagcca tcaacactga tatttactgc gtgatctgta ccaacgtcat cggtggttgc 2640

aacggttcca ccggacctgg ctccatgcat ccagatggaa atttctgggg taatcgcttc 2700

cccgccacgt ccattcgtga tcaggtaaac gccgaaaaac aattcctcga cgcactcggc 2760

atcaccacgg tcgccgcagt acttggtggt tccatgggtg gtgcccgcac cctagagtgg 2820

gccgcaatgt acccagaaac tgttggcgca gctgctgttc ttgcagtttc tgcacgcgcc 2880

agcgcctggc aaatcggcat tcaatccgcc caaattaagg cgattgaaaa cgaccaccac 2940

tggcacgaag gcaactacta cgaatccggc tgcaacccag ccaccggact cggcgccgcc 3000

cgacgcatcg cccacctcac ctaccgtggc gaactagaaa tcgacgaacg cttcggcacc 3060

aaagcccaaa agaacgaaaa cccactcggt ccctaccgca agcccgacca gcgcttcgcc 3120

gtggaatcct acttggacta ccaagcagac aagctagtac agcgtttcga cgccggctcc 3180

tacgtcttgc tcaccgacgc cctcaaccgc cacgacattg gtcgcgaccg cggaggcctc 3240

aacaaggcac tcgaatccat caaagttcca gtccttgtcg caggcgtaga taccgatatt 3300

ttgtacccct accaccagca agaacacctc tccagaaacc tgggaaatct actggcaatg 3360

gcaaaaatcg tatcccctgt cggccacgat gctttcctca ccgaaagccg ccaaatggat 3420

cgcatcgtga ggaacttctt cagcctcatc tccccagacg aagacaaccc ttcgacctac 3480

atcgagttct acatctaaca tatgactagt tcggacctag ggatatcgtc gacatcgatg 3540

ctcttctgcg ttaattaaca attgggatcc tctagacccg ggatttaaat cgctagcggg 3600

ctgctaaagg aagcggaaca cgtagaaagc cagtccgcag aaacggtgct gaccccggat 3660

gaatgtcagc tactgggcta tctggacaag ggaaaacgca agcgcaaaga gaaagcaggt 3720

agcttgcagt gggcttacat ggcgatagct agactgggcg gttttatgga cagcaagcga 3780

accggaattg ccagctgggg cgccctctgg taaggttggg aagccctgca aagtaaactg 3840

gatggctttc ttgccgccaa ggatctgatg gcgcagggga tcaagatctg atcaagagac 3900

aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc 3960

ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc 4020

cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc 4080

cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg 4140

cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt 4200

gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc 4260

catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga 4320

ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga 4380

tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct 4440

caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc 4500

gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt 4560

ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg 4620

cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat 4680

cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc 4740

gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa 4800

aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat 4860

ctcatgctgg agttcttcgc ccacgctagc ggcgcgccgg ccggcccggt gtgaaatacc 4920

gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga 4980

ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 5040

acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 5100

aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 5160

tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 5220

aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 5280

gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 5340

acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 5400

accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 5460

ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 5520

gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 5580

gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 5640

ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 5700

gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 5760

cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 5820

cttcacctag atccttttaa aggccggccg cggccgcgca aagtcccgct tcgtgaaaat 5880

tttcgtgccg cgtgattttc cgccaaaaac tttaacgaac gttcgttata atggtgtcat 5940

gaccttcacg acgaagtact aaaattggcc cgaatcatca gctatggatc tctctgatgt 6000

cgcgctggag tccgacgcgc tcgatgctgc cgtcgattta aaaacggtga tcggattttt 6060

ccgagctctc gatacgacgg acgcgccagc atcacgagac tgggccagtg ccgcgagcga 6120

cctagaaact ctcgtggcgg atcttgagga gctggctgac gagctgcgtg ctcggccagc 6180

gccaggagga cgcacagtag tggaggatgc aatcagttgc gcctactgcg gtggcctgat 6240

tcctccccgg cctgacccgc gaggacggcg cgcaaaatat tgctcagatg cgtgtcgtgc 6300

cgcagccagc cgcgagcgcg ccaacaaacg ccacgccgag gagctggagg cggctaggtc 6360

gcaaatggcg ctggaagtgc gtcccccgag cgaaattttg gccatggtcg tcacagagct 6420

ggaagcggca gcgagaatta tcgcgatcgt ggcggtgccc gcaggcatga caaacatcgt 6480

aaatgccgcg tttcgtgtgc cgtggccgcc caggacgtgt cagcgccgcc accacctgca 6540

ccgaatcggc agcagcgtcg cgcgtcgaaa aagcgcacag gcggcaagaa gcgataagct 6600

gcacgaatac ctgaaaaatg ttgaacgccc cgtgagcggt aactcacagg gcgtcggcta 6660

acccccagtc caaacctggg agaaagcgct caaaaatgac tctagcggat tcacgagaca 6720

ttgacacacc ggcctggaaa ttttccgctg atctgttcga cacccatccc gagctcgcgc 6780

tgcgatcacg tggctggacg agcgaagacc gccgcgaatt cctcgctcac ctgggcagag 6840

aaaatttcca gggcagcaag acccgcgact tcgccagcgc ttggatcaaa gacccggaca 6900

cggagaaaca cagccgaagt tataccgagt tggttcaaaa tcgcttgccc ggtgccagta 6960

tgttgctctg acgcacgcgc agcacgcagc cgtgcttgtc ctggacattg atgtgccgag 7020

ccaccaggcc ggcgggaaaa tcgagcacgt aaaccccgag gtctacgcga ttttggagcg 7080

ctgggcacgc ctggaaaaag cgccagcttg gatcggcgtg aatccactga gcgggaaatg 7140

ccagctcatc tggctcattg atccggtgta tgccgcagca ggcatgagca gcccgaatat 7200

gcgcctgctg gctgcaacga ccgaggaaat gacccgcgtt ttcggcgctg accaggcttt 7260

ttcacatagg ctgagccgtg gccactgcac tctccgacga tcccagccgt accgctggca 7320

tgcccagcac aatcgcgtgg atcgcctagc tgatcttatg gaggttgctc gcatgatctc 7380

aggcacagaa aaacctaaaa aacgctatga gcaggagttt tctagcggac gggcacgtat 7440

cgaagcggca agaaaagcca ctgcggaagc aaaagcactt gccacgcttg aagcaagcct 7500

gccgagcgcc gctgaagcgt ctggagagct gatcgacggc gtccgtgtcc tctggactgc 7560

tccagggcgt gccgcccgtg atgagacggc ttttcgccac gctttgactg tgggatacca 7620

gttaaaagcg gctggtgagc gcctaaaaga caccaagggt catcgagcct acgagcgtgc 7680

ctacaccgtc gctcaggcgg tcggaggagg ccgtgagcct gatctgccgc cggactgtga 7740

ccgccagacg gattggccgc gacgtgtgcg cggctacgtc gctaaaggcc agccagtcgt 7800

ccctgctcgt cagacagaga cgcagagcca gccgaggcga aaagctctgg ccactatggg 7860

aagacgtggc ggtaaaaagg ccgcagaacg ctggaaagac ccaaacagtg agtacgcccg 7920

agcacagcga gaaaaactag ctaagtccag tcaacgacaa gctaggaaag ctaaaggaaa 7980

tcgcttgacc attgcaggtt ggtttatgac tgttgaggga gagactggct cgtggccgac 8040

aatcaatgaa gctatgtctg aatttagcgt gtcacgtcag accgtgaata gagcacttaa 8100

ggtctgcggg cattgaactt ccacgaggac gccgaaagct tcccagtaaa tgtgccatct 8160

cgtaggcaga aaacggttcc cccgtagggt ctctctcttg gcctcctttc taggtcgggc 8220

tgattgctct tgaagctctc taggggggct cacaccatag gcagataacg ttccccaccg 8280

gctcgcctcg taagcgcaca aggactgctc ccaaagatct tcaaagccac tgccgcgact 8340

gccttcgcga agccttgccc cgcggaaatt tcctccaccg agttcgtgca cacccctatg 8400

ccaagcttct ttcaccctaa attcgagaga ttggattctt accgtggaaa ttcttcgcaa 8460

aaatcgtccc ctgatcgccc ttgcgacgtt ggcgtcggtg ccgctggttg cgcttggctt 8520

gaccgacttg atcagcggcc gctcgattta aatc 8554

<210>12

<211>8431

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Nucleotide sequence

<400>12

tcgaccaaag gacgcaccga tgagctgagc tgctgcagtg gattggccca ccatacccaa 60

aggaatccat gatgttggtg ttgccatcag tgggatgccc tgcgccttgg ggccgaaata 120

gtagaaatac actcgggtaa aaactgctgg tgcagacgcc aaagttaaaa ggaagagccc 180

gaaagaaacc cacagcatcg ccggaagttc aaagtgctca tggagttgtg ctgccgaggt 240

ggaagcaacc atcggcgtga caagaggaag accccacgca aaagttggtg tgcccgcctt 300

agatcgcaaa atggccgtta tatataagga ataggcaaca agtcccacgg ctgtgccaat 360

agaccagcac acaaacataa atccccacag atcatcaccc aaaactacgg ggcttgcagt 420

tcccaatgcg atcaaaccca tggacagcat tgcccatgcc ggcatgactt cagttttgaa 480

tgaaggagag cggtagatta gccaaccgcc aataatgaca attgccacca caacagctaa 540

cgcgaagaag aaatctgcga cgactggaaa accatggatt ttcaacagtg atgacaacaa 600

tgagatgccc atgagggaac cagcccacga ggggccaggt ggaggtaaga ccgcagcgta 660

gcttttggtc gaagaaggag tgggcatgcc cattacttta agcctttggg gcagtgaaac 720

cgctaaatgg gagcgttgtg cgctcgatca ctggtctaga cctttgggct ccaaaagttg 780

caatttcgcg aatacttcaa cacttgtttg caatgtttgt taataaatgg gttcgctagt 840

ggattctgtc gttagtactg gccgtcgtgg tggggtcatg tatttaggta gggcaaagtt 900

aagatcagag cactttttga tacgactaac tggatataac ctttggggta acgtggggat 960

gtgtgtgagt aattttcaaa gtatttaaaa gggggatcta gggtaaaaat ttggcttcaa 1020

gtacatatct ttagttcggt agttgagggc gggtggtgac agtgcgggca tgcatgtgag 1080

tgtaaatgtt gttttaaaaa ggtgtgtact gacagtgggc cggtttgtgc tggtcggcca 1140

ctagcggagt gcttggattg tgatggcagg gtaagggaaa gggattacca ttaccgctgt 1200

tcttggcgtt ttgttgccta ttgtccgaat gttaagtgtt aatggtggga aaactgggaa 1260

agttgtcccc tggaatgtgt gagaattgcc caaatctgaa cccaatggcc atggacgggg 1320

aatgaactgt cggagaacgg ttgaggttaa ttcttgaaac cacccccaaa ataggctatt 1380

taaacgggtg ctctcatatt aaagaaagtg tgtagatgcg tgtgggcagg gggtaggtcc 1440

actggtaatg acaaatgtgt ccgttgtctc acctaaagct cgagcggctt aaagtttggc 1500

tgccatgtga atttttagca ccctcaacag ttgagtgctg gcactctcgg gggtagagtg 1560

ccaaataggt tgtttgacac acagttgttc acccgcgacg acggctgtgc tggaaaccca 1620

caaccggcac acacaaaatt tttctcatga agggattgca tatgacctca gcatctgccc 1680

caagctttaa ccccggcaag ggtcccggct cagcagtcgg aattgccctt ttaggattcg 1740

gaacagtcgg cactgaggtg atgcgtctga tgaccgagta cggtgatgaa cttgcgcacc 1800

gcattggtgg cccactggag gttcgtggca ttgctgtttc tgatatctca aagccacgtg 1860

aaggcgttgc acctgagctg ctcactgagg acgcttttgc actcatcgag cgcgaggatg 1920

ttgacatcgt cgttgaggtt atcggcggca ttgagtaccc acgtgaggta gttctcgcag 1980

ctctgaaggc cggcaagtct gttgttaccg ccaataaggc tcttgttgca gctcactctg 2040

ctgagcttgc tgatgcagcg gaagccgcaa acgttgacct gtacttcgag gctgctgttg 2100

caggcgcaat tccagtggtt ggcccactgc gtcgctccct ggctggcgat cagatccagt 2160

ctgtgatggg catcgttaac ggcaccacca acttcatctt ggacgccatg gattccaccg 2220

gcgctgacta tgcagattct ttggctgagg caactcgttt gggttacgcc gaagctgatc 2280

caactgcaga cgtcgaaggc catgacgccg catccaaggc tgcaattttg gcatccatcg 2340

ctttccacac ccgtgttacc gcggatgatg tgtactgcga aggtatcagc aacatcagcg 2400

ctgccgacat tgaggcagca cagcaggcag gccacaccat caagttgttg gccatctgtg 2460

agaagttcac caacaaggaa ggaaagtcgg ctatttctgc tcgcgtgcac ccgactctat 2520

tacctgtgtc ccacccactg gcgtcggtaa acaagtcctt taatgcaatc tttgttgaag 2580

cagaagcagc tggtcgcctg atgttctacg gaaacggtgc aggtggcgcg ccaaccgcgt 2640

ctgctgtgct tggcgacgtc gttggtgccg cacgaaacaa ggtgcacggt ggccgtgctc 2700

caggtgagtc cacctacgct aacctgccga tcgctgattt cggtgagacc accactcgtt 2760

accacctcga catggatgtg gaagatcgcg tgggggtttt ggctgaattg gctagcctgt 2820

tctctgagca aggaatcttc ctgcgtacaa tccgacagga agagcgcgat gatgatgcac 2880

gtctgatcgt ggtcacccac tctgcgctgg aatctgatct ttcccgcacc gttgaactgc 2940

tgaaggctaa gcctgttgtt aaggcaatca acagtgtgat ccgcctcgaa agggactaaa 3000

ctagtagttc acgcttaaga actgctaaat aacaagaaag gctcccaccg aaagtgggag 3060

cctttcttgt cgttaagcga tgaattcctc aaaacctcag tgctttttaa acaccaacac 3120

caagttactt accgcgaatt ctcggagcac tgggacttta accatccacc agacccaata 3180

cgggtggtag cggggaaaag cggcaaccaa ttccgcattg cccacggagg ctccccattc 3240

cagcccctcc cggcaggaca cattaaacag tgactccccg aaaacgttct taggcgggtg 3300

cccgtgtttc ttcgtgtagc gatcgcgggc aaattctccg ccaacgtagt gttcccacag 3360

tccggtttca tggccgccga agggccctaa ccaaatggtg tagctcagga ttgccaggcc 3420

gccgctgcgg gtgacgcgga gcatttcttc tcccaattcc cacggtgcgg agacatgttc 3480

tgcaacgttg gaggagtaca ccacgtcaaa ggaatcggga agaaacggca ggtcgaggcc 3540

ggatccgcgg actgatccgt ggacgtcgat gccagctgcg gacatttcgc caacgtcggg 3600

ttcgacggag aagtaggtgg cgcccagtgt ctcaaaggct tcggcgaagt atccgggtcc 3660

gccgccgacg tcgagaactt tcaggtcatt taatccggcg ccagaaatat cttcagacaa 3720

agccgccacc agactcgagg tatcgagggc caggtttccg taaaagatgt caggtcgggt 3780

ttgttcgtat ttgaaatcag acagtaaacc ccacgacctg cccaaggtag ccaagcgacg 3840

aagagccgga agctccggaa atgaggccat ttatgcgcgg gtccagttga ggtcgcggat 3900

gtcttcgccg ttcatccatc gcaaaatggt ggtgatggca tcgtcgatgg aggaaacaat 3960

ggtggtgttt tttactcctg cgacgccgaa ttctagggtc cagccgtcta cttctgcgag 4020

gctgcgcgcg aagatgtagg aggactcgtc ggcggggttg tctaggtcga agtgtgcaat 4080

cttcaaataa tccccggaat ccccggaatc tccagagccc ccggcaatga cgttcaactg 4140

gtgcagatcc tctagagttc tgtgaaaaac accgtggggc agtttctgct tcgcggtgtt 4200

ttttatttgt ggggcactag acccgggatt taaatcgcta gcgggctgct aaaggaagcg 4260

gaacacgtag aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg tcagctactg 4320

ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt gcagtgggct 4380

tacatggcga tagctagact gggcggtttt atggacagca agcgaaccgg aattgccagc 4440

tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc 4500

gccaaggatc tgatggcgca ggggatcaag atctgatcaa gagacaggat gaggatcgtt 4560

tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct 4620

attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct 4680

gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga 4740

actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc 4800

tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg 4860

gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc 4920

aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca 4980

tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga 5040

cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc 5100

cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga 5160

aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca 5220

ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg 5280

cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct 5340

tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc 5400

aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga 5460

atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc 5520

ttcgcccacg ctagcggcgc gccggccggc ccggtgtgaa ataccgcaca gatgcgtaag 5580

gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 5640

cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 5700

atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 5760

taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 5820

aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 5880

tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 5940

gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 6000

cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 6060

cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 6120

atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 6180

tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 6240

ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 6300

acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 6360

aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 6420

aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 6480

tttaaaggcc ggccgcggcc gccatcggca ttttcttttg cgtttttatt tgttaactgt 6540

taattgtcct tgttcaagga tgctgtcttt gacaacagat gttttcttgc ctttgatgtt 6600

cagcaggaag ctcggcgcaa acgttgattg tttgtctgcg tagaatcctc tgtttgtcat 6660

atagcttgta atcacgacat tgtttccttt cgcttgaggt acagcgaagt gtgagtaagt 6720

aaaggttaca tcgttaggat caagatccat ttttaacaca aggccagttt tgttcagcgg 6780

cttgtatggg ccagttaaag aattagaaac ataaccaagc atgtaaatat cgttagacgt 6840

aatgccgtca atcgtcattt ttgatccgcg ggagtcagtg aacaggtacc atttgccgtt 6900

cattttaaag acgttcgcgc gttcaatttc atctgttact gtgttagatg caatcagcgg 6960

tttcatcact tttttcagtg tgtaatcatc gtttagctca atcataccga gagcgccgtt 7020

tgctaactca gccgtgcgtt ttttatcgct ttgcagaagt ttttgacttt cttgacggaa 7080

gaatgatgtg cttttgccat agtatgcttt gttaaataaa gattcttcgc cttggtagcc 7140

atcttcagtt ccagtgtttg cttcaaatac taagtatttg tggcctttat cttctacgta 7200

gtgaggatct ctcagcgtat ggttgtcgcc tgagctgtag ttgccttcat cgatgaactg 7260

ctgtacattt tgatacgttt ttccgtcacc gtcaaagatt gatttataat cctctacacc 7320

gttgatgttc aaagagctgt ctgatgctga tacgttaact tgtgcagttg tcagtgtttg 7380

tttgccgtaa tgtttaccgg agaaatcagt gtagaataaa cggatttttc cgtcagatgt 7440

aaatgtggct gaacctgacc attcttgtgt ttggtctttt aggatagaat catttgcatc 7500

gaatttgtcg ctgtctttaa agacgcggcc agcgtttttc cagctgtcaa tagaagtttc 7560

gccgactttt tgatagaaca tgtaaatcga tgtgtcatcc gcatttttag gatctccggc 7620

taatgcaaag acgatgtggt agccgtgata gtttgcgaca gtgccgtcag cgttttgtaa 7680

tggccagctg tcccaaacgt ccaggccttt tgcagaagag atatttttaa ttgtggacga 7740

atcaaattca gaaacttgat atttttcatt tttttgctgt tcagggattt gcagcatatc 7800

atggcgtgta atatgggaaa tgccgtatgt ttccttatat ggcttttggt tcgtttcttt 7860

cgcaaacgct tgagttgcgc ctcctgccag cagtgcggta gtaaaggtta atactgttgc 7920

ttgttttgca aactttttga tgttcatcgt tcatgtctcc ttttttatgt actgtgttag 7980

cggtctgctt cttccagccc tcctgtttga agatggcaag ttagttacgc acaataaaaa 8040

aagacctaaa atatgtaagg ggtgacgcca aagtatacac tttgcccttt acacatttta 8100

ggtcttgcct gctttatcag taacaaaccc gcgcgattta cttttcgacc tcattctatt 8160

agactctcgt ttggattgca actggtctat tttcctcttt tgtttgatag aaaatcataa 8220

aaggatttgc agactacggg cctaaagaac taaaaaatct atctgtttct tttcattctc 8280

tgtatttttt atagtttctg ttgcatgggc ataaagttgc ctttttaatc acaattcaga 8340

aaatatcata atatctcatt tcactaaata atagtgaacg gcaggtatat gtgatgggtt 8400

aaaaaggatc ggcggccgct cgatttgaga g 8431

<210>13

<211>5863

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Nucleotide sequence

<400>13

tcgagaggcc tgacgtcggg cccggtacca cgcgtcatat gcaggtgagg taaccccaaa 60

agaggtaaaa cccgcgccac cgacttttca ggagcgggga cgcgggtttt tgccatgaat 120

ccgaagatac tacatcagat ttttaggcca acttgagggc tgcgcgaagt tcatcaacgc 180

ggtcgatagc ctcccaagga aggtccaaat cagtgcgacc aaagtggccg taggcagcag 240

tgtcagcgta gatcggacga agcagatcaa gctcacggat aattgctgct ggacgcaggt 300

caaagacctc caacacggca gcctgaatct gctcgtcgct caggccttcc ttgttggtgt 360

caaaggtttc aacgtaaagt ccgactggct ttgcgcgtcc aatggcgtat gcaacctgaa 420

cttcagcgcg atcagcaagg cctgctgcca cgatgttctt tgctacccaa cgcatggcgt 480

atgcagcaga gcggtccacc ttgcttggat ccttaccgga gaatgctcca ccaccatggc 540

gagccatgcc accgtaggta tccacgatga tcttgcggcc ggtcagaccc gcatcaccca 600

tggggccacc cagaatgaag gaacctgaag ggttgatcaa cacggtgatc tcaccggttg 660

ccagatcctc aatgcctgcg tctttgatta cccaatcaat gacgtgttcg cgcagttggg 720

tttccaacca tgcacggtca acttctgggt cgtgctgggt ggagatgaca acggtatcca 780

ggtggctagg gcggtcttgc gcatcgtatg cgaaggtgac ctgggttttt ccgtctggac 840

gcaggtgagg aacgatgccc tctttacgaa cctgggtcag acgacgtgac agtcggtgcg 900

ccaacgcgat aggaagaggc atgtactctt cggtttcgtt ggtggcgtag ccgaacatca 960

ggccctggtc gccagcacct gcgcggtcgt cttcttcaac gtcgccgttg gtgcgggctt 1020

cgtcggagtt atccacgccg tcagcgattt cctgggactg ctcaccgatg gatactgaga 1080

cgccagcggt gcgtccgtcg aatccaacct cagaggagtt gaatccgatt tcgatgagct 1140

tgttgcggac taattgaggg atctctacgt aagcgctggt acggacctcg ccaacaacat 1200

ggacgattcc ggtggtgacc acagtttcca ctgcgacgcg cgactgcgga tctttttcga 1260

gcagcgcgtc caaaatggta tcggaaatag catcacatat tttgtctgga tgtccctcag 1320

ttacagattc actggtgaac aaacggacgg cggttggctg agccacaaat acccttcttt 1380

cgaagaagtt gagaataaat agtcttaaat acaaaaaacc aatatagacc aagctgtcta 1440

aaactgcaat gtcagtggtc tagctggatt tttctagact tcgcgatacg ccagtgccgc 1500

gtcccaaatt tgcgcagcaa cctcgatttt gctgccgtgc tccacatcga ctaccccacc 1560

gtgagcatcc aaaatccagc cctcattgtg cttttgccca aacactttgc ccatgcccac 1620

ctcattacac atgaggaggt cgcagccctt cttcccggga tttaaatcgc tagcgggctg 1680

ctaaaggaag cggaacacgt agaaagccag tccgcagaaa cggtgctgac cccggatgaa 1740

tgtcagctac tgggctatct ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc 1800

ttgcagtggg cttacatggc gatagctaga ctgggcggtt ttatggacag caagcgaacc 1860

ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag taaactggat 1920

ggctttcttg ccgccaagga tctgatggcg caggggatca agatctgatc aagagacagg 1980

atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg 2040

ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc 2100

cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg 2160

tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt 2220

tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg 2280

cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat 2340

catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca 2400

ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca 2460

ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa 2520

ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa 2580

tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc 2640

ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga 2700

atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc 2760

cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac 2820

caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg 2880

ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc 2940

atgctggagt tcttcgccca cgctagcggc gcgccggccg gcccggtgtg aaataccgca 3000

cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc 3060

gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 3120

gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 3180

ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 3240

cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 3300

ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 3360

taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 3420

ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 3480

ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 3540

aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 3600

tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac 3660

agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 3720

ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 3780

tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 3840

tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 3900

cacctagatc cttttaaagg ccggccgcgg ccgccatcgg cattttcttt tgcgttttta 3960

tttgttaact gttaattgtc cttgttcaag gatgctgtct ttgacaacag atgttttctt 4020

gcctttgatg ttcagcagga agctcggcgc aaacgttgat tgtttgtctg cgtagaatcc 4080

tctgtttgtc atatagcttg taatcacgac attgtttcct ttcgcttgag gtacagcgaa 4140

gtgtgagtaa gtaaaggtta catcgttagg atcaagatcc atttttaaca caaggccagt 4200

tttgttcagc ggcttgtatg ggccagttaa agaattagaa acataaccaa gcatgtaaat 4260

atcgttagac gtaatgccgt caatcgtcat ttttgatccg cgggagtcag tgaacaggta 4320

ccatttgccg ttcattttaa agacgttcgc gcgttcaatt tcatctgtta ctgtgttaga 4380

tgcaatcagc ggtttcatca cttttttcag tgtgtaatca tcgtttagct caatcatacc 4440

gagagcgccg tttgctaact cagccgtgcg ttttttatcg ctttgcagaa gtttttgact 4500

ttcttgacgg aagaatgatg tgcttttgcc atagtatgct ttgttaaata aagattcttc 4560

gccttggtag ccatcttcag ttccagtgtt tgcttcaaat actaagtatt tgtggccttt 4620

atcttctacg tagtgaggat ctctcagcgt atggttgtcg cctgagctgt agttgccttc 4680

atcgatgaac tgctgtacat tttgatacgt ttttccgtca ccgtcaaaga ttgatttata 4740

atcctctaca ccgttgatgt tcaaagagct gtctgatgct gatacgttaa cttgtgcagt 4800

tgtcagtgtt tgtttgccgt aatgtttacc ggagaaatca gtgtagaata aacggatttt 4860

tccgtcagat gtaaatgtgg ctgaacctga ccattcttgt gtttggtctt ttaggataga 4920

atcatttgca tcgaatttgt cgctgtcttt aaagacgcgg ccagcgtttt tccagctgtc 4980

aatagaagtt tcgccgactt tttgatagaa catgtaaatc gatgtgtcat ccgcattttt 5040

aggatctccg gctaatgcaa agacgatgtg gtagccgtga tagtttgcga cagtgccgtc 5100

agcgttttgt aatggccagc tgtcccaaac gtccaggcct tttgcagaag agatattttt 5160

aattgtggac gaatcaaatt cagaaacttg atatttttca tttttttgct gttcagggat 5220

ttgcagcata tcatggcgtg taatatggga aatgccgtat gtttccttat atggcttttg 5280

gttcgtttct ttcgcaaacg cttgagttgc gcctcctgcc agcagtgcgg tagtaaaggt 5340

taatactgtt gcttgttttg caaacttttt gatgttcatc gttcatgtct ccttttttat 5400

gtactgtgtt agcggtctgc ttcttccagc cctcctgttt gaagatggca agttagttac 5460

gcacaataaa aaaagaccta aaatatgtaa ggggtgacgc caaagtatac actttgccct 5520

ttacacattt taggtcttgc ctgctttatc agtaacaaac ccgcgcgatt tacttttcga 5580

cctcattcta ttagactctc gtttggattg caactggtct attttcctct tttgtttgat 5640

agaaaatcat aaaaggattt gcagactacg ggcctaaaga actaaaaaat ctatctgttt 5700

cttttcattc tctgtatttt ttatagtttc tgttgcatgg gcataaagtt gcctttttaa 5760

tcacaattca gaaaatatca taatatctca tttcactaaa taatagtgaa cggcaggtat 5820

atgtgatggg ttaaaaagga tcggcggccg ctcgatttaa atc 5863

<210>14

<211>5477

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Nucleotide sequence

<400>14

tcgagctctc caatctccac tgaggtactt aatccttccg gggaattcgg gcgcttaaat 60

cgagaaatta ggccatcacc ttttaataac aatacaatga ataattggaa taggtcgaca 120

cctttggagc ggagccggtt aaaattggca gcattcaccg aaagaaaagg agaaccacat 180

gcttgcccta ggttggatta catggatcat tattggtggt ctagctggtt ggattgcctc 240

caagattaaa ggcactgatg ctcagcaagg aattttgctg aacatagtcg tcggtattat 300

cggtggtttg ttaggcggct ggctgcttgg aatcttcgga gtggatgttg ccggtggcgg 360

cttgatcttc agcttcatca catgtctgat tggtgctgtc attttgctga cgatcgtgca 420

gttcttcact cggaagaagt aatctgcttt aaatccgtag ggcctgttga tatttcgata 480

tcaacaggcc ttttggtcat tttggggtgg aaaaagcgct agacttgcct gtggattaaa 540

actatacgaa ccggtttgtc tatattggtg ttagacagtt cgtcgtatct tgaaacagac 600

caacccgaaa ggacgtggcc gaacgtggct gctagctaat ccttgatggt ggacttgctg 660

gatctcgatt ggtccacaac atcagtcctc ttgagacggc tcgcgatttg gctcggcagt 720

tgttgtcggc tccacctgcg gactactcaa tttagtttct tcattttccg aaggggtatc 780

ttcgttgggg gaggcgtcga taagcccctt ctttttagct ttaacctcag cgcgacgctg 840

ctttaagcgc tgcatggcgg cgcggttcat ttcacgttgc gtttcgcgcc tcttgttcgc 900

gatttctttg cgggcctgtt ttgcttcgtt gatttcggca gtacgggttt tggtgagttc 960

cacgtttgtt gcgtgaagcg ttgaggcgtt ccatggggtg agaatcatca gggcgcggtt 1020

tttgcgtcgt gtccacagga agatgcgctt ttctttttgt tttgcgcggt agatgtcgcg 1080

ctgctctagg tggtgcactt tgaaatcgtc ggtaagtggg tatttgcgtt ccaaaatgac 1140

catcatgatg attgtttgga ggagcgtcca caggttgttg ctgacgcgtc atatgactag 1200

ttcggaccta gggatatcgt cgacatcgat gctcttctgc gttaattaac aattgggatc 1260

ctctagaccc gggatttaaa tcgctagcgg gctgctaaag gaagcggaac acgtagaaag 1320

ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag ctactgggct atctggacaa 1380

gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag tgggcttaca tggcgatagc 1440

tagactgggc ggttttatgg acagcaagcg aaccggaatt gccagctggg gcgccctctg 1500

gtaaggttgg gaagccctgc aaagtaaact ggatggcttt cttgccgcca aggatctgat 1560

ggcgcagggg atcaagatct gatcaagaga caggatgagg atcgtttcgc atgattgaac 1620

aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact 1680

gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc 1740

gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg 1800

cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg 1860

tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt 1920

catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc 1980

atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag 2040

cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg 2100

ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc 2160

tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt 2220

ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg 2280

ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt 2340

acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct 2400

tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg 2460

agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga 2520

cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccacgctag 2580

cggcgcgccg gccggcccgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca 2640

tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 2700

gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 2760

caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 2820

tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 2880

gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 2940

ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 3000

cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 3060

tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 3120

tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 3180

cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 3240

agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga 3300

agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 3360

gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 3420

aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 3480

ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aaggccggcc 3540

gcggccgcca tcggcatttt cttttgcgtt tttatttgtt aactgttaat tgtccttgtt 3600

caaggatgct gtctttgaca acagatgttt tcttgccttt gatgttcagc aggaagctcg 3660

gcgcaaacgt tgattgtttg tctgcgtaga atcctctgtt tgtcatatag cttgtaatca 3720

cgacattgtt tcctttcgct tgaggtacag cgaagtgtga gtaagtaaag gttacatcgt 3780

taggatcaag atccattttt aacacaaggc cagttttgtt cagcggcttg tatgggccag 3840

ttaaagaatt agaaacataa ccaagcatgt aaatatcgtt agacgtaatg ccgtcaatcg 3900

tcatttttga tccgcgggag tcagtgaaca ggtaccattt gccgttcatt ttaaagacgt 3960

tcgcgcgttc aatttcatct gttactgtgt tagatgcaat cagcggtttc atcacttttt 4020

tcagtgtgta atcatcgttt agctcaatca taccgagagc gccgtttgct aactcagccg 4080

tgcgtttttt atcgctttgc agaagttttt gactttcttg acggaagaat gatgtgcttt 4140

tgccatagta tgctttgtta aataaagatt cttcgccttg gtagccatct tcagttccag 4200

tgtttgcttc aaatactaag tatttgtggc ctttatcttc tacgtagtga ggatctctca 4260

gcgtatggtt gtcgcctgag ctgtagttgc cttcatcgat gaactgctgt acattttgat 4320

acgtttttcc gtcaccgtca aagattgatt tataatcctc tacaccgttg atgttcaaag 4380

agctgtctga tgctgatacg ttaacttgtg cagttgtcag tgtttgtttg ccgtaatgtt 4440

taccggagaa atcagtgtag aataaacgga tttttccgtc agatgtaaat gtggctgaac 4500

ctgaccattc ttgtgtttgg tcttttagga tagaatcatt tgcatcgaat ttgtcgctgt 4560

ctttaaagac gcggccagcg tttttccagc tgtcaataga agtttcgccg actttttgat 4620

agaacatgta aatcgatgtg tcatccgcat ttttaggatc tccggctaat gcaaagacga 4680

tgtggtagcc gtgatagttt gcgacagtgc cgtcagcgtt ttgtaatggc cagctgtccc 4740

aaacgtccag gccttttgca gaagagatat ttttaattgt ggacgaatca aattcagaaa 4800

cttgatattt ttcatttttt tgctgttcag ggatttgcag catatcatgg cgtgtaatat 4860

gggaaatgcc gtatgtttcc ttatatggct tttggttcgt ttctttcgca aacgcttgag 4920

ttgcgcctcc tgccagcagt gcggtagtaa aggttaatac tgttgcttgt tttgcaaact 4980

ttttgatgtt catcgttcat gtctcctttt ttatgtactg tgttagcggt ctgcttcttc 5040

cagccctcct gtttgaagat ggcaagttag ttacgcacaa taaaaaaaga cctaaaatat 5100

gtaaggggtg acgccaaagt atacactttg ccctttacac attttaggtc ttgcctgctt 5160

tatcagtaac aaacccgcgc gatttacttt tcgacctcat tctattagac tctcgtttgg 5220

attgcaactg gtctattttc ctcttttgtt tgatagaaaa tcataaaagg atttgcagac 5280

tacgggccta aagaactaaa aaatctatct gtttcttttc attctctgta ttttttatag 5340

tttctgttgc atgggcataa agttgccttt ttaatcacaa ttcagaaaat atcataatat 5400

ctcatttcac taaataatag tgaacggcag gtatatgtga tgggttaaaa aggatcggcg 5460

gccgctcgat ttaaatc 5477

<210>15

<211>8877

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Nucleotide sequence

<400>15

tcgatttaaa tctcgagagg cctgacgtcg ggcccggtac cgggcccccc ctcgaggtcg 60

agcggcttaa agtttggctg ccatgtgaat ttttagcacc ctcaacagtt gagtgctggc 120

actctcgggg gtagagtgcc aaataggttg tttgacacac agttgttcac ccgcgacgac 180

ggctgtgctg gaaacccaca accggcacac acaaaatttt tctcatggag ggattcatca 240

tgtcgacttc agttacttca ccagcccaca acaacgcaca ttcctccgaa tttttggatg 300

cgttggcaaa ccatgtgttg atcggcgacg gcgccatggg cacccagctc caaggctttg 360

acctggacgt ggaaaaggat ttccttgatc tggaggggtg taatgagatt ctcaacgaca 420

cccgccctga tgtgttgagg cagattcacc gcgcctactt tgaggcggga gctgacttgg 480

ttgagaccaa tacttttggt tgcaacctgc cgaacttggc ggattatgac atcgctgatc 540

gttgccgtga gcttgcctac aagggcactg cagtggctag ggaagtggct gatgagatgg 600

ggccgggccg aaacggcatg cggcgtttcg tggttggttc cctgggacct ggaacgaagc 660

ttccatcgct gggccatgca ccgtatgcag atttgcgtgg gcactacaag gaagcagcgc 720

ttggcatcat cgacggtggt ggcgatgcct ttttgattga gactgctcag gacttgcttc 780

aggtcaaggc tgcggttcac ggcgttcaag atgccatggc tgaacttgat acattcttgc 840

ccattatttg ccacgtcacc gtagagacca ccggcaccat gctcatgggt tctgagatcg 900

gtgccgcgtt gacagcgctg cagccactgg gtatcgacat gattggtctg aactgcgcca 960

ccggcccaga tgagatgagc gagcacctgc gttacctgtc caagcacgcc gatattcctg 1020

tgtcggtgat gcctaacgca ggtcttcctg tcctgggtaa aaacggtgca gaatacccac 1080

ttgaggctga ggatttggcg caggcgctgg ctggattcgt ctccgaatat ggcctgtcca 1140

tggtgggtgg ttgttgtggc accacacctg agcacatccg tgcggtccgc gatgcggtgg 1200

ttggtgttcc agagcaggaa acctccacac tgaccaagat ccctgcaggc cctgttgagc 1260

aggcctcccg cgaggtggag aaagaggact ccgtcgcgtc gctgtacacc tcggtgccat 1320

tgtcccagga aaccggcatt tccatgatcg gtgagcgcac caactccaac ggttccaagg 1380

cattccgtga ggcaatgctg tctggcgatt gggaaaagtg tgtggatatt gccaagcagc 1440

aaacccgcga tggtgcacac atgctggatc tttgtgtgga ttacgtggga cgagacggca 1500

ccgccgatat ggcgaccttg gcagcacttc ttgctaccag ctccactttg ccaatcatga 1560

ttgactccac cgagccagag gttattcgca caggccttga gcacttgggt ggacgaagca 1620

tcgttaactc cgtcaacttt gaagacggcg atggccctga gtcccgctac cagcgcatca 1680

tgaaactggt aaagcagcac ggtgcggccg tggttgcgct gaccattgat gaggaaggcc 1740

aggcacgtac cgctgagcac aaggtgcgca ttgctaaacg actgattgac gatatcaccg 1800

gcagctacgg cctggatatc aaagacatcg ttgtggactg cctgaccttc ccgatctcta 1860

ctggccagga agaaaccagg cgagatggca ttgaaaccat cgaagccatc cgcgagctga 1920

agaagctcta cccagaaatc cacaccaccc tgggtctgtc caatatttcc ttcggcctga 1980

accctgctgc acgccaggtt cttaactctg tgttcctcaa tgagtgcatt gaggctggtc 2040

tggactctgc gattgcgcac agctccaaga ttttgccgat gaaccgcatt gatgatcgcc 2100

agcgcgaagt ggcgttggat atggtctatg atcgccgcac cgaggattac gatccgctgc 2160

aggaattcat gcagctgttt gagggcgttt ctgctgccga tgccaaggat gctcgcgctg 2220

aacagctggc cgctatgcct ttgtttgagc gtttggcaca gcgcatcatc gacggcgata 2280

agaatggcct tgaggatgat ctggaagcag gcatgaagga gaagtctcct attgcgatca 2340

tcaacgagga ccttctcaac ggcatgaaga ccgtgggtga gctgtttggt tccggacaga 2400

tgcagctgcc attcgtgctg caatcggcag aaaccatgaa aactgcggtg gcctatttgg 2460

aaccgttcat ggaagaggaa gcagaagcta ccggatctgc gcaggcagag ggcaagggca 2520

aaatcgtcgt ggccaccgtc aagggtgacg tgcacgatat cggcaagaac ttggtggaca 2580

tcattttgtc caacaacggt tacgacgtgg tgaacttggg catcaagcag ccactgtccg 2640

ccatgttgga agcagcggaa gaacacaaag cagacgtcat cggcatgtcg ggacttcttg 2700

tgaagtccac cgtggtgatg aaggaaaacc ttgaggagat gaacaacgcc ggcgcatcca 2760

attacccagt cattttgggt ggcgctgcgc tgacgcgtac ctacgtggaa aacgatctca 2820

acgaggtgta caccggtgag gtgtactacg cccgtgatgc tttcgagggc ctgcgcctga 2880

tggatgaggt gatggcagaa aagcgtggtg aaggacttga tcccaactca ccagaagcta 2940

ttgagcaggc gaagaagaag gcggaacgta aggctcgtaa tgagcgttcc cgcaagattg 3000

ccgcggagcg taaagctaat gcggctcccg tgattgttcc ggagcgttct gatgtctcca 3060

ccgatactcc aaccgcggca ccaccgttct ggggaacccg cattgtcaag ggtctgccct 3120

tggcggagtt cttgggcaac cttgatgagc gcgccttgtt catggggcag tggggtctga 3180

aatccacccg cggcaacgag ggtccaagct atgaggattt ggtggaaact gaaggccgac 3240

cacgcctgcg ctactggctg gatcgcctga agtctgaggg cattttggac cacgtggcct 3300

tggtgtatgg ctacttccca gcggtcgcgg aaggcgatga cgtggtgatc ttggaatccc 3360

cggatccaca cgcagccgaa cgcatgcgct ttagcttccc acgccagcag cgcggcaggt 3420

tcttgtgcat cgcggatttc attcgcccac gcgagcaagc tgtcaaggac ggccaagtgg 3480

acgtcatgcc attccagctg gtcaccatgg gtaatcctat tgctgatttc gccaacgagt 3540

tgttcgcagc caatgaatac cgcgagtact tggaagttca cggcatcggc gtgcagctca 3600

ccgaagcatt ggccgagtac tggcactccc gagtgcgcag cgaactcaag ctgaacgacg 3660

gtggatctgt cgctgatttt gatccagaag acaagaccaa gttcttcgac ctggattacc 3720

gcggcgcccg cttctccttt ggttacggtt cttgccctga tctggaagac cgcgcaaagc 3780

tggtggaatt gctcgagcca ggccgtatcg gcgtggagtt gtccgaggaa ctccagctgc 3840

acccagagca gtccacagac gcgtttgtgc tctaccaccc agaggcaaag tactttaacg 3900

tctaatctag acccgggatt taaatcgcta gcgggctgct aaaggaagcg gaacacgtag 3960

aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg tcagctactg ggctatctgg 4020

acaagggaaa acgcaagcgc aaagagaaag caggtagctt gcagtgggct tacatggcga 4080

tagctagact gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc 4140

tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc gccaaggatc 4200

tgatggcgca ggggatcaag atctgatcaa gagacaggat gaggatcgtt tcgcatgatt 4260

gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat 4320

gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag 4380

gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac 4440

gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac 4500

gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc 4560

ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg 4620

ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag 4680

cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat 4740

caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag 4800

gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc 4860

ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg 4920

ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg 4980

ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag 5040

ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat 5100

cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc 5160

gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacg 5220

ctagcggcgc gccggccggc ccggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 5280

cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 5340

cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 5400

aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 5460

gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 5520

tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 5580

agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 5640

ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 5700

taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 5760

gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 5820

gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 5880

ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg 5940

ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 6000

gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 6060

caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 6120

taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaaggcc 6180

ggccgcggcc gcgcaaagtc ccgcttcgtg aaaattttcg tgccgcgtga ttttccgcca 6240

aaaactttaa cgaacgttcg ttataatggt gtcatgacct tcacgacgaa gtactaaaat 6300

tggcccgaat catcagctat ggatctctct gatgtcgcgc tggagtccga cgcgctcgat 6360

gctgccgtcg atttaaaaac ggtgatcgga tttttccgag ctctcgatac gacggacgcg 6420

ccagcatcac gagactgggc cagtgccgcg agcgacctag aaactctcgt ggcggatctt 6480

gaggagctgg ctgacgagct gcgtgctcgg ccagcgccag gaggacgcac agtagtggag 6540

gatgcaatca gttgcgccta ctgcggtggc ctgattcctc cccggcctga cccgcgagga 6600

cggcgcgcaa aatattgctc agatgcgtgt cgtgccgcag ccagccgcga gcgcgccaac 6660

aaacgccacg ccgaggagct ggaggcggct aggtcgcaaa tggcgctgga agtgcgtccc 6720

ccgagcgaaa ttttggccat ggtcgtcaca gagctggaag cggcagcgag aattatcgcg 6780

atcgtggcgg tgcccgcagg catgacaaac atcgtaaatg ccgcgtttcg tgtgccgtgg 6840

ccgcccagga cgtgtcagcg ccgccaccac ctgcaccgaa tcggcagcag cgtcgcgcgt 6900

cgaaaaagcg cacaggcggc aagaagcgat aagctgcacg aatacctgaa aaatgttgaa 6960

cgccccgtga gcggtaactc acagggcgtc ggctaacccc cagtccaaac ctgggagaaa 7020

gcgctcaaaa atgactctag cggattcacg agacattgac acaccggcct ggaaattttc 7080

cgctgatctg ttcgacaccc atcccgagct cgcgctgcga tcacgtggct ggacgagcga 7140

agaccgccgc gaattcctcg ctcacctggg cagagaaaat ttccagggca gcaagacccg 7200

cgacttcgcc agcgcttgga tcaaagaccc ggacacggag aaacacagcc gaagttatac 7260

cgagttggtt caaaatcgct tgcccggtgc cagtatgttg ctctgacgca cgcgcagcac 7320

gcagccgtgc ttgtcctgga cattgatgtg ccgagccacc aggccggcgg gaaaatcgag 7380

cacgtaaacc ccgaggtcta cgcgattttg gagcgctggg cacgcctgga aaaagcgcca 7440

gcttggatcg gcgtgaatcc actgagcggg aaatgccagc tcatctggct cattgatccg 7500

gtgtatgccg cagcaggcat gagcagcccg aatatgcgcc tgctggctgc aacgaccgag 7560

gaaatgaccc gcgttttcgg cgctgaccag gctttttcac ataggctgag ccgtggccac 7620

tgcactctcc gacgatccca gccgtaccgc tggcatgccc agcacaatcg cgtggatcgc 7680

ctagctgatc ttatggaggt tgctcgcatg atctcaggca cagaaaaacc taaaaaacgc 7740

tatgagcagg agttttctag cggacgggca cgtatcgaag cggcaagaaa agccactgcg 7800

gaagcaaaag cacttgccac gcttgaagca agcctgccga gcgccgctga agcgtctgga 7860

gagctgatcg acggcgtccg tgtcctctgg actgctccag ggcgtgccgc ccgtgatgag 7920

acggcttttc gccacgcttt gactgtggga taccagttaa aagcggctgg tgagcgccta 7980

aaagacacca agggtcatcg agcctacgag cgtgcctaca ccgtcgctca ggcggtcgga 8040

ggaggccgtg agcctgatct gccgccggac tgtgaccgcc agacggattg gccgcgacgt 8100

gtgcgcggct acgtcgctaa aggccagcca gtcgtccctg ctcgtcagac agagacgcag 8160

agccagccga ggcgaaaagc tctggccact atgggaagac gtggcggtaa aaaggccgca 8220

gaacgctgga aagacccaaa cagtgagtac gcccgagcac agcgagaaaa actagctaag 8280

tccagtcaac gacaagctag gaaagctaaa ggaaatcgct tgaccattgc aggttggttt 8340

atgactgttg agggagagac tggctcgtgg ccgacaatca atgaagctat gtctgaattt 8400

agcgtgtcac gtcagaccgt gaatagagca cttaaggtct gcgggcattg aacttccacg 8460

aggacgccga aagcttccca gtaaatgtgc catctcgtag gcagaaaacg gttcccccgt 8520

agggtctctc tcttggcctc ctttctaggt cgggctgatt gctcttgaag ctctctaggg 8580

gggctcacac cataggcaga taacgttccc caccggctcg cctcgtaagc gcacaaggac 8640

tgctcccaaa gatcttcaaa gccactgccg cgactgcctt cgcgaagcct tgccccgcgg 8700

aaatttcctc caccgagttc gtgcacaccc ctatgccaag cttctttcac cctaaattcg 8760

agagattgga ttcttaccgt ggaaattctt cgcaaaaatc gtcccctgat cgcccttgcg 8820

acgttggcgt cggtgccgct ggttgcgctt ggcttgaccg acttgatcag cggccgc 8877

<210>16

<211>7534

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Nucleotide sequence

<400>16

tcgatttaaa tctcgagagg cctgacgtcg ggcccggtac cacgcgtcat atgactagtt 60

cggacctagg gatatcgtcg accggcttaa agtttggctg ccatgtgaat ttttagcacc 120

ctcaacagtt gagtgctggc actctcgggg gtagagtgcc aaataggttg tttgacacac 180

agttgttcac ccgcgacgac ggctgtgctg gaaacccaca accggcacac acaaaatttt 240

tctcatggag ggattcatca tgacttccaa cttttcttcc actgtcgctg gtcttcctcg 300

catcggagcg aagcgtgaac tgaagttcgc gctcgaaggc tactggaatg gatcaattga 360

aggtcgcgaa cttgcgcaga ccgcccgcca attggtcaac actgcatcgg attctttgtc 420

tggattggat tccgttccgt ttgcaggacg ttcctactac gacgcaatgc tcgataccgc 480

cgctattttg ggtgtgctgc cggagcgttt tgatgacatc gctgatcatg aaaacgatgg 540

tctcccactg tggattgacc gctactttgg cgctgctcgc ggtactgaga ccctgcctgc 600

acaggcaatg accaagtggt ttgataccaa ctaccactac ctcgtgccgg agttgtctgc 660

ggatacacgt ttcgttttgg atgcgtccgc gctgattgag gatctccgtt gccagcaggt 720

tcgtggcgtt aatgcccgcc ctgttctggt tggtccactg actttccttt cccttgctcg 780

caccactgat ggttccaatc ctttggatca cctgcctgca ctgtttgagg tctacgagcg 840

cctcatcaag tctttcgata ctgagtgggt tcagatcgat gagcctgcgt tggtcaccga 900

tgttgctcct gaggttttgg agcaggtccg cgctggttac accactttgg ctaagcgcga 960

tggcgtgttt gtcaatactt acttcggctc tggcgatcag gcgctgaaca ctcttgcggg 1020

catcggcctt ggcgcgattg gcgttgactt ggtcacccat ggcgtcactg agcttgctgc 1080

gtggaagggt gaggagctgc tggttgcggg catcgttgat ggtcgtaaca tttggcgcac 1140

cgacctgtgt gctgctcttg cttccctgaa gcgcctggca gctcgcggcc caatcgcagt 1200

gtctacctct tgttcactgc tgcacgttcc ttacaccctc gaggctgaga acattgagcc 1260

tgaggtccgc gactggcttg ccttcggctc ggagaagatc accgaggtca agctgcttgc 1320

cgacgcccta gccggcaaca tcgacgcggc tgcgttcgat gcggcgtccg cagcaattgc 1380

ttctcgacgc acctccccac gcaccgcacc aatcacgcag gaactccctg gccgtagccg 1440

tggatccttc gacactcgtg ttacgctgca ggagaagtca ctggagcttc cagctctgcc 1500

aaccaccacc attggttctt tcccacagac cccatccatt cgttctgctc gcgctcgtct 1560

gcgcaaggaa tccatcactt tggagcagta cgaagaggca atgcgcgaag aaatcgatct 1620

ggtcatcgcc aagcaggaag aacttggtct tgatgtgttg gttcacggtg agccagagcg 1680

caacgacatg gttcagtact tctctgaact tctcgacggt ttcctctcaa ccgccaacgg 1740

ctgggtccaa agctacggct cccgctgtgt tcgtcctcca gtgttgttcg gaaacgtttc 1800

ccgcccagcg ccaatgactg tcaagtggtt ccagtacgca cagagcctga cccagaagca 1860

tgtcaaggga atgctcaccg gtccagtcac catccttgca tggtccttcg ttcgcgatga 1920

tcagccgctg gctaccactg ctgaccaggt tgcactggca ctgcgcgatg aaattaacga 1980

tctcatcgag gctggcgcga agatcatcca ggtggatgag cctgcgattc gtgaactgtt 2040

gccgctacga gacgtcgata agcctgccta cctgcagtgg tccgtggact ccttccgcct 2100

ggcgactgcc ggcgcacccg acgacgtcca aatccacacc cacatgtgct actccgagtt 2160

caacgaagtg atctcctcgg tcatcgcgtt ggatgccgat gtcaccacca tcgaagcagc 2220

acgttccgac atgcaggtcc tcgctgctct gaaatcttcc ggcttcgagc tcggcgtcgg 2280

acctggtgtg tgggatatcc actccccgcg cgttccttcc gcgcagaaag tggacggtct 2340

cctcgaggct gcactgcagt ccgtggatcc tcgccagctg tgggtcaacc cagactgtgg 2400

tctgaagacc cgtggatggc cagaagtgga agcttcccta aaggttctcg ttgagtccgc 2460

taagcaggct cgtgagaaaa tcggagcaac tatctaatct agagttctgt gaaaaacacc 2520

gtggggcagt ttctgcttcg cggtgttttt tatttgtggg gcactagacc cgggatttaa 2580

atcgctagcg ggctgctaaa ggaagcggaa cacgtagaaa gccagtccgc agaaacggtg 2640

ctgaccccgg atgaatgtca gctactgggc tatctggaca agggaaaacg caagcgcaaa 2700

gagaaagcag gtagcttgca gtgggcttac atggcgatag ctagactggg cggttttatg 2760

gacagcaagc gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg 2820

caaagtaaac tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagatc 2880

tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg 2940

ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg 3000

ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa 3060

gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct 3120

ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga 3180

ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc 3240

cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac 3300

ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc 3360

cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact 3420

gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga 3480

tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg 3540

ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga 3600

agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga 3660

ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg 3720

ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc 3780

gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc 3840

cagcgcgggg atctcatgct ggagttcttc gcccacgcta gcggcgcgcc ggccggcccg 3900

gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc 3960

ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 4020

aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 4080

aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 4140

gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 4200

gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 4260

tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 4320

ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 4380

ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 4440

tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 4500

tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 4560

ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 4620

aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 4680

ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 4740

tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 4800

atcaaaaagg atcttcacct agatcctttt aaaggccggc cgcggccgcg caaagtcccg 4860

cttcgtgaaa attttcgtgc cgcgtgattt tccgccaaaa actttaacga acgttcgtta 4920

taatggtgtc atgaccttca cgacgaagta ctaaaattgg cccgaatcat cagctatgga 4980

tctctctgat gtcgcgctgg agtccgacgc gctcgatgct gccgtcgatt taaaaacggt 5040

gatcggattt ttccgagctc tcgatacgac ggacgcgcca gcatcacgag actgggccag 5100

tgccgcgagc gacctagaaa ctctcgtggc ggatcttgag gagctggctg acgagctgcg 5160

tgctcggcca gcgccaggag gacgcacagt agtggaggat gcaatcagtt gcgcctactg 5220

cggtggcctg attcctcccc ggcctgaccc gcgaggacgg cgcgcaaaat attgctcaga 5280

tgcgtgtcgt gccgcagcca gccgcgagcg cgccaacaaa cgccacgccg aggagctgga 5340

ggcggctagg tcgcaaatgg cgctggaagt gcgtcccccg agcgaaattt tggccatggt 5400

cgtcacagag ctggaagcgg cagcgagaat tatcgcgatc gtggcggtgc ccgcaggcat 5460

gacaaacatc gtaaatgccg cgtttcgtgt gccgtggccg cccaggacgt gtcagcgccg 5520

ccaccacctg caccgaatcg gcagcagcgt cgcgcgtcga aaaagcgcac aggcggcaag 5580

aagcgataag ctgcacgaat acctgaaaaa tgttgaacgc cccgtgagcg gtaactcaca 5640

gggcgtcggc taacccccag tccaaacctg ggagaaagcg ctcaaaaatg actctagcgg 5700

attcacgaga cattgacaca ccggcctgga aattttccgc tgatctgttc gacacccatc 5760

ccgagctcgc gctgcgatca cgtggctgga cgagcgaaga ccgccgcgaa ttcctcgctc 5820

acctgggcag agaaaatttc cagggcagca agacccgcga cttcgccagc gcttggatca 5880

aagacccgga cacggagaaa cacagccgaa gttataccga gttggttcaa aatcgcttgc 5940

ccggtgccag tatgttgctc tgacgcacgc gcagcacgca gccgtgcttg tcctggacat 6000

tgatgtgccg agccaccagg ccggcgggaa aatcgagcac gtaaaccccg aggtctacgc 6060

gattttggag cgctgggcac gcctggaaaa agcgccagct tggatcggcg tgaatccact 6120

gagcgggaaa tgccagctca tctggctcat tgatccggtg tatgccgcag caggcatgag 6180

cagcccgaat atgcgcctgc tggctgcaac gaccgaggaa atgacccgcg ttttcggcgc 6240

tgaccaggct ttttcacata ggctgagccg tggccactgc actctccgac gatcccagcc 6300

gtaccgctgg catgcccagc acaatcgcgt ggatcgccta gctgatctta tggaggttgc 6360

tcgcatgatc tcaggcacag aaaaacctaa aaaacgctat gagcaggagt tttctagcgg 6420

acgggcacgt atcgaagcgg caagaaaagc cactgcggaa gcaaaagcac ttgccacgct 6480

tgaagcaagc ctgccgagcg ccgctgaagc gtctggagag ctgatcgacg gcgtccgtgt 6540

cctctggact gctccagggc gtgccgcccg tgatgagacg gcttttcgcc acgctttgac 6600

tgtgggatac cagttaaaag cggctggtga gcgcctaaaa gacaccaagg gtcatcgagc 6660

ctacgagcgt gcctacaccg tcgctcaggc ggtcggagga ggccgtgagc ctgatctgcc 6720

gccggactgt gaccgccaga cggattggcc gcgacgtgtg cgcggctacg tcgctaaagg 6780

ccagccagtc gtccctgctc gtcagacaga gacgcagagc cagccgaggc gaaaagctct 6840

ggccactatg ggaagacgtg gcggtaaaaa ggccgcagaa cgctggaaag acccaaacag 6900

tgagtacgcc cgagcacagc gagaaaaact agctaagtcc agtcaacgac aagctaggaa 6960

agctaaagga aatcgcttga ccattgcagg ttggtttatg actgttgagg gagagactgg 7020

ctcgtggccg acaatcaatg aagctatgtc tgaatttagc gtgtcacgtc agaccgtgaa 7080

tagagcactt aaggtctgcg ggcattgaac ttccacgagg acgccgaaag cttcccagta 7140

aatgtgccat ctcgtaggca gaaaacggtt cccccgtagg gtctctctct tggcctcctt 7200

tctaggtcgg gctgattgct cttgaagctc tctagggggg ctcacaccat aggcagataa 7260

cgttccccac cggctcgcct cgtaagcgca caaggactgc tcccaaagat cttcaaagcc 7320

actgccgcga ctgccttcgc gaagccttgc cccgcggaaa tttcctccac cgagttcgtg 7380

cacaccccta tgccaagctt ctttcaccct aaattcgaga gattggattc ttaccgtgga 7440

aattcttcgc aaaaatcgtc ccctgatcgc ccttgcgacg ttggcgtcgg tgccgctggt 7500

tgcgcttggc ttgaccgact tgatcagcgg ccgc 7534

<210>17

<211>7322

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Nucleotide sequence

<400>17

ggccgctcga tttaaatctc gagctctgga gtgcgacagg tttgatgata aaaaattagc 60

gcaagaagac aaaaatcacc ttgcgctaat gctctgttac aggtcactaa taccatctaa 120

gtagttgatt catagtgact gcatatgtaa gtatttcctt agataacaat tgattgaatg 180

tatgcaaata aatgcataca ccataggtgt ggtttaattt gatgcccttt ttcagggctg 240

gaatgtgtaa gagcggggtt atttatgctg ttgttttttt gttactcggg aagggcttta 300

cctcttccgc ataaacgctt ccatcagcgt ttatagttaa aaaaatcttt cggggggatg 360

gggagtaagc ttgtgttatc cgctcgggcc caatccgcaa gctccaccga ctcgttggcg 420

tgcgactcta gataaatatc aagcagctgg ccgccaataa cctcagtacg catgccacgc 480

caagcatccc tcgtgcgggc caatgcctct gcactcaaac cggaatcctg cagcatgtct 540

tctgcccaca ccaatgccat atcgccagcc aaaatcgaga ctgaaacgcc aaagtgctcg 600

ggatcgcctt cgaaattatt ggcgcggtga tcagcttcca cagcccggtg aactgtgggg 660

gctccgcgcc gggtatcaga agaatcgata atatcgtcat gaatcaaggc acaagcctgg 720

atgaattcga gactcgctgc ggcgtcaagg acggactcaa gtttttcaga agaattctta 780

tggccttgcg ccgccaggaa accagcccac gcataaagag gacggattcg ctttcctcca 840

ttgagcacga aactgcgaag atgggccaca gcatctgtga caggagcgcc gatatcagca 900

attgttagct cttgagcatc gaggaactgc gtcaaacgat ctcgcacgac ctccggaaat 960

ttgtcgaggt caaggtcatg ggcatcgaaa ctgctcaagg agacgtcctt caatcgaata 1020

gggggatgcg ggctgaattt tggtggaggt gaataaatgc cagaggcagt cccaacaaaa 1080

cactctcatc acactaagat acccgtcgac tcatacgtta aatctatcac cgcaagggat 1140

aaatatctaa caccgtgcgt gttgactatt ttacctctgg cggtgataat ggttgcatgt 1200

actaaggagg attaattaat gtccctaacg aacatcccag cctcatctca atgggcaatt 1260

agcgacgttt tgaagcgtcc ttcacccggc cgagtacctt tttctgtcga gtttatgcca 1320

ccccgcgacg atgcagctga agagcgtctt taccgcgcag cagaggtctt ccatgacctc 1380

ggtgcatcgt ttgtctccgt gacttatggt gctggcggat caacccgtga gagaacctca 1440

cgtattgctc gacgattagc gaaacaaccg ttgaccactc tggtgcacct gaccctggtt 1500

aaccacactc gcgaagagat gaaggcaatt cttcgggaat acctagagct gggattaaca 1560

aacctgttgg cgcttcgagg agatccgcct ggagacccat taggcgattg ggtgagcacc 1620

gatggaggac tgaactatgc ctctgagctc atcgatctta ttaagtccac tcctgagttc 1680

cgggaattcg acctcggtat cgcctccttc cccgaagggc atttccgggc gaaaactcta 1740

gaagaagaca ccaaatacac tctggcgaag ctgcgtggag gggcagagta ctccatcacg 1800

cagatgttct ttgatgtgga agactacctg cgacttcgtg atcgccggat cctgttttgg 1860

cggatgagag aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat 1920

aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc 1980

agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa 2040

ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct 2100

gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg 2160

ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc 2220

aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttggta 2280

cgggatttaa atgatccgct agcgggctgc taaaggaagc ggaacacgta gaaagccagt 2340

ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg gacaagggaa 2400

aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg atagctagac 2460

tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc ctctggtaag 2520

gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat ctgatggcgc 2580

aggggatcaa gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat 2640

ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca 2700

caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg 2760

gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg 2820

cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact 2880

gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct 2940

caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg 3000

cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt 3060

actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc 3120

gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc 3180

gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga 3240

ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc 3300

cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt 3360

atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga 3420

gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt 3480

tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg 3540

gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac gctagcggcg 3600

cgccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt 3660

gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct 3720

gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa 3780

gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat 3840

gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct 3900

gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt 3960

ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca 4020

tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac 4080

aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg 4140

agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc 4200

acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct 4260

gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 4320

aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt 4380

cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact 4440

gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat 4500

caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 4560

agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 4620

aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 4680

gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 4740

tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 4800

cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 4860

ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 4920

cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 4980

atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 5040

agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 5100

gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa 5160

gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 5220

tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 5280

agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 5340

gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa aggccggccg 5400

cggccgccat cggcattttc ttttgcgttt ttatttgtta actgttaatt gtccttgttc 5460

aaggatgctg tctttgacaa cagatgtttt cttgcctttg atgttcagca ggaagctcgg 5520

cgcaaacgtt gattgtttgt ctgcgtagaa tcctctgttt gtcatatagc ttgtaatcac 5580

gacattgttt cctttcgctt gaggtacagc gaagtgtgag taagtaaagg ttacatcgtt 5640

aggatcaaga tccattttta acacaaggcc agttttgttc agcggcttgt atgggccagt 5700

taaagaatta gaaacataac caagcatgta aatatcgtta gacgtaatgc cgtcaatcgt 5760

catttttgat ccgcgggagt cagtgaacag gtaccatttg ccgttcattt taaagacgtt 5820

cgcgcgttca atttcatctg ttactgtgtt agatgcaatc agcggtttca tcactttttt 5880

cagtgtgtaa tcatcgttta gctcaatcat accgagagcg ccgtttgcta actcagccgt 5940

gcgtttttta tcgctttgca gaagtttttg actttcttga cggaagaatg atgtgctttt 6000

gccatagtat gctttgttaa ataaagattc ttcgccttgg tagccatctt cagttccagt 6060

gtttgcttca aatactaagt atttgtggcc tttatcttct acgtagtgag gatctctcag 6120

cgtatggttg tcgcctgagc tgtagttgcc ttcatcgatg aactgctgta cattttgata 6180

cgtttttccg tcaccgtcaa agattgattt ataatcctct acaccgttga tgttcaaaga 6240

gctgtctgat gctgatacgt taacttgtgc agttgtcagt gtttgtttgc cgtaatgttt 6300

accggagaaa tcagtgtaga ataaacggat ttttccgtca gatgtaaatg tggctgaacc 6360

tgaccattct tgtgtttggt cttttaggat agaatcattt gcatcgaatt tgtcgctgtc 6420

tttaaagacg cggccagcgt ttttccagct gtcaatagaa gtttcgccga ctttttgata 6480

gaacatgtaa atcgatgtgt catccgcatt tttaggatct ccggctaatg caaagacgat 6540

gtggtagccg tgatagtttg cgacagtgcc gtcagcgttt tgtaatggcc agctgtccca 6600

aacgtccagg ccttttgcag aagagatatt tttaattgtg gacgaatcaa attcagaaac 6660

ttgatatttt tcattttttt gctgttcagg gatttgcagc atatcatggc gtgtaatatg 6720

ggaaatgccg tatgtttcct tatatggctt ttggttcgtt tctttcgcaa acgcttgagt 6780

tgcgcctcct gccagcagtg cggtagtaaa ggttaatact gttgcttgtt ttgcaaactt 6840

tttgatgttc atcgttcatg tctccttttt tatgtactgt gttagcggtc tgcttcttcc 6900

agccctcctg tttgaagatg gcaagttagt tacgcacaat aaaaaaagac ctaaaatatg 6960

taaggggtga cgccaaagta tacactttgc cctttacaca ttttaggtct tgcctgcttt 7020

atcagtaaca aacccgcgcg atttactttt cgacctcatt ctattagact ctcgtttgga 7080

ttgcaactgg tctattttcc tcttttgttt gatagaaaat cataaaagga tttgcagact 7140

acgggcctaa agaactaaaa aatctatctg tttcttttca ttctctgtat tttttatagt 7200

ttctgttgca tgggcataaa gttgcctttt taatcacaat tcagaaaata tcataatatc 7260

tcatttcact aaataatagt gaacggcagg tatatgtgat gggttaaaaa ggatcggcgg 7320

cc 7322

<210>18

<211>7520

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Nucleotide sequence

<400>18

ctggagtgcg acaggtttga tgataaaaaa ttagcgcaag aagacaaaaa tcaccttgcg 60

ctaatgctct gttacaggtc actaatacca tctaagtagt tgattcatag tgactgcata 120

tgtaagtatt tccttagata acaattgatt gaatgtatgc aaataaatgc atacaccata 180

ggtgtggttt aatttgatgc cctttttcag ggctggaatg tgtaagagcg gggttattta 240

tgctgttgtt tttttgttac tcgggaaggg ctttacctct tccgcataaa cgcttccatc 300

agcgtttata gttaaaaaaa tctttcgggg ggatggggag taagcttgtg ttatccgctc 360

gggcccggta ccacgcgtga gttctttgag ttcctgtggg gtgaacttga cctgtgctgg 420

gccacgacgt ccgaaaacgt gcacttcagt ggccttgttt tctttgaggg agtcgtagac 480

gttgtcggaa atttcggtga ctttgagctc gtcgcctgtc ttagccagga tgcgggctac 540

gtcgaggccg acgttaccaa cgccgataac agcgacggac tgtgcagaca gatcccagga 600

gcgctcgaag cgtgggttgc cgtcgtagaa gccaacgaac tcgccggcac cgaaggagcc 660

ttctgcttca attccgggga tgttgaggtc gcggtctgca actgcgccgg tggagaacac 720

gactgcatcg tagtagtcgc ggagttcttc gacggtgatg tctttgccga tttcaatgtt 780

accgagcagg cgcaggcgtg gcttgtccaa cacgttgtgc agggacttaa cgatgccctt 840

gatgcgtggg tggtctggag caacgccgta acggatgagt ccgaacggtg caggcatttg 900

ctcgaaaagg tcaacgaaca cttcgcgctc ttcattgcgg atgaggaggt cggatgcgta 960

aatgccagca gggccagctc cgatgacggc tacgcgcagg ggagttgtca taatattaca 1020

cctccttaga taacaattga ttgaatgtat gcaaataaat gcccttcttc agggcttaat 1080

ttttaagagc gtcaccttca tggtggtcag tgcgtcctgc tgatgtgctc agtatcaccg 1140

ccagtggtat ttatgtcaac accgccagag ataatttatc accgcagatg gttatctgta 1200

tgttttttat atgaatttat tttttgcagg ggggcattgt ttggtaggtg agagatcaat 1260

tctgcgtcga ctcatacgtt aaatctatca ccgcaaggga taaatatcta acaccgtgcg 1320

tgttgactat tttacctctg gcggtgataa tggttgcatg tactaaggag gattaattaa 1380

tgacaacaac caccggaagt gcccggccag cacgtgccgc caggaagcct aagcccgaag 1440

gccaatggaa aatcgacggc accgagccgc ttaaccatgc cgaggaaatt aagcaagaag 1500

aacccgcttt tgctgtcaag cagcgggtca ttgatattta ctccaagcag ggtttttctt 1560

ccattgcacc ggatgacatt gccccacgct ttaagtggtt gggcatttac acccagcgta 1620

agcaggatct gggcggtgaa ctgaccggtc agcttcctga tgatgagctg caggatgagt 1680

acttcatgat gcgtgtgcgt tttgatggcg gactggcttc ccctgagcgc ctgcgtgccg 1740

tgggtgaaat ttctagggat tatgctcgtt ccaccgcgga cttcaccgac cgccagaaca 1800

ttcagctgca ctggattcgt attgaagatg tgcctgcgat ctgggagaag ctagaaaccg 1860

tcggactgtc caccatgctt ggttgcggtg acgttccacg tgttatcttg ggctccccag 1920

tttctggcgt agctgctgaa gagctgatcg atgccacccc ggctatcgat gcgattcgtg 1980

agcgctacct agacaaggaa gagttccaca accttcctcg taaggatcct gttttggcgg 2040

atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa 2100

acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga 2160

agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag tagggaactg 2220

ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt 2280

gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg 2340

cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa 2400

ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact cttggtacgg 2460

gatttaaatg atccgctagc gggctgctaa aggaagcgga acacgtagaa agccagtccg 2520

cagaaacggt gctgaccccg gatgaatgtc agctactggg ctatctggac aagggaaaac 2580

gcaagcgcaa agagaaagca ggtagcttgc agtgggctta catggcgata gctagactgg 2640

gcggttttat ggacagcaag cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt 2700

gggaagccct gcaaagtaaa ctggatggct ttcttgccgc caaggatctg atggcgcagg 2760

ggatcaagat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga 2820

ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa 2880

cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt 2940

ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg 3000

ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa 3060

gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac 3120

cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt 3180

gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact 3240

cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg 3300

ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg 3360

acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc 3420

atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt 3480

gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc 3540

gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg 3600

ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg 3660

attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct 3720

ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacgct agcggcgcgc 3780

cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc 3840

ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca 3900

aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc 3960

tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct 4020

gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag 4080

tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca 4140

gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg 4200

gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg 4260

aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga 4320

aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg 4380

ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac 4440

acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag 4500

cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac 4560

gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag 4620

agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag 4680

gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 4740

ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 4800

aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 4860

ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 4920

gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 4980

cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 5040

gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 5100

tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 5160

cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 5220

cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 5280

gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 5340

agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 5400

cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 5460

tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 5520

tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaagg ccggccgcgg 5580

ccgccatcgg cattttcttt tgcgttttta tttgttaact gttaattgtc cttgttcaag 5640

gatgctgtct ttgacaacag atgttttctt gcctttgatg ttcagcagga agctcggcgc 5700

aaacgttgat tgtttgtctg cgtagaatcc tctgtttgtc atatagcttg taatcacgac 5760

attgtttcct ttcgcttgag gtacagcgaa gtgtgagtaa gtaaaggtta catcgttagg 5820

atcaagatcc atttttaaca caaggccagt tttgttcagc ggcttgtatg ggccagttaa 5880

agaattagaa acataaccaa gcatgtaaat atcgttagac gtaatgccgt caatcgtcat 5940

ttttgatccg cgggagtcag tgaacaggta ccatttgccg ttcattttaa agacgttcgc 6000

gcgttcaatt tcatctgtta ctgtgttaga tgcaatcagc ggtttcatca cttttttcag 6060

tgtgtaatca tcgtttagct caatcatacc gagagcgccg tttgctaact cagccgtgcg 6120

ttttttatcg ctttgcagaa gtttttgact ttcttgacgg aagaatgatg tgcttttgcc 6180

atagtatgct ttgttaaata aagattcttc gccttggtag ccatcttcag ttccagtgtt 6240

tgcttcaaat actaagtatt tgtggccttt atcttctacg tagtgaggat ctctcagcgt 6300

atggttgtcg cctgagctgt agttgccttc atcgatgaac tgctgtacat tttgatacgt 6360

ttttccgtca ccgtcaaaga ttgatttata atcctctaca ccgttgatgt tcaaagagct 6420

gtctgatgct gatacgttaa cttgtgcagt tgtcagtgtt tgtttgccgt aatgtttacc 6480

ggagaaatca gtgtagaata aacggatttt tccgtcagat gtaaatgtgg ctgaacctga 6540

ccattcttgt gtttggtctt ttaggataga atcatttgca tcgaatttgt cgctgtcttt 6600

aaagacgcgg ccagcgtttt tccagctgtc aatagaagtt tcgccgactt tttgatagaa 6660

catgtaaatc gatgtgtcat ccgcattttt aggatctccg gctaatgcaa agacgatgtg 6720

gtagccgtga tagtttgcga cagtgccgtc agcgttttgt aatggccagc tgtcccaaac 6780

gtccaggcct tttgcagaag agatattttt aattgtggac gaatcaaatt cagaaacttg 6840

atatttttca tttttttgct gttcagggat ttgcagcata tcatggcgtg taatatggga 6900

aatgccgtat gtttccttat atggcttttg gttcgtttct ttcgcaaacg cttgagttgc 6960

gcctcctgcc agcagtgcgg tagtaaaggt taatactgtt gcttgttttg caaacttttt 7020

gatgttcatc gttcatgtct ccttttttat gtactgtgtt agcggtctgc ttcttccagc 7080

cctcctgttt gaagatggca agttagttac gcacaataaa aaaagaccta aaatatgtaa 7140

ggggtgacgc caaagtatac actttgccct ttacacattt taggtcttgc ctgctttatc 7200

agtaacaaac ccgcgcgatt tacttttcga cctcattcta ttagactctc gtttggattg 7260

caactggtct attttcctct tttgtttgat agaaaatcat aaaaggattt gcagactacg 7320

ggcctaaaga actaaaaaat ctatctgttt cttttcattc tctgtatttt ttatagtttc 7380

tgttgcatgg gcataaagtt gcctttttaa tcacaattca gaaaatatca taatatctca 7440

tttcactaaa taatagtgaa cggcaggtat atgtgatggg ttaaaaagga tcggcggccg 7500

ctcgatttaa atctcgagct 7520

<210>19

<211>5697

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Nucleotide sequence

<400>19

tcgaggcgtc ttccggtgtc atggttgaac cgaattccag cacaatattt tccggtttaa 60

agcaatcgat cacatagtcg attttgtcca accactgaaa acctgcaagg accacccaat 120

cccctgcagc atgttcagca accattggca gcggcggata gcgaacttcc cccttttctc 180

ccgttgccat tttcgcgtca ctgatcaggt gactgagctt tttgtagcct tccggatttt 240

tacacaagac tgtcaacacg ccttcttgca gactcagctc cgcaccataa acggtatgca 300

ttccagcttc cgcggcagct tccgcaaatc tcactgcacc ataaaaacca tccctatcca 360

tgactgatag agcaacaagt cctaactttt tggcctgcac aaccacatca gacggatccg 420

atgcgccagt gagaaagtta taactgctgg tggcatgcag ctcggcaaaa ggaaccgacg 480

cttccccctg catggcagat gaaggcgcct gcgcatccgg ctcatgcagc accggacgca 540

gagattcgac ctttttacct gagaggattc tttccaattt ggaccacgat aatggcctgc 600

cgttaaagct tcccccgcca ttccattcca taatgatagg atacattttt agaacaaatt 660

ttccaataag ttttccacgc cagccggaga aggaaataga ccaagctgta cagatcgacg 720

cgtcctggct gagtacaacg tcggctccgg cgcagacctc accccagttg gctccagcga 780

aatcgtgcca ctggcactat tctggaagga ccacgactcc atcgacggca ttgacggcga 840

gtccgttgcc atccctaacg atccttccaa ccagggccgc gccatcaacg ttctcgttca 900

ggcaggtctg gtcaccctga agaccccagg tctggtcacc ccagctccag tcgatatcga 960

cgaggcagct tccaaggttt ccgtcatccc agtcgacgca gctcaggcac caaccgctta 1020

ccaggagggt cgcccagcga tcatcaacaa ctccttcctt gaccgcgcag gcatcgatcc 1080

aaacctcgcg gtcttcgaag atgatcctga gtctgaagaa gcagagccat acatcaacgt 1140

cttcgtcacc aaggctgagg acaaggacga tgccaacatc gcccgcctcg ttgagctgtg 1200

gcacgaccca gaggttctgg ctgcagtaga ccgcgactct gagggcacct ccgtcccagt 1260

tgatcgtcca ggagctgacc ttcaggaaat ccttgatcgc cttgaggctg atcaggaaaa 1320

cgcataatct cttttgagtt ctttgcatac ccatgtgcag atttctttgc acaatcacag 1380

cctgaaaatc agactgtgaa cttcaaacgc atatgactag ttcggaccta gggatatcgt 1440

cgacatcgat gctcttctgc gttaattaac aattgggatc ctctagaccc gggatttaaa 1500

tcgctagcgg gctgctaaag gaagcggaac acgtagaaag ccagtccgca gaaacggtgc 1560

tgaccccgga tgaatgtcag ctactgggct atctggacaa gggaaaacgc aagcgcaaag 1620

agaaagcagg tagcttgcag tgggcttaca tggcgatagc tagactgggc ggttttatgg 1680

acagcaagcg aaccggaatt gccagctggg gcgccctctg gtaaggttgg gaagccctgc 1740

aaagtaaact ggatggcttt cttgccgcca aggatctgat ggcgcagggg atcaagatct 1800

gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt 1860

tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc 1920

tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag 1980

accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg 2040

gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac 2100

tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc 2160

gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc 2220

tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc 2280

ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg 2340

ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat 2400

gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc 2460

cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa 2520

gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat 2580

tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt 2640

tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg 2700

ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc 2760

agcgcgggga tctcatgctg gagttcttcg cccacgctag cggcgcgccg gccggcccgg 2820

tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc 2880

tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 2940

aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 3000

aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 3060

ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 3120

acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 3180

ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 3240

tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 3300

tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 3360

gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 3420

agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 3480

tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 3540

agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 3600

tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 3660

acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 3720

tcaaaaagga tcttcaccta gatcctttta aaggccggcc gcggccgcca tcggcatttt 3780

cttttgcgtt tttatttgtt aactgttaat tgtccttgtt caaggatgct gtctttgaca 3840

acagatgttt tcttgccttt gatgttcagc aggaagctcg gcgcaaacgt tgattgtttg 3900

tctgcgtaga atcctctgtt tgtcatatag cttgtaatca cgacattgtt tcctttcgct 3960

tgaggtacag cgaagtgtga gtaagtaaag gttacatcgt taggatcaag atccattttt 4020

aacacaaggc cagttttgtt cagcggcttg tatgggccag ttaaagaatt agaaacataa 4080

ccaagcatgt aaatatcgtt agacgtaatg ccgtcaatcg tcatttttga tccgcgggag 4140

tcagtgaaca ggtaccattt gccgttcatt ttaaagacgt tcgcgcgttc aatttcatct 4200

gttactgtgt tagatgcaat cagcggtttc atcacttttt tcagtgtgta atcatcgttt 4260

agctcaatca taccgagagc gccgtttgct aactcagccg tgcgtttttt atcgctttgc 4320

agaagttttt gactttcttg acggaagaat gatgtgcttt tgccatagta tgctttgtta 4380

aataaagatt cttcgccttg gtagccatct tcagttccag tgtttgcttc aaatactaag 4440

tatttgtggc ctttatcttc tacgtagtga ggatctctca gcgtatggtt gtcgcctgag 4500

ctgtagttgc cttcatcgat gaactgctgt acattttgat acgtttttcc gtcaccgtca 4560

aagattgatt tataatcctc tacaccgttg atgttcaaag agctgtctga tgctgatacg 4620

ttaacttgtg cagttgtcag tgtttgtttg ccgtaatgtt taccggagaa atcagtgtag 4680

aataaacgga tttttccgtc agatgtaaat gtggctgaac ctgaccattc ttgtgtttgg 4740

tcttttagga tagaatcatt tgcatcgaat ttgtcgctgt ctttaaagac gcggccagcg 4800

tttttccagc tgtcaataga agtttcgccg actttttgat agaacatgta aatcgatgtg 4860

tcatccgcat ttttaggatc tccggctaat gcaaagacga tgtggtagcc gtgatagttt 4920

gcgacagtgc cgtcagcgtt ttgtaatggc cagctgtccc aaacgtccag gccttttgca 4980

gaagagatat ttttaattgt ggacgaatca aattcagaaa cttgatattt ttcatttttt 5040

tgctgttcag ggatttgcag catatcatgg cgtgtaatat gggaaatgcc gtatgtttcc 5100

ttatatggct tttggttcgt ttctttcgca aacgcttgag ttgcgcctcc tgccagcagt 5160

gcggtagtaa aggttaatac tgttgcttgt tttgcaaact ttttgatgtt catcgttcat 5220

gtctcctttt ttatgtactg tgttagcggt ctgcttcttc cagccctcct gtttgaagat 5280

ggcaagttag ttacgcacaa taaaaaaaga cctaaaatat gtaaggggtg acgccaaagt 5340

atacactttg ccctttacac attttaggtc ttgcctgctt tatcagtaac aaacccgcgc 5400

gatttacttt tcgacctcat tctattagac tctcgtttgg attgcaactg gtctattttc 5460

ctcttttgtt tgatagaaaa tcataaaagg atttgcagac tacgggccta aagaactaaa 5520

aaatctatct gtttcttttc attctctgta ttttttatag tttctgttgc atgggcataa 5580

agttgccttt ttaatcacaa ttcagaaaat atcataatat ctcatttcac taaataatag 5640

tgaacggcag gtatatgtga tgggttaaaa aggatcggcg gccgctcgat ttaaatc 5697

<210>20

<211>7767

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

Nucleotide sequence

<400>20

tcgatttaaa tctcgagctc tggagtgcga caggtttgat gataaaaaat tagcgcaaga 60

agacaaaaat caccttgcgc taatgctctg ttacaggtca ctaataccat ctaagtagtt 120

gattcatagt gactgcatat gtaagtattt ccttagataa caattgattg aatgtatgca 180

aataaatgca tacaccatag gtgtggttta atttgatgcc ctttttcagg gctggaatgt 240

gtaagagcgg ggttatttat gctgttgttt ttttgttact cgggaagggc tttacctctt 300

ccgcataaac gcttccatca gcgtttatag ttaaaaaaat ctttcggggg gatggggagt 360

aagcttgtgt tatccgctcg ggcccggtac cacgcgtcat atgactagtt cggacctagg 420

gatatcgtcg actcatacgt taaatctatc accgcaaggg ataaatatct aacaccgtgc 480

gtgttgacta ttttacctct ggcggtgata atggttgcat gtactaagga ggattaatta 540

atgtccctaa cgaacatccc agcctcatct caatgggcaa ttagcgacgt tttgaagcgt 600

ccttcacccg gccgagtacc tttttctgtc gagtttatgc caccccgcga cgatgcagct 660

gaagagcgtc tttaccgcgc agcagaggtc ttccatgacc tcggtgcatc gtttgtctcc 720

gtgacttatg gtgctggcgg atcaacccgt gagagaacct cacgtattgc tcgacgatta 780

gcgaaacaac cgttgaccac tctggtgcac ctgaccctgg ttaaccacac tcgcgaagag 840

atgaaggcaa ttcttcggga atacctagag ctgggattaa caaacctgtt ggcgcttcga 900

ggagatccgc ctggagaccc attaggcgat tgggtgagca ccgatggagg actgaactat 960

gcctctgagc tcatcgatct tattaagtcc actcctgagt tccgggaatt cgacctcggt 1020

atcgcctcct tccccgaagg gcatttccgg gcgaaaactc tagaagaaga caccaaatac 1080

actctggcga agctgcgtgg aggggcagag tactccatca cgcagatgtt ctttgatgtg 1140

gaagactacc tgcgacttcg tgatcgcctt gtcgctgcag accccattca tggtgcgaag 1200

ccaatcattc ctggcatcat gcccattacg agcctgcggt ctgtgcgtcg acaggtcgaa 1260

ctctctggtg ctcaattgcc gagccaacta gaagaatcac ttgttcgagc tgcaaacggc 1320

aatgaagaag cgaacaaaga cgagatccgc aaggtgggca ttgaatattc caccaatatg 1380

gcagagcgac tcattgccga aggtgcggaa gatctgcact tcatgacgct taacttcacc 1440

cgtgcaaccc aagaagtgtt gtacaacctt ggcatggcgc ctgcttgggg agcagagcac 1500

ggccaagacg cggtgcgtta aggatcctgt tttggcggat gagagaagat tttcagcctg 1560

atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt 1620

agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat 1680

ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa 1740

ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct 1800

gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg 1860

gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac 1920

ggatggcctt tttgcgtttc tacaaactct tggtacggga tttaaatgat ccgctagcgg 1980

gctgctaaag gaagcggaac acgtagaaag ccagtccgca gaaacggtgc tgaccccgga 2040

tgaatgtcag ctactgggct atctggacaa gggaaaacgc aagcgcaaag agaaagcagg 2100

tagcttgcag tgggcttaca tggcgatagc tagactgggc ggttttatgg acagcaagcg 2160

aaccggaatt gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact 2220

ggatggcttt cttgccgcca aggatctgat ggcgcagggg atcaagatct gatcaagaga 2280

caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg 2340

cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg 2400

ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt 2460

ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg 2520

gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat 2580

tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat 2640

ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg 2700

accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg 2760

atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc 2820

tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc 2880

cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg 2940

tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg 3000

gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca 3060

tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac 3120

cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga 3180

aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga 3240

tctcatgctg gagttcttcg cccacgctag cggcgcgcca cgggtgcgca tgatcgtgct 3300

cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc 3360

accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac 3420

aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc 3480

ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc 3540

tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg 3600

catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc 3660

agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag 3720

aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc 3780

ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga 3840

tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg 3900

cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt 3960

cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg 4020

tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac 4080

tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa 4140

ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc 4200

actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 4260

gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 4320

cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 4380

ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 4440

ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 4500

ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 4560

agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 4620

cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 4680

aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 4740

gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 4800

agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 4860

ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 4920

cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 4980

tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 5040

aggatcttca cctagatcct tttaaaggcc ggccgcggcc gcgcaaagtc ccgcttcgtg 5100

aaaattttcg tgccgcgtga ttttccgcca aaaactttaa cgaacgttcg ttataatggt 5160

gtcatgacct tcacgacgaa gtactaaaat tggcccgaat catcagctat ggatctctct 5220

gatgtcgcgc tggagtccga cgcgctcgat gctgccgtcg atttaaaaac ggtgatcgga 5280

tttttccgag ctctcgatac gacggacgcg ccagcatcac gagactgggc cagtgccgcg 5340

agcgacctag aaactctcgt ggcggatctt gaggagctgg ctgacgagct gcgtgctcgg 5400

ccagcgccag gaggacgcac agtagtggag gatgcaatca gttgcgccta ctgcggtggc 5460

ctgattcctc cccggcctga cccgcgagga cggcgcgcaa aatattgctc agatgcgtgt 5520

cgtgccgcag ccagccgcga gcgcgccaac aaacgccacg ccgaggagct ggaggcggct 5580

aggtcgcaaa tggcgctgga agtgcgtccc ccgagcgaaa ttttggccat ggtcgtcaca 5640

gagctggaag cggcagcgag aattatcgcg atcgtggcgg tgcccgcagg catgacaaac 5700

atcgtaaatg ccgcgtttcg tgtgccgtgg ccgcccagga cgtgtcagcg ccgccaccac 5760

ctgcaccgaa tcggcagcag cgtcgcgcgt cgaaaaagcg cacaggcggc aagaagcgat 5820

aagctgcacg aatacctgaa aaatgttgaa cgccccgtga gcggtaactc acagggcgtc 5880

ggctaacccc cagtccaaac ctgggagaaa gcgctcaaaa atgactctag cggattcacg 5940

agacattgac acaccggcct ggaaattttc cgctgatctg ttcgacaccc atcccgagct 6000

cgcgctgcga tcacgtggct ggacgagcga agaccgccgc gaattcctcg ctcacctggg 6060

cagagaaaat ttccagggca gcaagacccg cgacttcgcc agcgcttgga tcaaagaccc 6120

ggacacggag aaacacagcc gaagttatac cgagttggtt caaaatcgct tgcccggtgc 6180

cagtatgttg ctctgacgca cgcgcagcac gcagccgtgc ttgtcctgga cattgatgtg 6240

ccgagccacc aggccggcgg gaaaatcgag cacgtaaacc ccgaggtcta cgcgattttg 6300

gagcgctggg cacgcctgga aaaagcgcca gcttggatcg gcgtgaatcc actgagcggg 6360

aaatgccagc tcatctggct cattgatccg gtgtatgccg cagcaggcat gagcagcccg 6420

aatatgcgcc tgctggctgc aacgaccgag gaaatgaccc gcgttttcgg cgctgaccag 6480

gctttttcac ataggctgag ccgtggccac tgcactctcc gacgatccca gccgtaccgc 6540

tggcatgccc agcacaatcg cgtggatcgc ctagctgatc ttatggaggt tgctcgcatg 6600

atctcaggca cagaaaaacc taaaaaacgc tatgagcagg agttttctag cggacgggca 6660

cgtatcgaag cggcaagaaa agccactgcg gaagcaaaag cacttgccac gcttgaagca 6720

agcctgccga gcgccgctga agcgtctgga gagctgatcg acggcgtccg tgtcctctgg 6780

actgctccag ggcgtgccgc ccgtgatgag acggcttttc gccacgcttt gactgtggga 6840

taccagttaa aagcggctgg tgagcgccta aaagacacca agggtcatcg agcctacgag 6900

cgtgcctaca ccgtcgctca ggcggtcgga ggaggccgtg agcctgatct gccgccggac 6960

tgtgaccgcc agacggattg gccgcgacgt gtgcgcggct acgtcgctaa aggccagcca 7020

gtcgtccctg ctcgtcagac agagacgcag agccagccga ggcgaaaagc tctggccact 7080

atgggaagac gtggcggtaa aaaggccgca gaacgctgga aagacccaaa cagtgagtac 7140

gcccgagcac agcgagaaaa actagctaag tccagtcaac gacaagctag gaaagctaaa 7200

ggaaatcgct tgaccattgc aggttggttt atgactgttg agggagagac tggctcgtgg 7260

ccgacaatca atgaagctat gtctgaattt agcgtgtcac gtcagaccgt gaatagagca 7320

cttaaggtct gcgggcattg aacttccacg aggacgccga aagcttccca gtaaatgtgc 7380

catctcgtag gcagaaaacg gttcccccgt agggtctctc tcttggcctc ctttctaggt 7440

cgggctgatt gctcttgaag ctctctaggg gggctcacac cataggcaga taacgttccc 7500

caccggctcg cctcgtaagc gcacaaggac tgctcccaaa gatcttcaaa gccactgccg 7560

cgactgcctt cgcgaagcct tgccccgcgg aaatttcctc caccgagttc gtgcacaccc 7620

ctatgccaag cttctttcac cctaaattcg agagattgga ttcttaccgt ggaaattctt 7680

cgcaaaaatc gtcccctgat cgcccttgcg acgttggcgt cggtgccgct ggttgcgctt 7740

ggcttgaccg acttgatcag cggccgc 7767

Claims

1. recombinant microorganism, it comprises and is selected from ask ^Fbr, hom ^Fbr, metX, metY, metB, metH, metE, metF and zwf each of at least five kinds of genes in hereditary change, wherein said hereditary change causes cross expressing of described at least five kinds of genes, thereby the methionine(Met) that causes the methionine(Met) output of this microorganism and produce when not having described hereditary change in described at least five kinds of genes increases.

2. recombinant microorganism, it comprises and is selected from ask ^Fbr, hom ^Fbr, metX, metY, metB, metH, metE, metF and zwf each of at least eight kinds of genes in hereditary change, wherein said hereditary change causes cross expressing of described at least eight kinds of genes, thereby the methionine(Met) that causes the methionine(Met) output of this microorganism and produce when not having described hereditary change in described at least eight kinds of genes increases.

3. recombinant microorganism, it comprises following (a) and combination (b):

(a) be selected from ask ^Fbr, hom ^Fbr, metX, metY, metB, metH, metE, metF and zwf each of at least five kinds of genes in hereditary change, thereby cause crossing of each of described at least five kinds of genes to be expressed; With

(b) be selected from hereditary change at least a gene of mcbR, hsk, metQ, metK and pepCK, thereby cause the expression that reduces of described at least a gene;

The methionine level of wherein said microorganisms increases with respect to the methionine(Met) that does not exist described when combination to produce.

4. recombinant microorganism, it comprises following (a) and combination (b):

(a) be selected from ask ^Fbr, hom ^Fbr, metH and ask ^Fbr, hom ^Fbr, the group formed of metE every kind of gene in hereditary change, thereby cause crossing of described every kind of gene to be expressed; With

(b) mcbR and the hsk hereditary change in each, thus the expression of the reduction of mcbR and hsk caused,

The methionine(Met) that the methionine level of wherein said microorganisms produces when not having this combination increases.

5. recombinant microorganism, it comprises following (a) and combination (b):

(a) be selected from ask ^Fbr, hom ^Fbr, metX, metY, metF, metH, metE and ask ^Fbr, hom ^Fbr, metX, metY, metF, metE each of at least six kinds of genes in hereditary change, thereby cause crossing of each of described at least six kinds of genes to be expressed; With

6. recombinant microorganism, it comprises following (a) and (b) and combination (c):

(a) be selected from ask ^Fbr, hom ^FbrMetX, metY, metF, metH and ask ^Fbr, hom ^FbrHereditary change in each of at least six kinds of genes of metX, metY, metF, metH, metE, thus cause crossing of each of described at least six kinds of genes to be expressed; With

(c) ethionine resistant mutation;

Wherein this microorganism produces 16g/l methionine(Met) at least under appropriate condition.

7. recombinant microorganism, it comprises the hereditary change in each of at least eight kinds of genes being selected from ask, hom, metX, metY, metB, metH, metE, metF, metC, zwf, frpA, pyc, asd, cysE, cysK, cysM, cysZ, cysC, cysG, cysN, cysD, cysH, cysJ, cysA, cysI and cysX, wherein said hereditary change causes cross expressing of described at least eight kinds of genes, thereby the methionine(Met) that causes the methionine(Met) output of this microorganism and produce when not having described hereditary change increases.

8. recombinant microorganism, it comprises following (a) and combination (b):

(a) be selected from hereditary change in each of at least five kinds of genes of ask, hom, metX, metY, metB, metH, metE, metF, metC, zwf, wherein said hereditary change causes crossing of described at least five kinds of genes to be expressed; With

(b) be selected from hereditary change in each of at least six kinds of genes of cysM, cysA, cysZ, cysC, cysG, cysJ, cysE, cysK, cysN, cysD, cysH, cysI and cysX, wherein said hereditary change causes crossing of described at least six kinds of genes to be expressed

Increase with respect to the methionine(Met) that does not have described when combination and produce thereby cause the methionine(Met) output of this microorganism.

9. recombinant microorganism, it comprises following (a) and combination (b):

(a) be selected from ask ^Fbr, hom ^Fbr, metX, metY, metB, metH, metE, metF and zwf each of at least five kinds of genes in hereditary change, wherein said hereditary change causes crossing of described at least five kinds of genes to be expressed; With

(b) be selected from the hereditary change of at least a gene of mcbR, hsk, metQ, metK and pepCK, thereby change the expression that causes described at least a gene to reduce,

Wherein said combination causes under appropriate condition the methionine(Met) output of 8g/l at least.

10. each recombinant microorganism of claim 1 to 9, wherein said microorganism is gram-positive.

11. each recombinant microorganism of claim 1 to 9, wherein said microorganism is gram-negative.

12. each recombinant microorganism of claim 1 to 9, wherein said microorganism is to belong to the microorganism that is selected from bacillus, Corynebacterium, lactobacillus, lactococcus and streptomyces.

13. each recombinant microorganism of claim 1 to 9, wherein said microorganism belongs to Corynebacterium.

14. the recombinant microorganism of claim 13, wherein said microorganism is a Corynebacterium glutamicum.

15. the recombinant microorganism among the claim 1-9, it is selected from bacterial strain M2014, M1119, M1494, M1990, OM41, OM224, OM89, OM99, OM99 (H357), OM403, OM418, OM419, OM428, OM429, OM448, OM456, OM464, OM469, OM465 and OM508 or derivatives thereof.

16. recombinant microorganism, it is with DSMZ preserving number DSM17322 preservation.

17. recombinant microorganism; it comprises and is selected from: E.C. 2.7.2.4.; homoserine dehydrogenase; homoserine acetyltransferase; homoserine succinyltransferase; cystathionine gamma-synthase; cystathionine beta-lyase; O-ethanoyl homoserine sulfhydrylase; O-succinyl homoserine sulfhydrylase; vitamin B12 dependency methionine synthases; vitamin B12 is methionine synthases independently; N5; 10-methylene radical-tetrahydrofolate reductase; sulfate adenylyl transferase subunit 1; sulfate adenylyl transferase subunit 2; the APS kinases; the APS reductase enzyme; adenosine phosphate phosphinylidyne sulfate reduction enzyme; the NADP-ferredoxin reductase; sulfite reductase subunit 1; sulfite reductase subunit 2; the vitriol translocator; Serine O-Transacetylase; O-ethanoyl Serine (thiol)-lyase A; the uroporphyrinogen III synthase; glucose-6-phosphate dehydrogenase (G6PD); at least five kinds of proteinic imbalances of pyruvate carboxylase and aspartate-semialdehyde dehydrogenase; wherein said imbalance comprises described at least five kinds of proteinic cross to express, thereby causes under appropriate condition giving birth to methionine(Met) with the volume production of 8g/l at least.

18. produce the method for methionine(Met), it is included in and makes to give birth to the volume production of 8g/l at least and cultivate each recombinant microorganism of claim 1-5 under the condition of methionine(Met).

19. produce the method for methionine(Met), it comprises:

(a) cultivate coryneform bacterial strains make producing under the condition of methionine(Met), this bacterial strain comprises the hereditary change in each of at least eight kinds of genes being selected from ask, hom, metX, metY, metB, metC, metH, metE, metF, metK, ilvA, metQ, fprA, asd, cysD, cysN, cysC, pyc, cysH, cysI, cys Y, cysX, cysZ, cysE, cysK, cysG, zwf, hsk, mcbR and pepCK; With

(b) reclaim methionine(Met).

20. the method for claim 19, wherein said coryneform bacterial strains is from Corynebacterium glutamicum.

21. the method for claim 19, wherein with every liter of culture at least the volume production of 16g give birth to methionine(Met).

22. the method for claim 19 is wherein given birth to methionine(Met) with the volume production of 25g/l culture at least.