CN101223280B

CN101223280B - Use of dimethyl disulfide for methionine production in microorganisms

Info

Publication number: CN101223280B
Application number: CN200680026206.5A
Authority: CN
Inventors: O·策尔德尔; S·哈夫纳; A·赫罗尔德; C·克洛普罗格; H·施罗德; R·R·约库姆; M·K·威廉姆斯
Original assignee: Evonik Degussa GmbH
Current assignee: Evonik Operations GmbH
Priority date: 2005-07-18
Filing date: 2006-07-18
Publication date: 2013-05-15
Anticipated expiration: 2026-07-18
Also published as: CN101223280A; ZA200801496B; ES2355942T3

Abstract

The present invention features improved processes and organisms for the production of methionine. The invention demonstrates that a Delta metF organism or a Delta metE AmetH organism, for example, mutants of C. glutamicum or E. coli, can use a methyl capped sulfide source, e.g., dimethyl disulfide (DMDS), as a source of both sulfur and a methyl group, bypassing the need for MetH/MetE and MetF activity and the need to reduce sulfate, for the synthesis of methionine. Also described in this patent are data implicating MetY (also called MetZ) as an enzyme that incorporates a methyl capped sulfidesource, e.g., DMDS, into methionine. A Delta metF Delta metB strain of C. glutamicum can use a methyl capped sulfide source, e.g., DMDS, as a source of both sulfide and a methyl group. Furthermore, methionine production by engineered prototrophic organisms that overproduce O-acetyl-homoserine was improved by the addition of a methyl capped sulfide source, e.g., DMDS.

Description

Dimethyl disulfide is used for methionine(Met) production in microorganism

Related application

The application requires the title submitted on July 18th, 2005 to be the U.S. Provisional Patent Application of " dimethyl disulfide is used for methionine(Met) production in microorganism " (Use of Dimethyl Disulfide for Methionine Productionin Microrganisms) number 60/700, the title of submitting on September 1st, 698 and 2005 be the right of priority of the U.S. Provisional Patent Application number 60/713,907 of " dimethyl disulfide in microorganism for methionine(Met) production ".

The title that the application relates on July 18th, 2005 and submits to is the U.S. Provisional Patent Application of " using genus bacillus MetI gene to improve methionine(Met) output in microorganism " (Use of a Bacillus MetI Gene to ImproveMethionine Production in Microorganisms) number 60/700, the title of submitting on September 1st, 557 and 2005 be the U.S. Provisional Patent Application number 60/60/713,905 of " using the methionine(Met) output in genus bacillus MetI gene raising microorganism ".

The title that the application also relates on September 1st, 2005 and submits to is the U.S. Provisional Patent Application of " producing the recombinant microorganism of methionine(Met) " number 60/714, the title of submitting on July 18th, 042 and 2005 be the U.S. Provisional Patent Application number 60/700,699 of " recombinant microorganism of generation methionine(Met) ".

The complete content of these patent applications all clearly is incorporated herein by reference, and described content comprises specification sheets, claims and summary, with and accompanying drawing, table or figure.

Background of invention

Current chemical process by good foundation produces methionine(Met) with DL-methionine(Met) racemic mixture, and described method relates to toxicity, danger, inflammable, unsettled and toxicant or intermediate.The starting material that are used for the chemical production of methionine(Met) are propenal, thiomethyl alcohol and prussic acid.The chemosynthesis of methionine(Met) relates to thiomethyl alcohol and acrolein reaction, produces intermediate 3-methyl mercapto propionic aldehyde (MMP).Other processing relate to MMP and prussic acid reaction formation 5-(2-methylmercaptoethyl) glycolylurea, and then it use escharotic such as NaOH and Na 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 ₂The method is compared with the amount of the methionine(Met) of generation and is produced excessive obsolete compound, and it causes economic and ecological challenge.

Be used for the fermentation process of methionine(Met) production usually based on using nutrition, comprise sugared source, for example, sugar; as glucose, fructose or sucrose, nitrogenous source, for example, ammonium and sulphur source; for example, vitriol or thiosulphate, and other unnecessary nutrient media components culturing micro-organisms.The method produces METHIONINE and as the biomass of by product, using does not have toxicity risk, inflammable, unsettled and/or harmful starting material.

Yet from the vitriol generation methionine(Met) as the sulphur source, at first sulphur atom must be reduced into sulfide for biological (for example, microorganism).This process is energy-intensive, therefore, further will improve this process in the sulphur source of reduction to microorganism delivery ratio vitriol.A kind of sulphur source of this type of reduction is thiosulphate, and wherein one of two sulphur atoms is reduced.Another source of sulphur through reducing is thiomethyl alcohol, and it contains the sulphur atom of reduction fully.

Use thiomethyl alcohol to produce methionine(Met) two advantages are provided.At first, As mentioned above, produced sulphur atom.Secondly, provide methyl, it can potentially be avoided the common two kinds of enzymes that need of methionine(Met) biosynthesizing: the needs of methyl tetrahydrofolate reductase enzyme (MetF) and methionine synthases (MetE and/or MetH).There is bibliographical information to disclose some microorganisms; can be by thiomethyl alcohol and the reaction of O-ethanoyl homoserine be directly mixed (Yamagata, S.1971.J.Biochem. (Tokyo) 70:1035) in methionine(Met) with thiomethyl alcohol as yeast saccharomyces cerevisiae (Saccharomyces cerevisiae).Use the method for thiomethyl alcohol also to be disclosed in WO 93/17112 and WO 2004/076659 in methionine(Met) production.

Yet, use thiomethyl alcohol to produce methionine(Met) and also have shortcoming.It is toxicity, explosion gas, easily oxidation in air, and be poisonous.The chemical process that produces methionine(Met) also uses thiomethyl alcohol as one of substrate, and therefore, the slip-stick artist has known in technical scale and processes this compound.But not using thiomethyl alcohol to produce improving one's methods of methionine(Met) will have very large benefit.

Summary of the invention

The present invention relates to improve one's methods (for example, microorganism is synthetic) for generation of methionine(Met).The inventor has been found that the sulphur that is different from thiomethyl alcohol and/or methyl source can be for generation of methionine(Met)s.Particularly, the inventor have been found that can be with dimethyl disulfide (DMDS) (also referred to as methyl disulfide or CH ₃-S-S-CH ₃) add substratum and by microorganism as sulfide and methyl source, avoid the needs of MetH/MetE and MetF activity and the sulphate reducing needs with synthetic methionine(Met).

In addition; the present invention illustrates the methionine(Met) biosynthetic pathway with imbalance; the O-ethanoyl homoserine sulfhydrylase of for example lacking of proper care; and/or the microorganism of the homoserine dehydrogenase of the homoserine acetyltransferase of imbalance and/or imbalance can be used the sulfide compound of methyl blocking; for example sulphur and/or methyl the source, as dimethyl disulfide (DMDS) for the synthesis of methionine(Met).In addition, the contriver finds to accumulate the methionine(Met) output of prototroph bacterial strain of through engineering approaches of O-ethanoyl homoserine by adding DMDS to be improved.

Therefore, on the one hand, the invention describes the method for producing methionine(Met), it is included in the sulfide compound of methyl blocking, and for example sulphur and/or methyl source, as culturing micro-organisms under the existence of dimethyl disulfide (DMDS), make the generation methionine(Met).In one embodiment, the sulfide compound of methyl blocking, for example sulphur and/or methyl the source, as DMDS with 0.02% or greater concn be present in culture.In another embodiment, the sulfide compound of methyl blocking, for example sulphur and/or methyl the source, as DMDS with 0.06% or greater concn be present in culture.In other embodiments, the sulfide compound of methyl blocking, for example sulphur and/or methyl source is selected from dimethyltrisulfide (DMTS) or CH ₃-S-S-S-CH ₃, dimethyl four sulphur (DMTTS) or CH ₃-S-S-S-S-CH ₃, or the more high-molecular weight polymer of thioether, its end is by methyl blocking.

Another aspect of the present invention has been described the method that produces methionine(Met), it is included in the thioether delivery system of slowly-releasing methyl blocking, for example sulphur and/or methyl delivery system, for example there is the lower microorganism that produces methionine(Met) of cultivating in dimethyl disulfide (DMDS) delivery system, thereby produces methionine(Met).In one embodiment, the thioether delivery system of slowly-releasing methyl blocking, for example, and slowly-releasing sulphur and/or methyl delivery system, for example, the slowly-releasing delivery system of DMDS is Amberlite ^TMXAD4.In one embodiment, the slowly-releasing delivery system with in substratum altogether 0.1% or higher level discharge DMDS.In a further embodiment, the slowly-releasing delivery system with in substratum altogether 0.3% or higher level discharge DMDS.In one embodiment, but slowly-releasing DMDS delivery system comprises with water immiscibility dissolves the liquid of DMDS.In one embodiment, the thioether delivery system of slowly-releasing methyl blocking, for example sulphur and/or methyl delivery system, for example the slowly-releasing delivery system of DMDS comprises the liquid that is selected from animal oil, mineral oil, chemistry oil, vegetables oil, synthetic oil, organic solvent, chlorocarbon, fluorocarbon, Chlorofluorocarbons (CFCs) or its combination.In another embodiment, the thioether delivery system of slowly-releasing methyl blocking, for example sulphur and/or methyl delivery system, for example but the slowly-releasing delivery system of DMDS comprises with water immiscibility dissolves the sulfide compound of methyl blocking, for example sulphur and/or methyl are originated, for example the liquid of DMDS.In a further embodiment, the thioether delivery system of slowly-releasing methyl blocking, for example sulphur and/or methyl delivery system, for example the slowly-releasing delivery system of DMDS is the thioether of methyl blocking, the for example slow controlled charging in sulphur and/or methyl source, for example slowly controlled DMDS charging.In another embodiment, the thioether delivery system of slowly-releasing methyl blocking, for example sulphur and/or methyl delivery system, for example DMDS slowly-releasing delivery system is film, it is for the thioether of methyl blocking, sulphur and/or methyl source for example, for example DMDS is permeable.In another embodiment, the DMDS slow-released system is sent the DMDS of gaseous state, for example, and by evaporation or boiling liquid DMDS, perhaps by for example sending leading on the road of fermenting container by liquid D MDS drum air or oxygen bubbles.In one embodiment, the microorganism of generation methionine(Met) belongs to Corynebacterium (Corynebacterium).In another embodiment, the microorganism of generation methionine(Met) is Corynebacterium glutamicum (Corynebacterium glutamicum).In a further embodiment, the microorganism that produces methionine(Met) (for example is selected from gram negative bacterium, intestinal bacteria or relevant enterobacteria), gram positive bacterium (for example, subtilis (Bacillus subtilis) or relevant genus bacillus), yeast (for example, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) or relevant yeast strain), and archeobacteria.In one embodiment, the microorganism of generation methionine(Met) has the methionine(Met) biosynthetic enzyme of at least a imbalance.In another embodiment, described microorganism has the O-ethanoyl homoserine sulfhydrylase of imbalance.In a further embodiment, the microorganism of generation methionine(Met) has the methionine(Met) biosynthetic enzyme of at least two kinds of imbalances.In one embodiment, described microorganism has the homoserine acetyltransferase of imbalance and the homoserine dehydrogenase of imbalance.In another embodiment, microorganism has the O-ethanoyl homoserine sulfhydrylase of imbalance and the homoserine acetyltransferase of imbalance.

Another aspect of the present invention has been described the method that produces methionine(Met), it is included in the thioether of methyl blocking, for example sulphur and/or methyl are originated, for example, cultivate the microorganism of the methionine(Met) biosynthetic pathway with imbalance under the existence of dimethyl disulfide (DMDS), thereby produce methionine(Met).In one embodiment, described microorganism belongs to excellent bacillus.In another embodiment, microorganism is Corynebacterium glutamicum.In a further embodiment, described microorganism (for example is selected from gram negative bacterium, intestinal bacteria or relevant enterobacteria), gram positive bacterium (for example, subtilis (Bacillus subtilis) or relevant genus bacillus), yeast (for example, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) or relevant yeast strain), and archeobacteria.In one embodiment, described microorganism has the O-ethanoyl homoserine sulfhydrylase of imbalance.In another embodiment, described microorganism has the homoserine acetyltransferase of imbalance and the homoserine dehydrogenase of imbalance.In an embodiment again, microorganism has the O-ethanoyl homoserine sulfhydrylase of imbalance and the homoserine acetyltransferase of imbalance.

The product that synthesizes according to above-mentioned either method of having described on the one hand more of the present invention.

Another aspect of the present invention has been described the thioether that is used at methyl blocking, for example sulphur and/or methyl are originated, for example, produce the recombinant microorganism of methionine(Met) under the existence of dimethyl disulfide (DMDS), described microorganism has the methionine(Met) biosynthetic pathway of imbalance.In one embodiment, described microorganism belongs to excellent bacillus.In another embodiment, microorganism is Corynebacterium glutamicum.In a further embodiment, described microorganism (for example is selected from gram negative bacterium, intestinal bacteria or relevant enterobacteria), gram positive bacterium (for example, subtilis (Bacillus subtilis) or relevant genus bacillus), yeast (for example, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) or relevant yeast strain), and archeobacteria.In one embodiment, described microorganism has the O-ethanoyl homoserine sulfhydrylase of imbalance.In another embodiment, described microorganism has the homoserine acetyltransferase of imbalance and the homoserine dehydrogenase of imbalance.In an embodiment again, microorganism has the O-ethanoyl homoserine sulfhydrylase of imbalance and the homoserine acetyltransferase of imbalance.

O-ethanoyl homoserine sulfhydrylase and/or the OSHS sulfhydrylase of identifying methionine(Met) feedback resistance described and/or the method for the gene (for example, mutator gene or allelotrope) of the described methionine(Met) feedback resistance enzyme of encoding in another aspect of the present invention.in one embodiment, the invention describes the method for the mutation allele of the coding O-ethanoyl homoserine sulfhydrylase of identifying opposing methionine(Met) feedback inhibition and/or OSHS sulfhydrylase, it comprises: a) will depend on DMDS and contact with the methionine analogs that suppresses described microorganism growth with the microorganism that grows on the substratum without methionine(Met) with plasmid-encoded O-ethanoyl homoserine sulfhydrylase and/or OSHS sulfhydrylase, b) select the mutation variants of the anti-described analogue of described microorganism, c) separate described mutation variants, wherein said resistant phenotype is by described plasmid-encoded, and d) DNA sequence dna of the relevant portion of the described plasmid of mensuration is to identify mutant plasmid, this plasmid has the sequence of change in the coding region of described O-ethanoyl homoserine sulfhydrylase or OSHS sulfhydrylase.The present invention has also described O-ethanoyl homoserine sulfhydrylase or the OSHS sulfhydrylase of the new sudden change that separates by the method, the gene of the described mutant enzyme of encoding, and the biology that contains described mutant enzyme.

The accompanying drawing summary

Fig. 1 is the diagram of methionine(Met) biosynthetic pathway.The methionine(Met) biological enzyme is described with runic and the gene of their correspondences is pointed out with italic.

Fig. 2 is the schematic diagram of pH273 carrier.

Fig. 3 is the schematic diagram of pH373 carrier.

Fig. 4 is the schematic diagram of pH304 carrier.

Fig. 5 is the schematic diagram of pH399 carrier.

Fig. 6 is the schematic diagram of pH484 carrier.

Fig. 7 is the schematic diagram of pH491 carrier.

Fig. 8 A-8B uses plasmid H215, before disappearance metY part (8A) and (8B) afterwards, and the chromosomal structural representation of the metY excellent bacillus of district's Glutamic Acid.

Fig. 9 is the schematic diagram of carrier pOM86, and this carrier is that design is used for the plasmid of spectinomycin resistance box destruction Corynebacterium glutamicum metF gene.

Figure 10 is the schematic diagram of pH469 carrier.

Figure 11 is the schematic diagram of pH300 carrier.

Detailed Description Of The Invention

At least part of improve one's methods (for example, microorganism is synthetic) based on finding for generation of methionine(Met) of the present invention.As described herein, produce the poisonous and hazardous chemicals of the current use of methionine(Met) by chemical process, as thiomethyl alcohol as the sulphur source.Have been found that dangerous and toxicity lower sulphur source can be used for the biosynthesizing generation of methionine(Met).Particularly, the inventor has been found that the thioether of methyl blocking, and for example sulphur and/or methyl source, as dimethyl disulfide (DMDS), can join in substratum and by microorganism also referred to as methyl disulfide and use.Described in appended embodiment, the Δ metF bacterial strain of Corynebacterium glutamicum or Δ metE Δ metH bacterial strain can use the thioether of methyl blocking, for example sulphur and/or methyl are originated, as the source of dimethyl disulfide (DMDS) as thioether and methyl, avoided as synthetic methionine(Met) to the needs of MetH/MetE and MetF activity with to the needs of sulphate reducing.

The thioether of methyl blocking, for example sulphur and/or methyl source, provide most advantages of thiomethyl alcohol still there is no its most shortcomings as dimethyl disulfide for generation of methionine(Met).DMDS is the disulfide dimer of the oxidation of thiomethyl alcohol, and it is the relatively inexpensive by product of petroleum distillation industry.It is liquid in room temperature, and boiling point is approximately 109 ℃.DMDS is insoluble in water; If with 0.1% or greater concn, especially with 0.3% or greater concn join growth medium, so most DMDS are retained in bottom or the side of container as oil.

In addition, the present invention illustrates MetY (also referred to as MetZ; O-ethanoyl-homoserine sulfhydrylase) be the thioether with methyl blocking; for example sulphur and/or methyl are originated; directly or indirectly mix the enzyme of methionine(Met) as DMDS; because the Δ metF Δ metB bacterial strain of Corynebacterium glutamicum can use the thioether of methyl blocking; for example sulphur and/or methyl are originated, as the source of DMDS as disulfide and methyl.In addition, by adding the thioether of methyl blocking, for example sulphur and/or methyl are originated, and have improved the methionine(Met) output of the through engineering approaches prototroph bacterial strain of accumulation O-ethanoyl homoserine as DMDS.

Therefore, the invention provides method and microorganism for generation of methionine(Met).

In order more easily to understand the present invention, at first define some terms at this paper.

Term " methionine(Met) biosynthetic pathway " comprise relate to the formation of methionine(Met) and synthetic in utilization the methionine(Met) biosynthetic enzyme (for example, the polypeptide of biosynthesizing enzyme coding gene coding), the biosynthetic pathway of compound (for example, precursor, substrate, intermediate or product), cofactor etc.Term " methionine(Met) biosynthetic pathway " comprises the biosynthetic pathway of synthetic (for example, in the body) that cause methionine production in microorganisms and causes the synthetic biosynthetic pathway of external methionine(Met).

Term " methionine(Met) biosynthetic enzyme " comprises any enzyme that utilizes in compound (for example, intermediate or the product) formation of methionine(Met) biological approach." methionine(Met) biosynthetic enzyme " comprises the enzyme that for example relates to the synthetic alternative route of " transsulfuration approach " and " directly sulfhydrylation approach ", methionine(Met).For example, intestinal bacteria utilize the transsulfuration approach, and the close relative of other microorganisms such as yeast saccharomyces cerevisiae, Corynebacterium glutamicum and subtilis and these microorganisms has been developed direct sulfhydrylation approach.Although many microorganisms are used one of transsulfuration approach or direct sulfhydrylation approach, rather than two kinds of approach, some microorganisms use two kinds of approach to be used for methionine(Met) as Corynebacterium glutamicum synthetic.

" methionine(Met) biosynthetic enzyme " comprises all enzymes of the promotion methionine(Met) generation of usually finding in microorganism.Table 1 has been listed plurality of enzymes and their gene of correspondence of coding in the methionine(Met) biosynthetic pathway, and Fig. 1 has described the diagram of methionine(Met) biosynthetic pathway.Be appreciated that in some microorganisms, the title of the gene of the corresponding enzyme of coding can be different from the listed title of this paper.

Table 1: the enzyme in the methionine(Met) biosynthetic pathway and their gene of coding

According to Fig. 1, methionine(Met) is undertaken by intermediate aspartic acid, phosphoaspartate and aspartic acid semialdehyde from the synthetic of oxaloacetic acid (OAA).The aspartic acid semialdehyde by homoserine dehydrogenase (product of hom gene, in other biological also referred to as thrA, metL, hdh etc.) be transformed into homoserine.Later step during methionine(Met) is synthetic can be undertaken by transsulfuration approach and/or direct sulfhydrylation approach.

In the transsulfuration approach, homoserine is by the homoserine acetyltransferase (product of metX gene, sometimes also referred to as metA) be transformed into O-acetylhomoserine by adding acetyl-CoA, perhaps by the product of metA gene (homoserine succinyltransferase) by adding succinyl CoA to be transformed into OSHS.By cystathionine v-synthase (product of metB gene), supply with O-acetylhomoserine or OSHS has produced cystathionine from the methylthio group of halfcystine.Then cystathionine is transformed into homocysteine by the product cystathionine beta-lyase of metC gene (in some biologies also referred to as the aecD gene).

In direct sulfhydrylation approach, the product O-acetylhomoserine sulfhydrylase catalysis thioether of metY gene (sometimes being called the metZ gene) directly adds O-acetylhomoserine and forms homocysteine.Product OSHS sulfhydrylase by metZ gene gene to directly the adding of OSHS, also can directly form homocysteine in the sulfhydrylation approach with-----thioether group.

No matter use transsulfuration approach or the sort of approach of direct sulfhydrylation approach, pass through vitamins B ₁₂Methionine synthases (product of metH gene) or the B of-dependence ₁₂-methionine synthases (product of metE gene) independently produces methionine(Met) by adding methyl subsequently from homocysteine.(supply with, and methyl-tetrahydrofolic acid (THFA) reduces again methylene radical-THF in the reaction by the catalysis of metF gene product and produces by methyl-THF) by methyl-tetrahydrofolic acid (THFA) for the methyl of methionine(Met).

I. exist lower culturing micro-organisms with the generation methionine(Met) in dimethyl disulfide (DMDS) Method and be used for the recombinant microorganism of the inventive method

On the one hand, the invention describes the method that produces methionine(Met), it is included in the thioether of methyl blocking, for example sulphur and/or methyl source, there is the lower microorganism that produces methionine(Met) of cultivating in preferred dimethyl disulfide (DMDS), thereby produces the salt of METHIONINE or METHIONINE." producing the microorganism of methionine(Met) " is any microorganism that can produce methionine(Met), for example, and bacterium, yeast, fungi, archeobacteria etc.In one embodiment, the microorganism of generation methionine(Met) belongs to Corynebacterium.In another embodiment, the microorganism of generation methionine(Met) is Corynebacterium glutamicum.In a further embodiment, the microorganism that produces methionine(Met) (for example is selected from gram negative bacterium, intestinal bacteria or relevant enterobacteria), gram positive bacterium (for example, subtilis (Bacillussubtilis) or relevant genus bacillus), yeast (for example, yeast saccharomyces cerevisiae (Saccharomycescerevisiae) or relevant yeast strain), and archeobacteria, for example, be suitable for use in the microorganism in the inventive method.In one embodiment, the described microorganism that belongs to enterobacteria group is intestinal bacteria.In another embodiment, the microorganism that belongs to bacillus is subtilis.In a further embodiment, yeast microorganism is yeast saccharomyces cerevisiae or its close relative.

In more than one embodiment of the present invention, comprising the thioether of methyl blocking, for example sulphur and/or methyl source, cultivate microorganism of the present invention in the substratum of preferred dimethyl disulfide (DMDS).In one embodiment, the thioether of methyl blocking, for example sulphur and/or methyl the source, for example DMDS with 0.02% or greater concn be present in substratum.In another embodiment, the thioether of methyl blocking, for example sulphur and/or methyl the source, for example DMDS with 0.04% or greater concn be present in substratum.In a further embodiment, the thioether of methyl blocking, for example sulphur and/or methyl the source, for example DMDS with 0.06% or greater concn be present in substratum.

In more than one embodiment of the present invention, the thioether of methyl blocking, for example sulphur and/or methyl source is the polymkeric substance of the more high molecular of dimethyltrisulfide, dimethyl four sulphur or thioether, its end is by methyl blocking.H by the example of this type of disulfide polymer of methyl blocking ₃C-(S) _n-CH ₃, wherein n is 2-50.In one embodiment, n is 40-50.In another embodiment, n is 30-40.In another embodiment, n is 20-30.In another embodiment, n is 10-20.In another embodiment, n is 5-10.In preferred embodiments, n is 5,6,7,8,9 or 10.In another preferred embodiment, n is 2,3 or 4.Other examples that contain the polymkeric substance of thioether are poly-(oxyethane thioethers), and it (for example sees Lee et al., Biomacromolecules.2005 Jan-Feb by inner loop oxidative ethane oligomer and disulfide linkage; 6 (1): 24-6) form with poly-(diphenyl sulfide).

In one embodiment, use the thioether delivery system of slowly-releasing methyl blocking, for example, slowly-releasing sulphur and/or methyl delivery system, for example, slowly-releasing dimethyl disulfide (DMDS) delivery system is sent the thioether of methyl blocking to culture, for example, sulphur and/or methyl source.As used herein, phrase " the thioether delivery system of slowly-releasing methyl blocking ", " slowly-releasing sulphur and/or methyl delivery system " and " slowly-releasing dimethyl disulfide (DMDS) delivery system " comprise any inert substance, it can add or contact substratum and make little hydrophobic organic compound, thioether as methyl blocking, originate as sulphur and/or methyl, can be discharged into water as DMDS, make the thioether of methyl blocking, originate as sulphur and/or methyl, remain on the sublethal concentrations of biology used as the Css of DMDS." the thioether delivery system of slowly-releasing methyl blocking ", for example, " slowly-releasing sulphur and/or methyl delivery system ", for example, " slowly-releasing dimethyl disulfide (DMDS) delivery system " also allow these compounds in time with and the horizontal extension, sustained release these compounds that can not adversely affect microorganism self growth nontoxic on microorganism in solution.In one embodiment, the thioether of methyl blocking as sulphur and/or methyl source, is liquid as the slowly-releasing delivery system of DMDS, and itself and water immiscibility, but can dissolve the thioether of methyl blocking are as sulphur and/or methyl source, as DMDS.In preferred embodiments, the thioether of methyl blocking as sulphur and/or methyl source, is the pearl macropore polystyrene resin (ps) as the slowly-releasing delivery system of DMDS, for example, and Amberlite ^TMXAD4.Amberlite ^TMXAD4 is comprised of insoluble globule, and the wet product of its water as infiltration sodium-chlor and sodium carbonate provides.At the thioether of methyl blocking, originate as sulphur and/or methyl, before the absorption as DMDS, as use ethanol and the water washing Amberlite of manufacturer's recommendation ^TMXAD4 produces suspension in water.The thioether of methyl blocking is originated as sulphur and/or methyl, absorbs Amberlite as DMDS ^TMAfter XAD4 also added substratum with this mixture, the thioether of methyl blocking was originated as sulphur and/or methyl, discharged from globule with the speed of enough keeping metF or metE, the growth of metH auxotroph as DMDS.In one embodiment, the thioether of methyl blocking, as sulphur and/or methyl source, as DMDS with 0.1% or higher being present in contain Amberlite ^TMIn the substratum of XAD4.In another embodiment, the thioether of methyl blocking, as sulphur and/or methyl source, as DMDS with 0.2% or higher being present in contain Amberlite ^TMIn the substratum of XAD4.In a further embodiment, the thioether of methyl blocking, as sulphur and/or methyl source, as DMDS with 0.3% or higher being present in contain Amberlite ^TMIn the substratum of XAD4.

In another preferred embodiment, the thioether delivery system of slowly-releasing methyl blocking, for example, slowly-releasing sulphur and/or methyl delivery system, for example, DMDS slowly-releasing delivery system is the thioether charging of slowly-releasing methyl blocking, for example, sulphur and/or methyl source charging, for example, the DMDS charging.Phrase " the thioether charging of slowly-releasing methyl blocking ", " slowly-releasing sulphur and/or methyl source charging " and " slowly-releasing DMDS charging " are the slowly-releasing chargings as used herein, it is with the thioether of methyl blocking, for example, sulphur and/or methyl are originated, for example DMDS is cumulative or be delivered to continuously culture with q.s, make to produce the purpose product, as methionine(Met), but avoid producing the virose level of microorganism.In another preferred embodiment, slowly-releasing delivery system of the present invention is the thioether to methyl blocking, originate as sulphur and/or methyl, film as permeable in DMDS, and allow the thioether of methyl blocking, originate as sulphur and/or methyl, spread or flow in growth medium by this film as DMDS.The limiting examples of film is included in organic solvent (as DMDS) and there is lower any film that can keep the structure and function integrity in aqueous culture medium.This type of film is Technical Reference Handbook (the Millipore Corporation of " 1994-1995 Millipore Direct " in Millipore company's catalogue (Millipore Corporation Catalog) and title, Bedford, MA, USA) describe in, with its complete being incorporated herein by reference.Suitable membrane comprises those films that comprise PVDF (polyvinylidene difluoride (PVDF)), PTFE (tetrafluoroethylene), polypropylene, polyvinyl chloride, polyethersulfone, nylon and polycarbonate, and it has or do not have hydrophilic coating.In addition, the limiting examples that can be used as the material of slowly-releasing dimethyl disulfide (DMDS) delivery system comprises the hydrophobic resin of other pearlizations, animal oil, mineral oil, chemistry oil, vegetables oil, synthetic oil, organic solvent, chlorocarbon, fluorocarbon, Chlorofluorocarbons (CFCs), perhaps its combination.

As described herein; wherein genetically engineered methionine(Met) biosynthetic pathway; for example the microorganism with excessive generation O-ethanoyl homoserine causes containing the thioether of methyl blocking, originates as sulphur and/or methyl, as the output raising of methionine(Met) in the substratum of DMDS.Therefore, the present invention also provides the method that produces methionine(Met), and it is included in the thioether of methyl blocking, originate as sulphur and/or methyl, under preferred dimethyl disulfide (DMDS) exists, cultivate the microorganism of the methionine(Met) biosynthetic pathway with imbalance, thereby produce methionine(Met).

Method of the present invention has been described microorganism, for example, recombinant microorganism, it preferably includes carrier as described herein or gene (for example, wild-type and/or mutator gene) and/or cultivates in the mode that causes producing purpose product (as methionine(Met)).Term " restructuring " microorganism comprises that microorganism (for example, bacterium, yeast cell, fungal cell etc.), its genetically engineered, modification or through engineering approaches are (for example, genetically engineered), make naturally occurring microbial that it and it originates than demonstrating change, modify or different genotype and/or phenotypes (for example, when genetic modification affects the nucleic acid sequence encoding of microorganism).

In another preferred embodiment, design or through engineering approaches recombinant microorganism make and express or cross and express at least a natural methionine(Met) biosynthetic enzyme.Term " is crossed and is expressed " and is included in expressing gene product in suitable growth medium (for example, biosynthetic enzyme), its expression level higher than the operation of this microorganism before or the expression level in the suitable microorganism of not operation.In one embodiment, can genetic design or the through engineering approaches microorganism express the gene product of certain level to cross, this level is higher than the expression level in there is no the suitable microorganism of through engineering approaches.Preferably, the biosynthesizing enzyme coding gene is included in recombinant vectors and/or from recombinant vectors and expresses biosynthetic enzyme.The technician will understand the microorganism of expressing or crossing the expressing gene product due to the expression of the nucleotide sequence of this gene product of coding and/or gene or cross to express and produce or this gene product of excessive generation.

Term " through the microorganism of operation " comprises such microorganism, it by through engineering approaches (for example, genetically engineered) or modify, make amount or the structure of at least a enzyme of the methionine(Met) biosynthetic pathway of this microorganism be modified, make methionine(Met) output increase.The modification of this quasi-microorganism or through engineering approaches can include but not limited to according to any method described herein, the expression excessively of the imbalance of biosynthetic pathway and/or at least a biosynthetic enzyme." through what operate " enzyme (for example, " through what operate " biosynthetic enzyme) comprise such enzyme, its expression, generation or activity are changed or modify, make at least a upstream of this enzyme or downstream precursor, substrate or product compare with corresponding wild-type or naturally occurring enzyme and be changed or modify (for example, the level of the change of precursor, substrate and/or product or modification, ratio etc.)." through operation " enzyme also comprises a kind of enzyme, wherein to for example inhibition of one or more products or intermediate, is enhanced as the resistance of feedback inhibition.For example, can effectively bring into play the enzyme of enzymatic functions under for example methionine(Met) exists.

The horizontal expression that term " is crossed and expressed " or " cross and express " comprises gene product (for example, methionine(Met) biosynthetic enzyme) higher than this microorganism operation before or the expression level in the suitable microorganism of not operation.In one embodiment, can express the gene product of certain level to cross by genetic manipulation (for example, genetically engineered) microorganism, this level operates expression level front or in the suitable microorganism of not operation higher than this microorganism.genetic manipulation can comprise, but be not limited to, the adjusting sequence that change or modification are relevant with the expression of specific gene or site are (for example, by adding strong promoter, inducible promoter or a plurality of promotor or regulate sequence and make that to express be composing type by removing), modify the chromosome position of specific gene, the nucleotide sequence that change and specific gene such as ribosome binding site or transcription terminator are adjacent, increase the copy number of specific gene, modification relate to that specific gene is transcribed and/or the protein of specific gene product translation (for example, regulate protein, suppress son, enhanser, transcriptional activator etc.), any other ordinary method that perhaps in this area, conventional imbalance specific gene is expressed (includes but not limited to, use antisense nucleic acid molecule, for example, expression and/or the use of blocking-up repressor increase change allelotrope, for example, the bacterium allelotrope that strengthens heritable variation and accelerate for example adaptive evolution).

In another embodiment, can physically or operate microorganism on environment and express the gene product of certain level to cross, this level operates expression level front or in the suitable microorganism that does not operate higher than microorganism.For example, can known or suspect to strengthen specific gene transcribing and/or the material of specific gene product translation is processed or culturing micro-organisms under existing, make to transcribe and/or translate be enhanced or increase.Alternatively, can through select with strengthen specific gene transcribe and/or the temperature of specific gene product translation under culturing micro-organisms, make to transcribe and/or translate be enhanced or increase.

Preferred " restructuring " microorganism of the present invention is to have the methionine(Met) biosynthetic pathway of imbalance or the microorganism of enzyme.Term " imbalance " or " imbalance " comprise at least a gene of the enzyme in the encoding human route of synthesis in change or modified microorganism, make the level of this biosynthetic enzyme in microorganism or activity be changed or modify.Preferably, at least a gene of the enzyme in change or modification encoding human route of synthesis makes this gene product be enhanced or increase.Phrase " approach of imbalance " can also comprise such biosynthetic pathway, and wherein the gene of the enzyme in more than one encoding human route of synthesis is changed or modifies, and makes level or the activity of more than one biosynthetic enzyme be changed or modify.In " imbalance " microorganism, the ability of approach (for example, lack of proper care simultaneously more than one gene in given biosynthetic pathway, for example, 2,3,4,5,6,7 kind of gene) caused by the particular phenomenon in microorganism, wherein more than one enzyme (for example, 2,3,4,5,6,7 etc. kind biosynthetic enzyme) of the genes encoding of mutual adjacent existence is known as " operon " on the continuous fragment of genetic material.

Term " operon " comprises at least two adjacent genes or ORF, chooses wantonly in sequence and holds overlapping at 5 ' or 3 ' of at least one gene or ORF.Term " operon " comprises the genetic expression unit of coordination, its contain promotor and possibly with one or more, the preferred regulatory element of at least two kinds of structure genes (for example, codase is as the gene of biosynthetic enzyme) combination.The expression of structure gene can be for example by in conjunction with the adjusting albumen of regulatory element or regulate synergistically by the anti-termination of transcribing.Structure gene can be transcribed the single mRNA of all structural proteins that obtain encoding.The collaborative adjusting of the gene that comprises due to operon, the change of single promotor and/or regulatory element or modify change or the modification of every kind of gene product that can cause the operon coding.the change of regulatory element or modification can include but not limited to, remove endogenesis promoter and/or regulatory element, add strong promoter, inducible promoter or a plurality of promotor or remove and regulate sequence and make the expression of gene product be modified, change the chromosome position of operon, change nucleotide sequence such as ribosome bind site in or operon adjacent with operon, increase the copy number of operon, modification relate to that operon is transcribed and/or the protein of the translation of operon gene product (for example, regulate albumen, suppress son, enhanser, transcriptional activator etc.), perhaps any other ordinary method of the imbalance genetic expression of this area routine (includes but not limited to, for example use the expression of antisense nucleic acid molecule blocking-up repressor).Imbalance can also relate to the coding region that changes one or more genes for example to produce, and feeds back resistance or inhibition anti-product or intermediate, perhaps has the enzyme of higher or lower specific activity.

Especially preferred " restructuring " of the present invention microorganism is expressed gene or the gene product of bacterial origin by genetically engineered to cross.Term " bacterial origin " or " from " bacterium for example; be included in the gene of natural discovery in bacterium; perhaps the gene product of bacterial gene coding (for example; homoserine acetyltransferase, homoserine dehydrogenase and O-ethanoyl homoserine sulfhydrylase) (such as respectively by metX, hom (also referred to as hsd etc.), and metY coding).

In one embodiment, at least a methionine(Met) biosynthetic enzyme that produces the microorganism of methionine(Met) is lacked of proper care.In preferred embodiments, the methionine(Met) biosynthetic enzyme of imbalance is O-ethanoyl homoserine sulfhydrylase.In another embodiment, at least two kinds of methionine(Met) biosynthetic enzymes that produce the microorganism of methionine(Met) are lacked of proper care.In a preferred embodiment, the methionine(Met) biosynthetic enzyme of imbalance is homoserine acetyltransferase and homoserine dehydrogenase.In another preferred embodiment, the methionine(Met) biosynthetic enzyme of imbalance is O-ethanoyl homoserine sulfhydrylase and homoserine acetyltransferase.

In one embodiment, the invention describes the multiple biosynthesizing modification of enzyme of methionine(Met) biosynthetic pathway.Particularly, the invention describes by modifying or change the genetic modification plurality of enzymes activity relevant with described approach of the described biosynthetic enzyme of coding.

Term " gene " comprises that nucleic acid molecule (for example as used herein, DNA molecular or its section), it can be by intergenic DNA (namely in biology, interleave or spacer DNA, it is natural is in gene in the chromosomal DNA of the flank of this gene and/or separating bio) separate with another gene or other genes.Alternatively, gene can overlapping a little with another gene (for example, 3 ' end of first gene and 5 ' end of second gene be overlapping), and overlapping gene is by intergenic DNA and other gene isolation.Gene is synthetic enzyme or other protein molecules (for example, can comprise encoding sequence, for example, the continuous open reading-frame (ORF) (ORF) of coded protein) or can be from function be arranged in biology directly.Gene in biology can cluster in operon, and as defined herein, described operon separates with other genes and/or operon by intergenic DNA." gene of separation " comprises such gene as used herein, its there is no this gene from the chromosomal DNA of biology in the natural sequence that is positioned at this gene flank (namely, do not encode the adjacent encoder sequence of another kind or different proteins, adjacent structure sequence etc.), and optional 5 ' and 3 ' the adjusting sequence that comprises, for example, promoter sequence and/or terminator sequence.In one embodiment, the gene of separation mainly comprises the encoding sequence (for example, the sequence of the excellent thuringiensis protein of coding) of protein.In another embodiment, the gene that separates comprises that protein (for example, the rod thuringiensis protein) encoding sequence and 5 ' and/or 3 ' adjacent adjusting sequence, the chromosomal DNA of the biology that this adjusting sequence is originated from this gene (for example, the 5 ' and/or 3 ' adjacent excellent bacillus is regulated sequence).Preferably, the gene of separation contains less than approximately 10kb, 5kb, 2kb, 1kb, 0.5kb, 0.2kb, 0.1kb, 50bp, 25bp or 10bp nucleotide sequence, and it is natural is arranged in the flank of this gene of chromosomal DNA of the biology that this gene originates.

" gene with sudden change " or " mutator gene " comprise having and (for example comprise at least one change as used herein, substitute, insert, disappearance) the gene of nucleotide sequence, described change makes the polypeptide of described mutant code or protein demonstrate the activity different from the polypeptide of wild-type nucleic acid molecule or genes encoding or protein.In one embodiment, for example, when measuring (for example, measuring in the microorganism of cultivating at the same temperature) under similar condition, have the polypeptide of the gene of sudden change or mutator gene coding and wild type gene coding or protein and compare to have and increase active polypeptide or protein." activity of increase " or " enzymic activity of increase " is such activity as used herein, it is than the activity of the polypeptide of wild-type nucleic acid molecule or genes encoding or protein greatly at least 5%, preferably than the activity of the polypeptide of wild-type nucleic acid molecule or genes encoding or protein 5-10% greatly at least, more preferably 10-25% or even more preferably 25-50%, 50-75% or 75-100% at least greatly at least.Scope in the middle of above-cited value, for example, 75-85%, 85-90%, 90-95% also are intended to be the present invention includes." activity of increase " or " enzymatic activity of increase " also comprises than at least 1.25 times greatly of the activity of the polypeptide of wild type gene coding or protein as used herein, preferably than at least 1.5 times greatly of the activity of the polypeptide of wild type gene coding or protein, more preferably at least 2 times, even more preferably at least 3 times, 4 times, 5 times, 10 times, 20 times, 50 times, 100 times.

In another embodiment, for example, when measuring (for example, measuring in the microorganism of cultivating at the same temperature) under similar condition, have the polypeptide of the gene of sudden change or mutator gene coding and wild type gene coding or protein and compare to have and fall SA polypeptide or protein.Mutator gene is the wild type peptide that reduces of coded protein or yield level not." activity of reduction " or " enzymic activity of reduction " is such activity as used herein, it is than the activity low at least 5% of polypeptide or the protein of wild-type nucleic acid molecule or genes encoding, preferably than the polypeptide of wild-type nucleic acid molecule or genes encoding or the low 5-10% at least of activity of protein, more preferably low 10-25% at least and even more preferably low 25-50%, 50-75% or 75-100% at least.Scope in the middle of above-cited value, for example, 75-85%, 85-90%, 90-95% also are intended to be the present invention includes." activity of reduction " or " enzymic activity of reduction " also comprises and being lacked or the activity of " knocking out " (for example, low by approximately 100% than the activity of the polypeptide of wild-type nucleic acid molecule or genes encoding or protein) as used herein.

In more than one embodiment, microorganisms methionine(Met) with imbalance assortment of genes of the present invention, the methionine level summation that its level produces when for example existing than the gene of every kind of imbalance is 1-2% greatly at least, or 3-5% greatly at least, or 5-10% greatly at least, or 10-20% greatly at least, or 20-30% greatly at least, or 30-40% greatly at least, or 40-50% greatly at least, or 50-60% greatly at least, or 60-70% greatly at least, or 70-80% greatly at least, or 80-90% greatly at least, or 90-95% greatly at least.

In some embodiments, 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 of the methionine level summation height that the level of methionine(Met) of microorganisms that comprises the combination of the gene of lacking of proper care produces when existing than the gene of every kind of imbalance.

The summation of the amount that in other embodiments, comprises the methionine(Met) that the microorganism of the assortment of genes of change produces under suitable fermentation condition amount of microorganisms than the gene that has every kind of change or when not having gene alteration is 5g or 7g or 8g or 9g or 10g or 11g or 12g or 13g or 14g or 15g or 16g or 17g or 18g or 19g or 20g or 25g or 30g or 40g or 50g/ liter 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 at least at least at least at least at least greatly at least.

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

Can measure active according to the assay method of any generally accepted activity be used to measuring concrete target protein matter.For example, by measuring from the activity of the protein of cell or microorganism separation or purifying, can directly measure or measure activity.Alternatively, can measure or measure in cell or microorganism or the activity in the substratum of extracellular.For example, the mensuration of mutator gene (namely, the described mutant of the enzymic activity that coding reduces) can pass through in microorganism, enzyme is to express the gene of described sudden change in stable responsive mutant microbial as described therein, and the ability of measuring the enzymic activity of complementary responsive to temperature type (Ts) mutant of this mutator gene is also completed.The mutator gene of coding " enzymic activity of increase " can be the wild type gene gene of complementary Ts mutant more effectively of ratio such as correspondence.The mutator gene of coding " enzymic activity of reduction " for example is, there is no the gene of the corresponding such effective supplement Ts of wild type gene mutant.

Even understanding single substitute (base of the amino acid change in the aminoacid sequence that for example, coding is corresponding substitutes) in nucleic acid or gene order, the technician compares also can remarkably influenced coded polypeptide or the activity of protein with corresponding wild type peptide or protein.Mutator gene as defined herein (for example, encoding mutant polypeptide or protein) can easily distinguish with nucleic acid or the gene of coded protein homologue, because having, the mutator gene coding changes active protein or polypeptide, choose wantonly in expressing described mutator gene or producing the microorganism (that is, mutant microbial) of described mutain or polypeptide from the microbial of corresponding expression wild type gene and be different or differentiable phenotype than can be observed.Compare, the protein homology thing can have identical or similar activity basically, and is optional when produce in microorganism, with the corresponding microbial of expression wild type gene than distinguishing on phenotype.Therefore, for example, not that sequence identity degree between nucleic acid molecule, gene, protein or polypeptide be used for to be distinguished homologue and mutant, but the activity of coded protein or polypeptide is distinguished homologue and mutant: homologue (for example for example has low sequence identity, 30-50% sequence identity) have the functionally active that basically is equal to, mutant for example has 99% sequence identity and has the functionally active of remarkable difference or change.

In one embodiment, recombinant microorganism of the present invention is Gram-positive biological (for example, due to the gram-positive cell wall that exists around microorganism, and keeping basic dyestuff, as the microorganism of Viola crystallina).In preferred embodiments, recombinant microorganism of the present invention is the Corynebacterium microorganism.In one embodiment, recombinant microorganism is bacillus.In another preferred embodiment, recombinant microorganism is selected from Bacillus licheniformis (Bacillus licheniformi), bacillus amyloliquefaciens (Bacillusamyloliquefaciens), Bacillus halodurans, subtilis (Bacillus subtilis) and bacillus pumilus (Bacillus pumilus).

In another embodiment, recombinant microorganism is Gram-negative (exclusion basic dyestuff) biology.In another embodiment, recombinant microorganism of the present invention is the microorganism that belongs to enterobacteria.In preferred embodiments, recombinant microorganism is the microorganism that belongs to the genus that is selected from Salmonella (Salmonella), Escherichia (Escherichia), Klebsiella (Klebsiella), Serratia (Serratia) and proteus (Proteus).In a more preferred embodiment, recombinant microorganism is Escherichia (Escherichia).In even preferred embodiment, recombinant microorganism is intestinal bacteria (Escherichia coli).In another embodiment, recombinant microorganism is yeast belong (for example, yeast saccharomyces cerevisiae) yeast and archeobacteria (Archaea).

An important aspect of the present invention relates to cultivates microorganism of the present invention, makes to produce purpose compound (for example, methionine(Met)).

Term " cultivation " comprises and keeping and/or the live microorganism of the present invention of growing (for example, maintenance and/or grown culture or bacterial strain).In one embodiment, cultivate microorganism of the present invention in liquid medium within.In another embodiment, cultivate microorganism of the present invention in solid medium or semisolid medium.In preferred embodiments, comprise for the maintenance of microorganism and/or growth must or useful nutrition (for example, carbon source or carbon substrate, for example, carbohydrate, hydrocarbon, oil, fat, lipid acid, organic acid and alcohol; Nitrogenous source, for example, peptone, yeast extract, meat extract, wort, urea, ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate; The phosphorus source is as 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 etc.) substratum (as aseptic liquid nutrient medium) in cultivate microorganism of the present invention.

The microorganism that produces according to the present invention can be continuously or batch fermentation or cultivate to produce methionine(Met) with fed-batch or repeated fed-batch method.The summary of known cultural method can be seen textbook (Bioprozelitechnik 1.Einfiihrung in dieBioverfahrenstechnik (the Gustav Fischer Verlag of Chmiel, Stuttgart, 1991)) or the textbook of Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

Preferably, cultivate microorganism of the present invention under controlled pH.Term " controlled pH " comprises any pH that causes producing purpose product (for example, methionine(Met)).In one embodiment, culturing micro-organisms under about 7 pH.In another embodiment, culturing micro-organisms under 6.0 to 8.5 pH.Can keep the pH of hope by any method well known by persons skilled in the art.

Also preferably, cultivate microorganism of the present invention under controlled ventilation.Term " controlled ventilation " comprises that enough ventilations (for example, oxygen) are to cause producing purpose product (for example, methionine(Met)).In one embodiment, by regulating the oxygen level in culture, for example, control ventilation by the amount of oxygen that adjusting is dissolved in substratum.Preferably, control the ventilation of culture by the stir culture thing.By water screw or similar mechanical stirring equipment, by rotation or wave and culture container (for example, pipe or bottle) or move equipment by various pumps stirring is provided.Can also pass sterile air or Oxygen control ventilation via substratum (for example, via fermenting mixture).Also preferably, do not having excess foam (for example, by adding defoamer) to have the lower microorganism of the present invention of cultivating.

In addition, microorganism of the present invention can be cultivated at controlled temperature.Term " controlled temperature " comprises any temperature that causes producing purpose product (for example, methionine(Met)).In one embodiment, controlled temperature comprises the temperature between 15 ℃ to 95 ℃.In another embodiment, controlled temperature comprises the temperature between 15 ℃ to 70 ℃.Preferred temperature is 20 ℃ to 55 ℃, more preferably 30 ℃ to 50 ℃.

Microorganism can (for example be cultivated in liquid medium within, keep and/or growth), and continuously preferred or intermittently cultivation, cultivate by ordinary method such as standing cultivation, test tube cultivation, wave and culture (for example, rotation wave and culture, shake-flask culture etc.), ventilation turn cultivation or fermentation.In preferred embodiments, culturing micro-organisms in shaking flask.In a more preferred embodiment, culturing micro-organisms in fermentor tank (for example, fermentation process).Fermentation process of the present invention includes, but not limited in batches, fed-batch and continuation method or fermentation process.Phrase " batch processes " or " batch fermentation " refer to such system, wherein substratum, nutrition, supplemented by additives etc. begin arrange and during fermentation do not changed in fermentation, yet, can attempt to control the factors such as pH and oxygen concentration to prevent excessive medium acidification 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 " continuation method " 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, be preferred for reclaiming purpose product (for example, methionine(Met)).Multiple these class methods have been developed and they are well known in the art.

The substratum that uses must be satisfied in suitable mode the needs of concrete bacterial strain.The description that is used for the substratum of multiple-microorganism is included in the handbook " Manual of Methodsfor General Bacteriology " (Washington D.C., USA, 1981) of U.S. bacteriology association.

The carbon source that is suitable for use in substratum is for for example, sugar and carbohydrate, as glucose, sucrose, lactose, fructose, maltose, molasses, starch and Mierocrystalline cellulose, oil ﹠ fat, as soybean oil, sunflower oil, peanut oil and Oleum Cocois, lipid acid, as palmitinic acid, stearic acid and linolic acid, alcohol, as glycerine and ethanol, and organic acid, as acetic acid.These materials can use separately or as mixture.

Nitrogen-containing organic compound as peptone, yeast extract, meat extract, wort, corn steep liquor, soyflour and urea or mineral compound, can be used as nitrogenous source as ammonium sulfate, ammonium chloride, ammonium phosphate, volatile salt and ammonium nitrate.Nitrogenous source can use separately or as mixture.

Phosphoric acid, potassium primary phosphate or dipotassium hydrogen phosphate or the corresponding sodium salt that contains can be as the phosphorus sources.

Except DMDS, end can be used as extra sulphur source with the dimethyltrisulfide of methyl blocking, dimethyl four sulphur or more high molecular disulfide polymer, organic and inorganic sulfocompound as sulfide, sulphite, vitriol and thiosulphate.

Substratum can also comprise the necessary metal-salt of growth, as sal epsom or ferric sulfate.At last, except above-mentioned substance, can also use essential and nonessential growth substance, as amino acid and VITAMIN.In addition, can add suitable precursor in substratum.The initial substance of mentioning can add with the form of single batch, perhaps can be in the training period with suitable mode charging.

Basic cpd, as sodium hydroxide, potassium hydroxide, ammonia or ammoniacal liquor, perhaps acidic cpd, can be used for controlling pH in suitable mode as phosphoric acid or sulfuric acid.Defoamer can be used for controlling the generation of foam as fatty acid polyglycol ester.Can will have the suitable substance of selective action, add the stability that keeps plasmid in substratum as microbiotic.In order to keep aerobic condition, import oxygen in the culture or contain the gaseous mixture of oxygen, as air.The temperature of culture is generally 20 ℃ to 45 ℃, preferred 25 ℃ to 40 ℃.Continue to cultivate until formed maximum purpose product.This target reached in 160 hours at 10 hours usually.

Phrase " is cultivated " to be included under the condition that produces the purpose product and is fit to or enough obtains the output of purpose compound or obtain wishing lower maintenance and/or the microorganism that grows of condition (as temperature, pressure, pH, time length etc.) of the compound concrete to be generated of output.For example, cultivate the lasting compound (for example, methionine(Met)) of time enough to produce desired amount.Preferably, cultivate the lasting suitable output (for example, enough reach the time of the methionine(Met) of suitable concn) of time enough basically to reach this compound.In one embodiment, cultivate lasting approximately 12 to 24 hours.In another embodiment, cultivate to continue approximately 24 to 36 hours, 36 to 48 hours, 48 to 72 hours, 72 to 96 hours, 96 to 120 hours, 120 to 144 hours or greater than 144 hours.In another embodiment, cultivate to continue time enough with the production output of the hope that reaches methionine(Met), for example, culturing micro-organisms makes generation arrive 10g/L or at least 10 at least about 7 and arrives 15g/L, or arrives 20g/L or arrive 25g/L or arrive 30g/L or arrive 35g/L or arrive 40g/L or arrive the 50g/L methionine(Met) at least about 40 at least about 35 at least about 30 at least about 25 at least about 20 at least about 15.In other embodiments, under certain condition culturing micro-organisms make approximately in 24 hours, approximately in 36 hours, approximately in 48 hours, approximately in 72 hours or approximately produce the preferred output of methionine(Met) in 96 hours, for example provide the output in scope in the above.

Method of the present invention can also comprise the step that reclaims purpose compound (for example, methionine(Met)).That term " recovery " purpose compound comprises is concentrated from substratum, extraction, results, isolated or purified compound.Can carry out the recovery of compound according to any conventional separation known in the art or purification process, include but not limited to, with conventional resin (for example, negatively charged ion or Zeo-karb, non-ionic adsorption resin etc.) process, process with conventional sorbent material (for example, gac, silicic acid, silica gel, Mierocrystalline cellulose, alumina etc.), change pH, solvent extraction (for example, with conventional solvent as alcohol, ethyl acetate, hexane etc.), dialysis, filter, concentrated, crystallization, recrystallization, pH regulator, freeze-drying, drying, evaporation etc.For example, by at first removing microorganism from culture, can reclaim methionine(Met) from substratum.

Preferably, " extraction ", " separation " or " purifying " purpose compound of the present invention make the gained prepared product there is no other nutrient media componentses (for example, there is no nutrient media components and/or fermentation byproduct).Phrase " essentially no other nutrient media componentses " comprises the prepared product of purpose compound, and wherein this compound separates with nutrient media components or the fermentation byproduct of the culture that produces this compound.in one embodiment, prepared product greater than the purpose compound of about 80% (dry weight) (for example has, less than approximately 20% other nutrient media componentses or fermentation byproduct), more preferably greater than about 90% purpose compound (for example, less than about 10% other nutrient media componentses or fermentation byproduct), more preferably greater than about 95% purpose compound (for example, less than about 5% other nutrient media componentses or fermentation byproduct), most preferably greater than the purpose compound of about 98-99% (for example, less than approximately other nutrient media componentses or the fermentation byproduct of 1-2%).

The disclosure also comprises bioconversion method, and it has described multiple recombinant microorganism as herein described.Term " bioconversion method " comprises in this article also referred to as " biotransformation method " biological method that causes the suitable substrate of generation (for example, transform or change) 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 their to carry out the form of their expectation functions (for example, producing the purpose compound).This quasi-microorganism can be full cell, can be only perhaps those cell parts (for example, gene and/or enzyme) that obtain desirable net result necessity.Can be with these microbial suspensions (for example, in suitable solution, in buffered soln or substratum), (for example rinse, the substratum of culturing micro-organisms is removed in flushing), acetone drying, immobilization (for example, with polyacrylamide gel or k-carrageenan or on synthetic support such as globule, matrix etc.), fixing, crosslinked or saturatingization (for example, the film of having and/or cell walls make compound can easily pass as substrate, intermediate or product as described in film or cell walls).

In alternative embodiment, for example when microorganism is biology harmless (for example safety), not from microorganism purifying purpose compound.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 product that obtains by the present invention can also comprise other components of fermentation culture except methionine(Met), as phosphoric acid salt, carbonate, remaining carbohydrate, biomass, complicated nutrient media components, etc.

II. recombinant nucleic acid molecules, carrier and polypeptide

The present invention has also described recombinant nucleic acid molecules (for example, recombinant DNA molecules), and it (for example comprises gene described herein, the gene that separates), preferred excellent bacillus gene, more preferably Corynebacterium glutamicum gene, even more preferably Corynebacterium glutamicum methionine(Met) biosynthesis gene.Term " recombinant nucleic acid molecules " comprise change, the nucleic acid molecule of modification or through engineering approaches (for example, DNA molecular), described change, modification or through engineering approaches make it list with this recombinant nucleic acid molecules at nucleotides sequence to originate the original or natural acid molecule of (for example, by adding, lack or substituting one or more Nucleotide) different.Preferably, recombinant nucleic acid molecules (for example, recombinant DNA molecules) comprises the gene of the separation of the present invention of effective connection adjusting sequence.The nucleotide sequence of the gene of phrase " effectively connect regulate sequence " feeling the pulse with the finger-tip with the expression that allows this gene (for example, that strengthen, that increase, composing type, the expression basis, that weaken, that reduce or that suppress), the mode of the expression of the gene product of preferred this genes encoding (for example, when recombinant nucleic acid molecules is included in as defined herein recombinant vectors and imports in microorganism) is connected to the adjusting sequence.

Term " heterologous nucleic acids " refers to usually not be present in the nucleotide sequence in target organism as used herein.They can also comprise and being present in target organism, but the nucleotide sequence of usually not finding in the heredity district of purpose target organism.Similarly, term " heterologous gene " refers to not to be present in the gene in the wild-type strain isolated of host living beings.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 disclosure also comprises the homologue of several genes described herein and protein." homologue " of gene refers to nucleotide sequence, itself and this gene at least part of or basically same with its complementary strand or its part, condition is that this nucleotide sequence coded protein has substantially the same activity/function with the protein of the homologue coding of this gene.Hydrogen base acid by the homologue of inferring and nucleotide sequence and described gene are (for example, the nucleotide sequence of Corynebacterium glutamicum gene ask, hom, metX, metY, metB, metH, metE, metF, metC and metK or their complementary strand) or the sequence of their coded proteins between per-cent identity, can identify the homologue of gene described herein.For example, by visual inspection or by using multiple computer program known in the art or as herein described can determine per-cent identity.For example, use Devereux et al. (1984) Nucl.Acids.Res., 12:387 described and can be from University of Wisconsin GeneticsComputer Group (UWGCG)) the GAP computer program that obtains, can determine two per-cent identity between nucleotide sequence by comparative sequences information.Use as Tatusova et al. (1999) FEMS Microbiol.Lett., the described basic Local Alignment research tool of 174:247 (BasicLocal Alignment Search Tool (BLAST.RTM.)) program, by comparing two nucleotide sequences, also can determine per-cent identity.For example, for using BLAST ^TMThe nucleotide sequence comparison that program is carried out, default setting is as follows: matching score 2, mispairing point penalty-2, make a breach and extend the breach point penalty and be respectively 5 and 2, breach time decreased (gap.times.dropoff) is 50, expected value is 10, word length 11, and filter (filter) is for cutting out (OFF).

Term " homology " and " homology " are not limited to specify the protein with theoretic common hereditary ancestors as used herein, but 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 to have remarkable identity in their aminoacid sequence, but the protein with function homology define as enzymatic activity by having similar or identical activity.Similarly, the protein that has a structural homology is defined as having similar three grades (perhaps level Four) structures and needn't needs amino acid identity or their nucleic acid homology of gene of encoding.In some cases, structural homology can comprise only at the avtive spot of protein or the protein of binding site maintenance structural homology.

Except the structure and function homology, the present invention also comprises the protein that has at least part of 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 aminoacid sequence the best of another protein in the aminoacid sequence of a protein).Then the amino-acid residue on more corresponding amino acid position.When same amino acid occupied on correspondence position in by another sequence when the position in a sequence, these two molecules were same on this position.Between two sequences, per-cent identity is the function (that is, % identity=same position number/total number of positions * 100) of the same position number enjoyed of these 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 nucleotide sequence or the aminoacid sequence of above identity.

The present invention also comprises the genetically engineered technology that has the enzyme of improved or decorative features with generation for protein as herein described as known in the art.For example, known modifying protein in instruction available in the art makes this protein have Binding Capacity affinity increase or that reduce.Also favourable and be the protein that design has the enzymatic speed that increases or reduce in the instruction of this area.Especially for multifunctional enzyme, usefully the various active of difference ground fine adjustments protein is to carry out best under specific environment.In addition, regulatory enzyme can participate in methionine(Met) or may participate in the combination of other cofactors of negative or positive regeeration or the amino acid of coordination is completed by selectively changing the ability of the susceptibility of feedback inhibition (for example, by methionine(Met)).In addition, genetically engineered comprise with transcribe with translation skill on the relevant event of the 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 pass through the activity identification of the protein of this homologue coding.For example, this homologue can be complementary sudden change in its homologue gene.

Term " adjusting sequence " comprises the nucleotide sequence that impact (for example, regulation and control or adjusting) other nucleotide sequences (being gene) are expressed as used herein.In one embodiment, regulating sequence is included in recombinant nucleic acid molecules, be in respect on specific purposes gene similar or identical position and/or direction, as for appearing at of regulating that sequence and goal gene observe natural, as on natural place and/or direction.For example, can comprise goal gene in recombinant nucleic acid molecules, it is effectively connected to the adjusting sequence, in natural biological, this adjusting sequence is followed or (for example, is effectively 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 recombinant nucleic acid molecules, is effectively connected to follow in natural biological or in abutting connection with the adjusting sequence of another (for example, different) gene.Alternatively, goal gene can be included in recombinant nucleic acid molecules, is effectively connected to from another biological regulation sequence.For example, the adjusting sequence (for example, other bacteriums are regulated sequence, phage is regulated sequence etc.) from other microorganisms can be effectively connected to the specific purposes gene.

In one embodiment, regulate sequence and be the sequence that non-natural or non-natural exist (for example, modified, sudden change, substitute, the sequence of derivative, disappearance, comprise the sequence of chemosynthesis).The preferred sequence of regulating (for example comprises promotor, enhanser, termination signal, antitermination signal and other expression controlling elementss, suppress the sequence of son or elicitor combination and/or for example in the mRNA that transcribes, transcribe and/or translate the binding site of regulating protein).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, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Patek, M.etal is in (2003) Journal of Biotechnology 104:311-323.Regulate sequence comprise instruct that nucleotides sequence is listed in constitutive expression in microorganism those (for example, constitutive promoter and strong constitutive promoter), instruct that nucleotides sequence is listed in inducible expression in 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 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 regulator of transcribing makes the expression of goal gene be enhanced.

In one embodiment, recombinant nucleic acid molecules of the present invention can comprise nucleotide sequence or the gene of at least a bacterial gene product of coding (for example, the methionine(Met) biosynthetic enzyme), and it effectively connects promotor or promoter sequence.Preferred promoter of the present invention comprises excellent bacillus promotor and/or phage promoter (for example, infecting the phage of excellent bacillus).In one embodiment, promotor is excellent bacillus promotor, preferred strong excellent bacillus promotor (promotor that for example, the biological chemistry house-keeping gene is combined in excellent bacillus).In another embodiment, promotor is phage promoter.For example, the extra preferred promotor that is used for the Gram-positive biology includes but not limited to, superoxide-dismutase, groEL, EF-T u, amy and SPO1 promotor.For example, the extra preferred promotor for 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 or λ-PL.

In another embodiment, recombinant nucleic acid molecules of the present invention comprises one or more terminator sequences (for example, transcription termination sequence).Term " terminator sequence " comprises the adjusting sequence of transcribing for stopping mRNA.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 a further embodiment, recombinant nucleic acid molecules of the present invention 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 a further embodiment, recombinant nucleic acid molecules of the present invention 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 DNA) the internal ribosome combination is (for example, to start translation) the site, itself and natural RBS (RBS that for example, finds in naturally occurring gene) differ at least one Nucleotide.Preferred artificial RBS comprises approximately 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 more Nucleotide, its approximately 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 has also described the carrier (for example, recombinant vectors) 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 or other purifying), that the original or natural acid molecule that described change, modification or through engineering approaches make this carrier originate than this recombinant vectors contains is more, still less or different nucleotide sequences.Preferably, recombinant vectors comprises the biosynthesizing enzyme coding gene or comprises the recombinant nucleic acid molecules of described gene, and it is effectively connected to the adjusting sequence, for example, promoter sequence, terminator sequence and/or artificial ribosome bind site (RBS), as defined herein.In another embodiment, recombinant vectors of the present invention comprises the sequence (for example, copying-enhancement sequences) that copies in the enhancing bacterium.In one embodiment, copy enhancement sequences and bring into play function in intestinal bacteria or Corynebacterium glutamicum.In another embodiment, copy enhancement sequences from pBR322.

In a further embodiment, 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 one embodiment, 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 of the present invention can also comprise homologous recombination sequence (for example, be designed for allow the recombinate chromosomal sequence of host living beings of goal gene).Those skilled in the art will understand also that the design of carrier can be according to such as expression level of the selection for the treatment of genetically engineered microorganism, goal gene product etc. 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 karyomit(e) by single homologous recombination event ((cross in) event 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 around the homologous recombination exchange spot of a copy.This title is from Alan Campbell professor, and he has proposed the restructuring of the type first.

" Campbell goes out " as used herein (Campbell out) refers to the progeny cell of " Campbell advances " transformant, wherein upper contained second DNA sequence dna of the DNA that the linearizing of " Campbell advances " DNA is inserted and and second DNA sequence dna in the karyomit(e) source of second DNA sequence dna homology of described linearizing Insert Fragment between homologous recombination event (hybridizing (cross out) event) has for the second time occured, this is recombination event DNA sequence dna of causing disappearance (a casting) part to be integrated for the second time, 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 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 Substitution, a plurality of Substitutions, 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 are usually but not necessary, by to " being advanced by Campbell " DNA sequence dna, carrying out anti-the selection as the gene of the part (this part is abandoned in hope) of subtilis sacB gene and obtain, is lethal during cells 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) 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, etc.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 event that causes " Campbell advances " or " Campbell goes out " can occur in the DNA base scope in homologous DNA sequence, and because homologous sequence will be mutually identical at least part of of this scope, so usually can not point out with precision, where the exchange event has occured.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 usually by part non-homogeneous zone separately and should be placed in the karyomit(e) of " Campbell goes out " cell in the maintenance of non-homogeneous zone.

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 shorter 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 use DNA A (also referred to as pH273) (SEQ IDNO:1) are transformed also " advanced " to produce " Campbell advances " bacterial strain by Campbell.Fig. 2 has shown the schematic 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 (also referred to as pH373) (SEQ ID NO:2) and obtain " Campbell advances " bacterial strain.Fig. 3 has described the schematic 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 approximately 17.4mM Methionin, but and the ATCC13032 bacterial strain does not produce the Methionin of measuring vol.In addition, the M603 bacterial strain produces approximately 0.5mM homoserine, compares the ATCC13032 bacterial strain and does not produce measurable amount, as general introduction in table 2.

The amount of homoserine, O-ethanoyl homoserine, methionine(Met) and Methionin that table 2: strains A TCC13032 and M603 produce

Transform bacterial strain M603 with DNA C (also referred to as pH304, its schematic diagram is described at Fig. 4) (SEQ ID NO:3), obtain " Campbell advances " bacterial strain, then it obtained " Campbell goes out " bacterial strain M690 by " Campbell goes out ".The M690 bacterial strain contains the metH gene (also referred to as P ₄₉₇MetH) the PgroES promotor (P of upstream ₄₉₇Nucleotide sequence provide in SEQ ID NO:12).The M690 bacterial strain produces approximately 77.2mM Methionin and approximately 41.6mM homoserine, as shown in following table 3.

Table 3: the amount of homoserine, O-ethanoyl homoserine, methionine(Met) and Methionin that bacterial strain M603 and M690 produce

Following mutagenesis M690 bacterial strain subsequently: will be grown in M603 overnight culture in BHI substratum (BECTONDICKINSON) with 50mM citrate buffer solution pH 5.5 washings, and process 20 minutes with N-methyl-N-nitrosoguanidine (10mg/ml in 50mM citrate buffer solution pH 5.5) at 30 ℃.After processing, cell again wash with 50mM citrate buffer solution pH 5.5 and the substratum of plating composition below containing in: (all amounts of mentioning are all 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, L-ethionine (SIGMA CHEMICALS, CATALOG#E5139) is adjusted to pH 7.0 with KOH.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 in 0.1M HCl.Add aseptic 50% glucose solution (40ml) of 40ml to final substratum degerming and in the substratum and without bacterio-agar to 1.5% final concentration by filtering.Pour into the final substratum that contains agar in 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 substratum and again streak culture 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 on the CM nutrient agar 30 ℃, 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.

After strain growth, scrape off cell and be resuspended in 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 10ml medium ii (seeing below) together, and shakes on platform with about 200 rev/mins (rpm) 30 ℃ of lower culturing cells at rail in there is no the 100ml shaking flask of baffle plate.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 120 ℃ of autoclavings approximately 20 minutes.Autoclaving and cooling after, add the 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 at the upper amino acid that produces of measuring of the Agilent1100 series LC HPLC of system (Series LC System HPLC), comprise methionine(Met).Allow at the rear quantitative amino acid that produces of the upper separation of Hypersil AA-post (AGILENT) the pre-column of sample is derivative with Phthalyldicarboxaldehyde.

Separate methionine(Met) titre and be in M690 the clone of twice at least.Be used for a kind of these 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 4 general introduction.

Table 4: the amount of homoserine, O-ethanoyl homoserine, methionine(Met) and Methionin that bacterial strain M690 and M1197 produce

Bacterial strain M1179 is transformed with DNA F (also referred to as pH399, its schematic diagram is described at Fig. 5) (SEQ IDNO:4) produce " Campbell advances " bacterial strain, 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 of bacterial strain M1494 generation and the output of bacterial strain M1197 are compared, as following table 5 general introduction.

Table 5: the amount of homoserine, O-ethanoyl homoserine, methionine(Met) and Methionin that bacterial strain LU11197 and M1494 produce

Bacterial strain M1494 is transformed with DNA D (also referred to as pH484, its schematic diagram is described at Fig. 6) (SEQ IDNO:5) produce " Campbell advances " bacterial strain, 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 ₁₂₈₄Sequence show in SEQ ID NO:6).The amino acid output of bacterial strain M1494 is compared with the output of bacterial strain M1990, as general introduction in following table 6.

Table 6: the amount of homoserine, O-ethanoyl homoserine, methionine(Met) and Methionin that bacterial strain M1494 and M1990 produce

Bacterial strain M1990 is transformed with DNA E (also referred to as pH 491, its schematic diagram is described at Fig. 7) (SEQID NO:7) produce " Campbell advances " bacterial strain, it is obtained " Campbell goes out " bacterial strain M2014 by " Campbell goes out " subsequently.The M2014 bacterial strain is crossed expression metA allelotrope, uses the superoxide-dismutase promotor (to be called P ₃₁₁₉MetA).The amino acid output of bacterial strain M2014 is compared with the output of bacterial strain M2014, as general introduction in following table 7.

Table 7: the amount of homoserine, O-ethanoyl homoserine, methionine(Met) and Methionin that bacterial strain M1494 and M1990 produce

Embodiment 2. Corynebacterium glutamicum methionine(Met) auxotrophs are mixed dimethyl disulfide in methionine(Met)

In order to determine whether Corynebacterium glutamicum can mix DMDS in methionine(Met), built the metF disappearance (describing in embodiment 1) in M2014.M2014 is transformed with plasmid pOM86 (Fig. 9) (SEQ ID NO:8) produce " Campbell advances " bacterial strain.Then " Campbell advances " bacterial strain " Campbell goes out " is obtained being called " Campbell goes out " bacterial strain of OM63.In order to determine whether this methionine(Met) auxotroph OM63 can utilize DMDS to synthesize methionine(Met), by measuring the OD of 600nm, measure the growth of the tube culture of OM63 and parent M2014.Culture is grown in substratum (formula sees below) without methionine(Met), replenish methionine(Met) without the methionine(Met) substratum or replenish different amounts DMDS (Aldrich catalog number (Cat.No.) 32,041-2) without in the methionine(Met) substratum.Design this experiment and be used for determining whether DMDS can pass the cytolemma of bacterium; just be reduced into thiomethyl alcohol in tenuigenin, and directly formed METHIONINE as substrate by MetY or MetB or another kind of enzyme such as MetC together with O-ethanoyl-homoserine subsequently.Also design this toxicity of testing to measure the DMDS cell growth (if existence).

As shown in table 8, Corynebacterium glutamicum metF auxotroph can utilize DMDS as the substrate of growth, and therefore is used for methionine(Met) production.In addition, contain replenish 0.02%, 0.04% or the test tube of 5ml without the methionine(Met) substratum of 0.06%DMDS in, the optical density(OD) of all bacterial strains is all similar.Contain replenish 0.08% or the 5ml of 0.1%DMDS have seldom growth or not growth without the test tube of methionine(Met) substratum, may be due to the toxicity of DMDS under these concentration.At last, as expected, what there is no DMDS keeps the growth of M2014 without the methionine(Met) substratum, but does not keep the growth of OM63.

Table 8: replenish or replenish methionine(Met) or dimethyl disulfide (DMDS) without the methionine(Met) substratum in 30 ℃ of growths Corynebacterium glutamicum tube cultures of 36 hours ¹Optical density(OD) under 600nm

¹Tube culture seals with Parafilm around crown cap.

²Substratum without methionine(Met)

³Replenish the 40mg/l methionine(Met) without the Met substratum.

The result of this experiment shows that DMDS can be absorbed and become thiomethyl alcohol by the Corynebacterium glutamicum reductive cleavage and enter the methionine(Met) approach to keep the auxotrophic growth of methionine(Met).Alternatively, DMDS is the direct substrate of O-ethanoyl-homoserine sulfhydrylase or O-O-succinyl--homoserine sulfhydrylase or another kind of enzyme.In other words, reductive cleavage that may a kind of enzyme catalysis DMDS and being incorporated in methionine(Met).

Without the substratum-1 of methionine(Met) liter

100ml Difco ^TMMethionine(Met) is measured substratum (Methionine AssayMedium) (105g/l)

100ml 10x Spizizen salt *

6ml glucose (50%)

4ml " 4B " solution ^*

The 100mg Threonine

The 40mg halfcystine

785ml dH ₂O

5ml 2％CaCl ₂

^*4B solution

VitB1 (B ₁)-0.25mg/ml

Cyanocobalamin (B ₁₂)-50 μ g/ml

Vitamin H-50mM KPO ₄28 μ g/ml in pH=7.0

Pyridoxine hydrochloride-1.25mg/ml

10x Snizizen salt *

20g/l ammonium sulfate

174g/l dipotassium hydrogen phosphate (trihydrate)

60g/l potassium primary phosphate (anhydrous)

The 10g/l Trisodium Citrate

2g/l sal epsom (heptahydrate)

After autoclaving, add 3.5ml 0.4%FeCl ₃6H ₂O and

1ml micronutrient cellulose solution ¹

¹Micronutrient cellulose solution-1 liter

0.15g Na ₂MoO ₄·2H ₂O

2.5g H ₃BO ₃

0.7g CuSO ₄·5H ₂O

1.6g MnCl ₂·4H ₂O

0.3g ZnSO ₄·7H ₂O

15 arrive 20g/L agar by comprising approximately, can prepare the solid form of this substratum.This can complete by standard step, as to approximately adding 20g agar in 750ml water, and autoclaving, and when still melting, add composition listed above with aseptic storage liquid.

Embodiment 3. exploitation DMDS to the delivery system of Corynebacterium glutamicum to be incorporated in methionine(Met)

As discussed above, if DMDS is poisonous directly to join in liquid culture higher than about 0.06% amount so.In order to overcome this problem, the delivery system that searching will allow DMDS slowly to discharge in the solution in time.Amberlite ^TMXAD4 is the macropore polystyrene resin (ps) of pearlization, hereinafter is called " XAD4 ", and it is selected as delivery system, is because it is inertia, can adsorb little hydrophobic organic compound, can be moistening by water, and have high surface-area and little aperture.

Carry out the test tube experiment in order to determine to be adsorbed by XAD4 and still allow the maximum of the DMDS of growth.For this reason, use the 5ml substratum to carry out the test tube experiment to OM63 and M2014.Each test tube contain 100 μ l XAD4 50% suspension (v/v) and without the substratum of methionine(Met), replenish methionine(Met) without the substratum of methionine(Met) or replenish the substratum without methionine(Met) of the DMDS of different amounts.

Add 5ml without the substratum of methionine(Met) in the aseptic 20mM * 20mM that covers by the complexed metal that unclamps to use * 150mM test tube, prepare test tube and measure.The sterile suspension of XAD4 in water (50%v/v) that adds 100 μ l in each test tube.Test tube is seeded in contains BHI substratum (Bacto ^TMBrain heart infusion (Brain Heart Infusion) (Becton, Dickinson andCompany, Sparks, MD)) then the cell of overnight growth in test tube rotates and uses the substratum without methionine(Met) to rinse twice.Cell is resuspended in substratum without methionine(Met) with 50% initial volume.Cell suspension (5 μ l) is as the inoculum of each test tube.After cell inoculation, to each test tube with shown in concentration (v/v) add DMDS.With test tube on the platform shaking table with 200rpm (rev/min) cultivated under 30 ℃ 24-48 hour.Use Genesys ^TM2 spectrophotometers are by the photo densitometry Growth of Cells under 600am.

As shown in table 9, contain replenish 0.1%, 0.2%, 0.3% or the test tube without the substratum of methionine(Met) of 0.4%DMDS in, the optical density(OD) of all bacterial strains is all quite similar.As if growth demonstrates negative interaction to the test tube of additional 0.5%DMDS to OM63, yet the DMDS of this level is tolerated by M2014.In a word, DMDS is adsorbed onto on the XAD4 globule and allows more DMDS to be added in the liquid tube culture of Corynebacterium glutamicum.Enough DMDS discharge from globule, allow the auxotrophic growth fully of methionine(Met).

Table 9. have or do not have shown in the amount DMDS without the substratum 1 of methionine(Met) at XAD4 ²Under existence in 30 ℃ of growth OM63 of 42 hours and the M2014 optical density(OD) at 600nm

¹Substratum without methionine(Met) ²Each test tube contains 50% suspension that the 5ml substratum adds 100 μ lAmberlite XAD4.Porosity=0.5ml/ml.

Add DMDS after the inoculation test tube.

Inoculum-cell is overnight growth in containing the test tube of BHI, then rotates and uses the substratum without Met to rinse twice.With cell with 50% initial volume resuspension.With the inoculum of this cell suspension (5 μ l) as each test tube.

In addition, only XAD4 does not have obvious unfavorable effect for Growth of Cells.As expected, contain have or not DMDS support the growth of M2014 without the test tube of methionine(Met) substratum, but do not support the growth of OM63.Containing additional 40mg/l methionine(Met) does not still have the test tube without the methionine(Met) substratum of DMDS to cause similar final optical density for M2014, but it is low to compare the desired optical density(OD) of OM63.This may be because XAD4 adsorbs the methionine(Met) that some replenish, thus restriction OM63 Growth of Cells.

Embodiment 4. gaseous state DMDS can support the growth of the synthetic and OM101 (Δ metB, Δ metF) of methionine(Met)

The analogue dimethyl diselenide ether (DMDSe) that had shown in the past DMDS is poisonous at gaseous state to microorganism; but can be used for selecting to lack mutant (Brzywczy, the J., and Paszewski of O-ethanoyl homoserine sulfhydrylase; A. (1994) Yeast 9:1335-1342 and Treichler; H.J., et al. (1978) is at FEMS Symposium No.5, pp 177-199; R.Hutter et al.; eds, Academic Press, New York).DMDSe is obtained the approximately final concentration of 5 μ M (if the supply fully of DMDSe is diffused into agar and is dissolved in wherein) as the bottom surface that drop imports the culture dish lid.Yet, do not mention Compound D MDS, and spreading by gaseous state the DMDS whether enough levels can be provided, to be used for growth (suppressing opposite with analogue) be not apparent.For relatively, in the above in the liquid growth experiment of embodiment 1-3, the concentration of DMDS is about 10mM (for example for 0.1% situation) or than high approximately 2000 times of DMDSe in above-mentioned reference.Yet the sending of gaseous state DMDS can prevent the toxicity seen with liquid D MDS.

In order to test this possibility, with approximately 10 ⁸The lawn of the OM101 of individual cell (Δ metB, Δ metF) rinses to relatively without methionine(Met), and is seeded on the agar plate without methionine(Met) that contains the 25ml nutrient agar of having an appointment.DMDS is by cutting a hole at dull and stereotyped center point sample 50 μ l or in by the agar at dull and stereotyped center and placing 50 μ l DMDS and send in the hole.If DMDS is diffused into flat board everywhere, final concentration will be about 25mM so.The contrast flat board is accepted the cell lawn, but without DMDS.Flat board is placed on together in the polypropylene plastic box of the sealing slightly larger than this heap flat board, and cultivated 48-60 hour at 30 ℃.With DMDS on lawn directly the flat board of point sample have from liquid D MDS point sample approximately 30mm kill the district, but dull and stereotyped remainder uniform fold growth lawn.The flat board that contains the liquid D MDS that is placed in the hole is not killed the district, but uniform fold comprises around flat board and until centre hole at the growth lawn of whole flat board.Usually, when the nutrition with needs is placed in nutraceutical auxotrophy lawn central authorities, see growth gradient, at nutrition, Fast Growth occurs the most nearby.At last, do not add DMDS but obtain the complete lawn of OM101 growth with the contrast flat board that the flat board of accepting DMDS is placed in identical sealed plastic box.These are observed the auxotrophic growth of prompting Corynebacterium glutamicum and can keep by the diffusion of gaseous state DMDS.

In order to prove that gaseous state shifts, carried out an experiment, wherein from before the agar center without the flat board of methionine(Met) of coating OM101 lawn cut out annular (25 * 25 * 5mm) holes.((20 * 20 * 5mm), it is from Sarstedt to place aseptic red polypropylene screw cap in these holes

(No.62.554.002)) conical tube, it is as the cup of 50 μ l liquid D MDS.The method is guaranteed that liquid D MDS does not directly contact with cell and is only spread the arrival cell by gaseous state, because DMDS is by the polypropylene rapid diffusion.Flat board is wrapped in airtight plastics casing under 30 ℃ cultivate.The contrast flat board that is used for this experiment is not existing under DMDS in 30 ℃ of cultivations without the dull and stereotyped of methionine(Met) and at independent airtight plastics casing of coating OM101 lawn.After 2 days, observe completely the bacterium lawn and cover and have the apileate flat board that contains liquid D MDS, and the contrast flat board that lacks DMDS in container does not separately show growth.These experiments show that together the direct contact of liquid D MDS is essential for the toxicity to OM101, and compare, and gaseous state DMDS is nontoxic and still can be used for growth by OM101, and it is synthetic therefore to be used for methionine(Met).Thereby, in fermentor tank, DMDS can be delivered in cell in order to overcome DMDS toxicity to cell under liquid state with gaseous state.This can and pump into DMDS steam in fermenting container by evaporation or boiling DMDS, perhaps completes by steeping fermenting container via liquid D MDS drum air or oxygen on its approach.

The structure of embodiment 5. Δ metE Δ metH bacterial strains

Build the Corynebacterium glutamicum strain of disappearance metE and metH.M2014 is transformed with plasmid pH469 (Figure 10) (SEQ ID NO:9) obtain " Campbell advances " bacterial strain.Then " Campbell advances " bacterial strain " Campbell goes out " is obtained " Campbell goes out " bacterial strain OM228C-2, it contains the metE disappearance.Then OM228C-2 is transformed with plasmid pH300 (Figure 11) (SEQ ID NO:10) and obtain " Campbell advances " bacterial strain.Then should obtain " Campbell goes out " bacterial strain OM246C by " Campbell advances " bacterial strain " Campbell goes out ", this bacterial strain contains Δ metE and Δ metH.As expected, two of two deletion mycopremnas kinds of isolate OM246C-1 and OM246C-2 are methionine(Met) auxotroph and do not produce methionine(Met) in molasses culture medium in test tube are cultivated.

Embodiment 6.MetY is responsible for reaction between catalysis thiomethyl alcohol and O-ethanoyl-homoserine to produce the main enzymatic activity of methionine(Met)

Because reported in literature proposes MetB (Flavin, M. and S.Slaughter 1967.Biochim.Biophys.Acta, 132:400-405; Kanzaki, H.et al.1987.Eur.J.Biochem.163:105-112; Kiene, R.P.et al.1999.Appl.Environ.Microbiol.65:4549-4558) or MetZ (Yamagata, S.1971.J.Biochem. (Tokyo) 70:1035) can participate in thiomethyl alcohol and mix, so test to identify and relate to the enzyme that in Corynebacterium glutamicum, thiomethyl alcohol mixes with containing the allelic OM63 derivative of Δ metB.Illustrate as top, Corynebacterium glutamicum can directly be incorporated into dimethyl disulfide (DMDS) in methionine(Met) by thiomethyl alcohol.In embodiment 3, if determine DMDS directly to join in liquid nutrient medium less than about 0.06% amount, so can be by cell tolerance, yet, if at delivery system such as adsorbent A mberlite ^TMXAD4 adds under existing, and adds so the amount of the DMDS of liquid culture to increase by 500 before observing toxicity.

Carry out " concept proves " experiment with methionine(Met) auxotroph OM63 (Δ metF), show that Corynebacterium glutamicum can utilize DMDS to keep the auxotrophic growth of Δ metF methionine(Met).In order further to identify which enzyme relates to thiomethyl alcohol in Corynebacterium glutamicum to the mixing of methionine(Met), to the ability of their growths under DMDS exists of bacterial strain test of containing disappearance in metB.

Cultivate OM101C (Δ metF at 50% suspension that contains 100 μ l Amberlite XAD4 with without the substratum of methionine(Met) or in replenishing the test tube without the methionine(Met) substratum of DMDS of different amounts, Δ metB), OM246c (the Δ metH that describes in embodiment 4, Δ metE), OM63 (Δ metF), and M2014, OM101C (Δ metF, Δ metB) OM63 that H216 transforms that uses by oneself, H216 contains (Hwang BJ, et al.J Bacteriol.2002Mar with pSH315; 184 (5): 1277-86) identical metB disappearance allelotrope is with disappearance metB.As shown in table 10, contain replenish 0.1 or the test tube without the methionine(Met) substratum of 0.2%DMDS in, the optical density(OD) of all bacterial strains is all similar.Except OM101C, all bacterial strains can both replenish 0.4%DMDS without the methionine(Met) substratum in grow, OM101C demonstrates growth-inhibiting under 0.3%DMDS.Only bacterial strain OM246C can growth under 0.5%DMDS exists.These results show together, and MetB, MetH/MetE and MetF are optional to mixing of methionine(Met) for thiomethyl alcohol.Therefore, MetY is enough for allowing DMDS to the enzymatic activity that mixes of methionine(Met).

Table 10. be grown in have in test tube shown in the amount DMDS and Amberlite XAD4 ²Under existing without the methionine(Met) substratum ¹In OM63, OM101C, OM246C and the M2014 optical density(OD) under 600nm

¹Substratum without methionine(Met).

²Each test tube contains 50% suspension that the 5ml substratum adds 100 μ l XAD4.Inoculate test tube after adding XAD4 and DMDS.Inoculum-cell is overnight growth in containing the test tube of BHI, then rotates and uses the substratum without Met to rinse twice.With cell with 50% initial volume resuspension.5 μ l cell suspending liquids are as the inoculum of each test tube.

Embodiment 7. identification of M etY are the enzyme with O-ethanoyl homoserine thiomethyl alcohol sulfhydrylase activity

Here we show that directly MetY rather than MetB are the active essential and enough enzymes of O-ethanoyl homoserine thiomethyl alcohol sulfhydrylase.M2014 derivative with the various combination that contains metY, metB and metF disappearance carries out the test tube experiment.Bacterial strain has Amberlite ^TMThe XAD4 globule and have or do not have 200mg/l DMDS or 100mg/l methionine(Met) without growing in the methionine(Met) substratum.Inoculum is each test tube approximately 5 * 10 ³Individual cell.Cell was grown 60 hours under on the platform shaking table 30 ℃, at OD ₆₀₀Lower measurement cell density.

As shown in table 11, DMDS can keep methionine(Met) auxotroph OM63 (Δ metF) and OM101 (Δ metF, Δ metB) growth, however DMDS does not keep any methionine(Met) auxotroph of containing metY disappearance such as the growth of OM158 or OM174.Obtain " Campbell advances " strain construction OM158 by transforming OM63 with plasmid pH215 (SEQ ID NO:11).The nucleotide sequence that shows in SEQ IDNO:11 is the zone of the metY gene that lacks in pH215,---residue 1-3 extends to the terminator codon of metA---residue 912-914 from the initiator codon of metY.Two bases around this disappearance are residue 609-610.Then should obtain " Campbell goes out " bacterial strain OM158 by " Campbell advances " bacterial strain " Campbell goes out ", it contains Δ metF, Δ metY.Obtain " Campbell advances " bacterial strain by transforming OM101 with plasmid pH215, build OM174.Then " Campbell advances " bacterial strain " Campbell goes out " is obtained " Campbell goes out " bacterial strain OM174, it contains Δ metF, Δ metB, Δ metY.Fig. 8 has shown before the part of using plasmid pH215 disappearance metY (8A) and (8B) afterwards, in the chromosomal structure of the metY excellent bacillus of zone Glutamic Acid.

These data show that MetY is unique enzyme of being responsible for O-ethanoyl homoserine thiomethyl alcohol sulfhydrylase activity, and MetB or MetC do not contain this enzymatic activity of enough levels with growth under these conditions.

The efficient that in the auxotrophic strain of embodiment 8. Corynebacterium glutamicums, methionine(Met) is produced

In case definite Δ metF or Δ metE Δ metH Corynebacterium glutamicum auxotroph can utilize DMDS to synthesize methionine(Met), importantly measure methionine(Met) production efficiency.Carry out shake flat experiment, wherein compare OM246C and M2014.Each shaking flask contains the Amberlite of 20ml molasses culture medium and 800 μ l ^TM50% suspension of XAD4 is with or without 0.4%DMDS.As shown in Table 12, do not have the OM246C of DMDS almost not accumulate methionine(Met).On the contrary, the OM246C accumulation that has a DMDS is the 0.3g/l methionine(Met) approximately.Thereby the generation of methionine(Met) directly occurs to the transformation of methionine(Met) from O-ethanoyl-homoserine, has walked around homocysteine.The most important thing is, the net increase of the methionine(Met) titre of the OM246C of growth is determined absolutely when having DMDS under DMDS exists, and can produce methionine(Met) by defective Corynebacterium glutamicum mutant in the synthetic final step of methionine(Met).Control strain M2014 is the similar aminogram of accumulation in the DMDS existence or not.What is interesting is, in M2014, DMDS stimulates methionine(Met) output from about 0.5g/l to about 0.7g/l slightly.This can be by the additive effect explanation that produces the methionine(Met) combination with the methionine(Met) biosynthetic pathway from routine of mixing of DMDS.

Table 12. has or does not have 0.4%DMDS ²The time in molasses culture medium ¹The shaking flask research of the M2014 of middle growth and OM246C

¹Molasses culture medium is replenished 1% yeast extract, vitamin H, B ₁, B ₁₂, B ₆With the 100mg/l Threonine.

²Each shaking flask contains 50% suspension that the 20ml substratum adds 800 μ l XAD4.Inoculate shaking flask after adding XAD4 and 80 μ l DMDS (being with or without).The final concentration of the DMDS that adds is 0.4%v/v.

Inoculum-cell is overnight growth in containing the test tube of BHI, then rotates and uses the substratum without Met to rinse twice.With cell with 50% initial volume resuspension.With the inoculum of 100 μ l cell suspending liquids as each shaking flask.

³Amino acid is reported with g/l.

Embodiment 9.DMDS sends to be incorporated into the further research and development of the delivery system of methionine(Met) to Corynebacterium glutamicum

In order further to study the potential delivery system of DMDS (except Amberlite ^TMOutside XAD4), studied heavy white mineral oil (Sigma catalog number (Cat.No.) 400-5) and vegetables oil, Canola oil.Because oil is hydrophobic, so they can dissolve DMDS and potential permission DMDS slowly-releasing in aqueous culture medium.There is or do not have to add in the test tube without the methionine(Met) substratum of 100mg/l methionine(Met) the oil (0.5ml) of the dissolving DMDS that contain different amounts to containing 5ml.Cultivate after 24 hours for 30 ℃ in the platform shaking table, measure the optical density(OD) of the substratum that contains 0,0.2,0.4,0.8 and 1.2% final concentration DMDS.As shown in table 13, the growth of OM246C occurs in containing up to the test tube of 0.4%DMDS, and is containing the growth that M2014 occurs in up to the test tube of 0.8%DMDS in being dissolved in mineral oil.Yet optical density(OD) has or not the optical density(OD) without the tube culture of methionine(Met) substratum of mineral oil to compare less than half with containing.With the Amberlite that allows Growth of Cells ^TMXAD4 compares, and in mineral oil, the maximum of DMDS for M2014 slightly high (0.8% pair 0.4%), is still similar (0.4% pair 0.4%) for OM63.During as delivery system, observe similar result when test oil rape oil; Yet, only Canola oil for the negative influence of Growth of Cells less than mineral oil large (table 14).Weakening of growth may be partly owing to lacking the destruction of enough ventilations, cytolemma or the two combination in test tube is cultivated under these oil exist.Yet obviously oil can be as the delivery system of DMDS in fermentation.Oil or its combination from animal, mineral, chemistry or plant origin can be sent for DMDS is cytotropic.Other possible delivery systems comprise synthetic oil, organic solvent, chlorocarbon, fluorocarbon, perhaps Chlorofluorocarbons (CFCs).Extra method will be controlled DMDS charging, and it provides steady-state level lower than toxic level to cell.Select the Corynebacterium glutamicum strain of DMDS resistance will also alleviate DMDS toxicity tissue.At last, utilize the host type of inherent more anti-DMDS toxicity will also alleviate this problem.

Embodiment 10. strain improvements

Intestinal bacteria metB gene has been suddenlyd change or has been evolved into utilizes thiomethyl alcohol (WO2004/076659 A2).Use methionine(Met) auxotroph, for example, lack the methionine(Met) auxotroph of MetE and MetH or MetF, but use DMDS rather than thiomethyl alcohol in selecting substratum, can similarly select step.Thereby; can be by beginning with methionine(Met) auxotroph; and select growth or growth faster on the minimum medium of DMDS lacking methionine(Met) but contain; and use or do not use chemical, mutagenesis, radiate or increase change allelotrope; selection has mixes the larger microorganism of methionine(Met) ability with DMDS; described methionine(Met) auxotroph for example can produce O-ethanoyl homoserine or OSHS, but can not utilize homoserine.The selection of the type can concentrate on specific gene, as metB gene, metY gene, metC gene or metI gene (Auger et al., 2002 Microbiology 148:507-518), lack (by disappearance or mutagenesis) and mix in the bacterial strain of endogenous capacity of DMDS by inserting described gene on plasmid and this plasmid being imported, and carry out selection described above and carry out the selection of this described type.The offspring of this type of microorganism through selecting, the gene that perhaps separates from this type of microorganism through selecting also can be used for building or derivative methionine production bacterin strain.

If embodiment 11. provides O-ethanoyl homoserine sulfhydrylase or OSHS sulfhydrylase, intestinal bacteria can be metabolized to methionine(Met) with DMDS

MetE::Tn10 with intestinal bacteria methionine(Met) auxotroph CGSC 3592 (metF64) and RY714B, MM294, Δ metH derivative (also referred to as ATCC 33625) and CGSC 6315 (endA1, thi-1, supE44, hsdR17) use pH357 (SEQ ID NO:13; Express the plasmid of Corynebacterium glutamicum metY and metX), pH309 (SEQ ID NO:14; Express the plasmid of Corynebacterium glutamicum metY), perhaps pCLIK transforms, and pCLIK is the empty carrier relevant with pH357 and pH309, copies (SEQ ID NO:15) in intestinal bacteria and Corynebacterium glutamicum.In the upper resistance of selecting the 25mg/L sulphuric acid kanamycin of rich medium (LuriaBroth agar).6 kinds of transformant platings on the nutrient agar without methionine(Met), are cut out a hole at the agar center, and Xiang Kongzhong adds 50 microlitre DMDS, and as described in example 4 above at 30 ℃ of incubation flat boards.Grow on this flat board with two kinds of bacterial strains that pH309 or pH357 transform, but all can not grow with the bacterial strain that empty carrier pCLIK transforms, show that it is essential with enough that metY utilizes DMDS to synthesize methionine(Met) for intestinal bacteria.These results also support following viewpoint: MetY to have O-ethanoyl homoserine sulfhydrylase and the OSHS sulfhydrylase is active; because for example RY714B/pH309 depends on intestinal bacteria MetA enzyme, known its mainly produces OSHS.

Thereby, if intestinal bacteria by through engineering approaches as described herein, can be inputted DMDS so, with its reduction, and be incorporated in methionine(Met).Because intestinal bacteria and Corynebacterium glutamicum are not closely-related biologies, and all have this ability, so we expect that multiple biology all has this ability, and can be with multiple Bioengineered to use DMDS to produce methionine(Met) as one of feed compound.

The selection of embodiment 12. feedback resistance O-ethanoyl homoserine sulfhydrylases or OSHS sulfhydrylase

In order to produce methionine(Met) by biosynthesizing, wish to use feedback resistance O-ethanoyl homoserine sulfhydrylase and/or OSHS sulfhydrylase.In many biologies, these enzymes are by the methionine(Met) feedback inhibition.For example, the MetY in Corynebacterium glutamicum (O-ethanoyl homoserine sulfhydrylase) is by the methionine(Met) feedback inhibition, and methionine(Met) is synthetic for methionine(Met) is counterproductive.Described the MetY (WO2004/108894 A2) of the mutant form of inferring that anti-methionine(Met) suppresses, but these forms may not to improve the biosynthetic best form of methionine(Met).Thereby, still need the suitable feedback resistance form of MetY.Because shown that at this paper MetY can give the enzyme of growing on DMDS, so developed following be used to selecting the allelic new departure of useful metY.To lack MetF or MetE and MetH and optional MetY, MetB and/or the MetC of also lacking; but through through engineering approaches with the synthetic Corynebacterium glutamicum strain of relatively high O-ethanoyl homoserine (for example; OM174 (Δ metF; Δ metB; Δ metY) or OM246C (Δ metE; Δ metH)) (U.S. Provisional Patent Application number 60/700; 699; on July 18th, 2005 application, title " Methionine Producing Recombinant Microorganism ") transform with the plasmid of expressing MetY such as pH357 or pH309.By the MetY that plasmid pH357 or pH309 produce, the bacterial strain of gained can contain DMDS without the methionine(Met) substratum on grow.Use methionine analogs, screening suppresses the methionine analogs of this strain growth as Alpha-Methyl methionine(Met), selenomethionine, nor-leucine, tetrafluoro methionine(Met), methionine(Met) hydroxamic acid ester (Methionine hydroxamate), ethionine, S-methyl halfcystine etc.In some cases, the analogue that suppresses this strain growth suppresses by the false feedback inhibition of MetY.In other words, this analogue is in connection with the methionine(Met) binding site on MetY, and suppresses the activity of described enzyme.Select anti-described analogue mutant (with or without mutagenesis) will cause the variant of MetY, combination and the methionine(Met) of its anti-described analogue.Also be transformed into again the host strain of inmature not sudden change from this type of mutant material standed for isolated plasmid dna, and determine whether such analogue resistance phenotype is plasmid-encoded by what introduce.Plasmid by this screening will contain one or more sudden changes, and they some will be given desirable feedback resistance to methionine(Met).

Above-mentioned generation also is suitable for separating feedback resistance OSHS sulfhydrylase variant with the scheme of identifying feedback resistance O-ethanoyl homoserine sulfhydrylase variant.The method is similarly, but initial biological OSHS rather than the O-ethanoyl homoserine of producing be as intermediate, and this plasmid-encoded OSHS sulfhydrylase rather than O-ethanoyl homoserine sulfhydrylase.In other words, this plasmid contains metZ gene rather than metY gene.

Above-mentioned selection to feedback resistance MetY or MetZ also can be carried out in being different from the biology of Corynebacterium glutamicum.For example, as shown in top embodiment 11, intestinal bacteria RY714B/pH309 or intestinal bacteria CGSC 3592/pH357 etc. also can grow on the substratum without methionine(Met) of DMDS having, thereby this type of bacterial strain also can be used for selecting desirable MetY variant by growing on the substratum without methionine(Met) of DMDS and select resistance to methionine analogs containing.Because intestinal bacteria MetA is also responsive (Usuda Y, Kurahashi O., Appl.Environ.Microbiol., 2005June for the inhibition of methionine(Met) and some analogues such as Alpha-Methyl methionine(Met); 71 (6): 3228-34), so by using intestinal bacteria metA-mutant strain, and for example provide feedback resistance MetA or MetX with pH357, and perhaps use the metA allelotrope of having selected this similar deposits yields resistance, can strengthen the allelic selection of desirable metY.Usually, the method can be used for various bacteria, yeast, fungi, archeobacteria and plant.

Equivalent

Those skilled in the art will recognize that or only use normal experiment just can determine many equivalents of specific embodiments of the present invention as herein described.This type of equivalent is intended to be comprised by following claim.

Sequence table

＜110〉BASF Aktiengesellchaft

＜120〉dimethyl disulfide is used for methionine(Met) production in microorganism

<130>BGI-177PC

<140>

<141>

<150>60/713,907

<151>2005-09-01

<150>60/700,698

<151>2005-07-18

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＜170〉PatentIn version 3 .3

<210>1

<211>7070

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic plasmid

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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〉artificial sequence description: synthetic plasmid

<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〉artificial sequence description: synthetic plasmid

<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>7070

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic plasmid

<400>4

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>5

<211>6625

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic plasmid

<400>5

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>6

<211>363

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic promotor

<400>6

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>7

<211>6350

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic plasmid

<400>7

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>8

<211>6440

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic plasmid

<400>8

tcgagaggcc tgacgtcggg cccgacgtcg catgctcccg gccgccatgg ccgcgggata 60

tcactagtgc ggccgcctgc aggtcgacca tatgggagag ccactagtag gagcacaaac 120

acatgtccct aacgaacatc ccagcctcat ctcaatgggc aattagcgac gttttgaagc 180

gtccttcacc cggccgagta cctttttctg tcgagtttat gccaccccgc gacgatgcag 240

ctgaagagcg tctttaccgc gcagcagagg tcttccatga cctcggtgca tcgtttgtct 300

ccgtgactta tggtgctggc ggatcaaccc gtgagagaac ctcacgtatt gctcgacgat 360

tagcgaaaca accgttgacc actctggtgc acctgaccct ggttctgcgg ccgcacagcg 420

atcccagagg aaatatcctc tggggtcgct gtgtcgacct taaagtttgg ctgccatgtg 480

aatttttagc accctcaaca gttgagtgct ggcactctcg ggggtagagt gccaaatagg 540

ttgtttgaca cacagttgtt cacccgcgac gacggctgtg ctggaaaccc acaaccggca 600

cacacaaaat ttttctagag gagggattca tcatgaatac atacgaacaa attaataaag 660

tgaaaaaaat acttcggaaa catttaaaaa ataaccttat tggtacttac atgtttggat 720

caggagttga gagtggacta aaaccaaata gtgatcttga ctttttagtc gtcgtatctg 780

aaccattgac agatcaaagt aaagaaatac ttatacaaaa aattagacct atttcaaaaa 840

aaataggaga taaaagcaac ttacgatata ttgaattaac aattattatt cagcaagaaa 900

tggtaccgtg gaatcatcct cccaaacaag aatttattta tggagaatgg ttacaagagc 960

tttatgaaca aggatacatt cctcagaagg aattaaattc agatttaacc ataatgcttt 1020

accaagcaaa acgaaaaaat aaaagaatat acggaaatta tgacttagag gaattactac 1080

ctgatattcc attttctgat gtgagaagag ccattatgga ttcgtcagag gaattaatag 1140

ataattatca ggatgatgaa accaactcta tattaacttt atgccgtatg attttaacta 1200

tggacacggg taaaatcata ccaaaagata ttgcgggaaa tgcagtggct gaatcttctc 1260

cattagaaca tagggagaga attttgttag cagttcgtag ttatcttgga gagaatattg 1320

aatggactaa tgaaaatgta aatttaacta taaactattt aaataacaga ttaaaaaaat 1380

tataaaaaaa ttgaaaaaat ggtggaaaca cttttttcaa tttttttgtt ttattattta 1440

atatttggga aatattcatt ctaattggta atcagatttt agaaaacaat aaacccttgc 1500

atagggggat cgatatccgt ttaggctggg cggatccgcc ctcccgcacg ctttgcggga 1560

gggcggtacc agctcatcga tcttattaag tccactcctg agttccggga attcgacctc 1620

ggtatcgcct ccttccccga agggcatttc cgggcgaaaa ctctagaaga agacaccaaa 1680

tacactctgg cgaagctgcg tggaggggca gagtactcca tcacgcagat gttctttgat 1740

gtggaagact acctgcgact tcgtgatcgc cttgtcgctg cagaccccat tcatggtgcg 1800

aagccaatca ttcctggcat catgcccatt acgagcctgc ggtctgtgcg tcgacaggtc 1860

gaactctctg gtgctcaatt gccgagccaa ctagaagaat cacttgttcg agctgcaaac 1920

ggcaatgaag aagcgaacaa agacgagatc cgcaaggtgg gcattgaata ttccaccaat 1980

atggcagagc gactcattgc cgaaggtgcg gaagatctgc acttcatgac gcttaacttc 2040

acccgtgcaa cccaagaagt gttgtacaac cttggcatgg cgcctgcttg gggagcagag 2100

cacggccaag acgcggtgcg ttaagggatc cttaggaagg ctcccaacgc gtcatatgac 2160

tagttcggac ctagggatat cgtcgacatc gatgctcttc tgcgttaatt aacaattggg 2220

atcctctaga cccgggattt aaatcgctag cgggctgcta aaggaagcgg aacacgtaga 2280

aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga 2340

caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat 2400

agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct 2460

ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct 2520

gatggcgcag gggatcaaga tctgatcaag agacaggatg aggatcgttt cgcatgattg 2580

aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 2640

actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 2700

ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg 2760

aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 2820

ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 2880

tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 2940

tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 3000

gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 3060

aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg 3120

atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 3180

tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 3240

tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 3300

tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 3360

tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc 3420

acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg 3480

ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccacgc 3540

tagcggcgcg ccggccggcc cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 3600

gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 3660

ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 3720

acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 3780

cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 3840

caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 3900

gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 3960

tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 4020

aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 4080

ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 4140

cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 4200

tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 4260

tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 4320

ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 4380

aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 4440

aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaaggccg 4500

gccgcggccg ccatcggcat tttcttttgc gtttttattt gttaactgtt aattgtcctt 4560

gttcaaggat gctgtctttg acaacagatg ttttcttgcc tttgatgttc agcaggaagc 4620

tcggcgcaaa cgttgattgt ttgtctgcgt agaatcctct gtttgtcata tagcttgtaa 4680

tcacgacatt gtttcctttc gcttgaggta cagcgaagtg tgagtaagta aaggttacat 4740

cgttaggatc aagatccatt tttaacacaa ggccagtttt gttcagcggc ttgtatgggc 4800

cagttaaaga attagaaaca taaccaagca tgtaaatatc gttagacgta atgccgtcaa 4860

tcgtcatttt tgatccgcgg gagtcagtga acaggtacca tttgccgttc attttaaaga 4920

cgttcgcgcg ttcaatttca tctgttactg tgttagatgc aatcagcggt ttcatcactt 4980

ttttcagtgt gtaatcatcg tttagctcaa tcataccgag agcgccgttt gctaactcag 5040

ccgtgcgttt tttatcgctt tgcagaagtt tttgactttc ttgacggaag aatgatgtgc 5100

ttttgccata gtatgctttg ttaaataaag attcttcgcc ttggtagcca tcttcagttc 5160

cagtgtttgc ttcaaatact aagtatttgt ggcctttatc ttctacgtag tgaggatctc 5220

tcagcgtatg gttgtcgcct gagctgtagt tgccttcatc gatgaactgc tgtacatttt 5280

gatacgtttt tccgtcaccg tcaaagattg atttataatc ctctacaccg ttgatgttca 5340

aagagctgtc tgatgctgat acgttaactt gtgcagttgt cagtgtttgt ttgccgtaat 5400

gtttaccgga gaaatcagtg tagaataaac ggatttttcc gtcagatgta aatgtggctg 5460

aacctgacca ttcttgtgtt tggtctttta ggatagaatc atttgcatcg aatttgtcgc 5520

tgtctttaaa gacgcggcca gcgtttttcc agctgtcaat agaagtttcg ccgacttttt 5580

gatagaacat gtaaatcgat gtgtcatccg catttttagg atctccggct aatgcaaaga 5640

cgatgtggta gccgtgatag tttgcgacag tgccgtcagc gttttgtaat ggccagctgt 5700

cccaaacgtc caggcctttt gcagaagaga tatttttaat tgtggacgaa tcaaattcag 5760

aaacttgata tttttcattt ttttgctgtt cagggatttg cagcatatca tggcgtgtaa 5820

tatgggaaat gccgtatgtt tccttatatg gcttttggtt cgtttctttc gcaaacgctt 5880

gagttgcgcc tcctgccagc agtgcggtag taaaggttaa tactgttgct tgttttgcaa 5940

actttttgat gttcatcgtt catgtctcct tttttatgta ctgtgttagc ggtctgcttc 6000

ttccagccct cctgtttgaa gatggcaagt tagttacgca caataaaaaa agacctaaaa 6060

tatgtaaggg gtgacgccaa agtatacact ttgcccttta cacattttag gtcttgcctg 6120

ctttatcagt aacaaacccg cgcgatttac ttttcgacct cattctatta gactctcgtt 6180

tggattgcaa ctggtctatt ttcctctttt gtttgataga aaatcataaa aggatttgca 6240

gactacgggc ctaaagaact aaaaaatcta tctgtttctt ttcattctct gtatttttta 6300

tagtttctgt tgcatgggca taaagttgcc tttttaatca caattcagaa aatatcataa 6360

tatctcattt cactaaataa tagtgaacgg caggtatatg tgatgggtta aaaaggatcg 6420

gcggccgctc gatttaaatc 6440

<210>9

<211>5106

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic plasmid

<400>9

tcgaggtcga cggtatcgat ggtttgatgt tggttccgat gggcatcatc tctgtggtga 60

tgtcaccagt aattggacga ttggtggatc gcctggcacc aggaatgatc tccaagatcg 120

gattcggcgc gctgattttc tcgatggcgt tgatggctgt ctttatgatc gccaacctat 180

cgccgtggtg gctactcatc ccgattattt tgttcggtag ctccaacgcg atgagttttg 240

caccgaactc tgtgattgct ctgcgtgatg ttccgcagga tttagtgggc tctgcttctg 300

gtttttacaa cacctcacgc caggtgggcg ctgttttggg cgccgctacc ttgggcgctg 360

tgatgcaaat aggagtgggc acggtgtcct tcggtgttgc catgggtgcg gcaatcctgg 420

tgacactcgt gcccttaatc tttgggttcc tagcggtaac ccaatgctag ctcttctgac 480

gatgtttcaa ggttcgcgaa atcaccgggt ggcagaaagc tgccccgggg gtttcctcgc 540

ctttttgtaa tcgaggtcgt cccacttaac gcttaaaatt tccattcaga aagctttggc 600

gttcctcatc atccggggct ccagggagag gattaaaagt gaaccgattg cgttttcaac 660

caaaccctag actgctcggt ctgtcaagaa gttccgggga tttaaattca tggtgccgtt 720

ttgggctcct gttgtctgcg tcgggtgggg aagtggcgta aaggtgtgca acctcatagt 780

caagttgacg gaaaagggga gatcgcattt tacccccgca gattttgggg aacctgtttt 840

gaactggggt tttgcaaaat gcaacgcggt gacgtgtggt tacaactagt tctagacccg 900

ggatttaaat cgctagcggg ctgctaaagg aagcggaaca cgtagaaagc cagtccgcag 960

aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca 1020

agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg 1080

gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg 1140

aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga 1200

tcaagatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg 1260

cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag 1320

acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt 1380

tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta 1440

tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg 1500

ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt 1560

gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat 1620

ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg 1680

atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca 1740

gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc 1800

catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc 1860

gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat 1920

attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc 1980

gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga 2040

ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt 2100

ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga 2160

tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccacgctagc ggcgcgccgg 2220

ccggcccggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct 2280

tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 2340

gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 2400

atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 2460

ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 2520

cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 2580

tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 2640

gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 2700

aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 2760

tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 2820

aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 2880

aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 2940

ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 3000

ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 3060

atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 3120

atgagattat caaaaaggat cttcacctag atccttttaa aggccggccg cggccgccat 3180

cggcattttc ttttgcgttt ttatttgtta actgttaatt gtccttgttc aaggatgctg 3240

tctttgacaa cagatgtttt cttgcctttg atgttcagca ggaagctcgg cgcaaacgtt 3300

gattgtttgt ctgcgtagaa tcctctgttt gtcatatagc ttgtaatcac gacattgttt 3360

cctttcgctt gaggtacagc gaagtgtgag taagtaaagg ttacatcgtt aggatcaaga 3420

tccattttta acacaaggcc agttttgttc agcggcttgt atgggccagt taaagaatta 3480

gaaacataac caagcatgta aatatcgtta gacgtaatgc cgtcaatcgt catttttgat 3540

ccgcgggagt cagtgaacag gtaccatttg ccgttcattt taaagacgtt cgcgcgttca 3600

atttcatctg ttactgtgtt agatgcaatc agcggtttca tcactttttt cagtgtgtaa 3660

tcatcgttta gctcaatcat accgagagcg ccgtttgcta actcagccgt gcgtttttta 3720

tcgctttgca gaagtttttg actttcttga cggaagaatg atgtgctttt gccatagtat 3780

gctttgttaa ataaagattc ttcgccttgg tagccatctt cagttccagt gtttgcttca 3840

aatactaagt atttgtggcc tttatcttct acgtagtgag gatctctcag cgtatggttg 3900

tcgcctgagc tgtagttgcc ttcatcgatg aactgctgta cattttgata cgtttttccg 3960

tcaccgtcaa agattgattt ataatcctct acaccgttga tgttcaaaga gctgtctgat 4020

gctgatacgt taacttgtgc agttgtcagt gtttgtttgc cgtaatgttt accggagaaa 4080

tcagtgtaga ataaacggat ttttccgtca gatgtaaatg tggctgaacc tgaccattct 4140

tgtgtttggt cttttaggat agaatcattt gcatcgaatt tgtcgctgtc tttaaagacg 4200

cggccagcgt ttttccagct gtcaatagaa gtttcgccga ctttttgata gaacatgtaa 4260

atcgatgtgt catccgcatt tttaggatct ccggctaatg caaagacgat gtggtagccg 4320

tgatagtttg cgacagtgcc gtcagcgttt tgtaatggcc agctgtccca aacgtccagg 4380

ccttttgcag aagagatatt tttaattgtg gacgaatcaa attcagaaac ttgatatttt 4440

tcattttttt gctgttcagg gatttgcagc atatcatggc gtgtaatatg ggaaatgccg 4500

tatgtttcct tatatggctt ttggttcgtt tctttcgcaa acgcttgagt tgcgcctcct 4560

gccagcagtg cggtagtaaa ggttaatact gttgcttgtt ttgcaaactt tttgatgttc 4620

atcgttcatg tctccttttt tatgtactgt gttagcggtc tgcttcttcc agccctcctg 4680

tttgaagatg gcaagttagt tacgcacaat aaaaaaagac ctaaaatatg taaggggtga 4740

cgccaaagta tacactttgc cctttacaca ttttaggtct tgcctgcttt atcagtaaca 4800

aacccgcgcg atttactttt cgacctcatt ctattagact ctcgtttgga ttgcaactgg 4860

tctattttcc tcttttgttt gatagaaaat cataaaagga tttgcagact acgggcctaa 4920

agaactaaaa aatctatctg tttcttttca ttctctgtat tttttatagt ttctgttgca 4980

tgggcataaa gttgcctttt taatcacaat tcagaaaata tcataatatc tcatttcact 5040

aaataatagt gaacggcagg tatatgtgat gggttaaaaa ggatcggcgg ccgctcgatt 5100

taaatc 5106

<210>10

<211>5512

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic plasmid

<400>10

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 gtacccttgg tgtatggcta cttcccagcg gtcgcggaag 720

gcgatgacgt ggtgatcttg gaatccccgg atccacacgc agccgaacgc atgcgcttta 780

gcttcccacg ccagcagcgc ggcaggttct tgtgcatcgc ggatttcatt cgcccacgcg 840

agcaagctgt caaggacggc caagtggacg tcatgccatt ccagctggtc accatgggta 900

atcctattgc tgatttcgcc aacgagttgt tcgcagccaa tgaataccgc gagtacttgg 960

aagttcacgg catcggcgtg cagctcaccg aagcattggc cgagtactgg cactcccgag 1020

tgcgcagcga actcaagctg aacgacggtg gatctgtcgc tgattttgat ccagaagaca 1080

agaccaagtt cttcgacctg gattaccgcg gcgcccgctt ctcctttggt tacggttctt 1140

gccctgatct ggaagaccgc gcaaagctgg tggaattgct cgagccaggc cgtatcggcg 1200

tggagttgtc cgaggaactc cagctgcacc cagagcagtc cacagacgcg tttgtgctct 1260

accacccaga ggcaaagtac tttaacgtct aatctagacc cgggatttaa atgatccgct 1320

agcgggctgc taaaggaagc ggaacacgta gaaagccagt ccgcagaaac ggtgctgacc 1380

ccggatgaat gtcagctact gggctatctg gacaagggaa aacgcaagcg caaagagaaa 1440

gcaggtagct tgcagtgggc ttacatggcg atagctagac tgggcggttt tatggacagc 1500

aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt 1560

aaactggatg gctttcttgc cgccaaggat ctgatggcgc aggggatcaa gatctgatca 1620

agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc 1680

ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc 1740

tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga 1800

cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac 1860

gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct 1920

gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa 1980

agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc 2040

attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct 2100

tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc 2160

caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg 2220

cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct 2280

gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct 2340

tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca 2400

gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa 2460

atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc 2520

tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 2580

ggggatctca tgctggagtt cttcgcccac gctagcggcg cgccggccgg cccggtgtga 2640

aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct 2700

cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 2760

ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 2820

ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 2880

cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 2940

actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 3000

cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 3060

tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 3120

gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 3180

caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 3240

agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 3300

tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 3360

tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 3420

gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 3480

gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 3540

aaggatcttc acctagatcc ttttaaaggc cggccgcggc cgccatcggc attttctttt 3600

gcgtttttat ttgttaactg ttaattgtcc ttgttcaagg atgctgtctt tgacaacaga 3660

tgttttcttg cctttgatgt tcagcaggaa gctcggcgca aacgttgatt gtttgtctgc 3720

gtagaatcct ctgtttgtca tatagcttgt aatcacgaca ttgtttcctt tcgcttgagg 3780

tacagcgaag tgtgagtaag taaaggttac atcgttagga tcaagatcca tttttaacac 3840

aaggccagtt ttgttcagcg gcttgtatgg gccagttaaa gaattagaaa cataaccaag 3900

catgtaaata tcgttagacg taatgccgtc aatcgtcatt tttgatccgc gggagtcagt 3960

gaacaggtac catttgccgt tcattttaaa gacgttcgcg cgttcaattt catctgttac 4020

tgtgttagat gcaatcagcg gtttcatcac ttttttcagt gtgtaatcat cgtttagctc 4080

aatcataccg agagcgccgt ttgctaactc agccgtgcgt tttttatcgc tttgcagaag 4140

tttttgactt tcttgacgga agaatgatgt gcttttgcca tagtatgctt tgttaaataa 4200

agattcttcg ccttggtagc catcttcagt tccagtgttt gcttcaaata ctaagtattt 4260

gtggccttta tcttctacgt agtgaggatc tctcagcgta tggttgtcgc ctgagctgta 4320

gttgccttca tcgatgaact gctgtacatt ttgatacgtt tttccgtcac cgtcaaagat 4380

tgatttataa tcctctacac cgttgatgtt caaagagctg tctgatgctg atacgttaac 4440

ttgtgcagtt gtcagtgttt gtttgccgta atgtttaccg gagaaatcag tgtagaataa 4500

acggattttt ccgtcagatg taaatgtggc tgaacctgac cattcttgtg tttggtcttt 4560

taggatagaa tcatttgcat cgaatttgtc gctgtcttta aagacgcggc cagcgttttt 4620

ccagctgtca atagaagttt cgccgacttt ttgatagaac atgtaaatcg atgtgtcatc 4680

cgcattttta ggatctccgg ctaatgcaaa gacgatgtgg tagccgtgat agtttgcgac 4740

agtgccgtca gcgttttgta atggccagct gtcccaaacg tccaggcctt ttgcagaaga 4800

gatattttta attgtggacg aatcaaattc agaaacttga tatttttcat ttttttgctg 4860

ttcagggatt tgcagcatat catggcgtgt aatatgggaa atgccgtatg tttccttata 4920

tggcttttgg ttcgtttctt tcgcaaacgc ttgagttgcg cctcctgcca gcagtgcggt 4980

agtaaaggtt aatactgttg cttgttttgc aaactttttg atgttcatcg ttcatgtctc 5040

cttttttatg tactgtgtta gcggtctgct tcttccagcc ctcctgtttg aagatggcaa 5100

gttagttacg cacaataaaa aaagacctaa aatatgtaag gggtgacgcc aaagtataca 5160

ctttgccctt tacacatttt aggtcttgcc tgctttatca gtaacaaacc cgcgcgattt 5220

acttttcgac ctcattctat tagactctcg tttggattgc aactggtcta ttttcctctt 5280

ttgtttgata gaaaatcata aaaggatttg cagactacgg gcctaaagaa ctaaaaaatc 5340

tatctgtttc ttttcattct ctgtattttt tatagtttct gttgcatggg cataaagttg 5400

cctttttaat cacaattcag aaaatatcat aatatctcat ttcactaaat aatagtgaac 5460

ggcaggtata tgtgatgggt taaaaaggat cggcggccgc tcgatttaaa tc 5512

<210>11

<211>914

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic plasmid

<400>11

atgccaaagt acgacaattc caatgctgac cagtggggct ttgaaacccg ctccattcac 60

gcaggccagt cagtagacgc acagaccagc gcacgaaacc ttccgatcta ccaatccacc 120

gctttcgtgt tcgactccgc tgagcacgcc aagcagcgtt tcgcacttga ggatctaggc 180

cctgtttact cccgcctcac caacccaacc gttgaggctt tggaaaaccg catcgcttcc 240

ctcgaaggtg gcgtccacgc tgtagcgttc tcctccggac aggccgcaac caccaacgcc 300

attttgaacc tggcaggagc gggcgaccac atcgtcacct ccccacgcct ctacggtggc 360

accgagactc tattccttat cactcttaac cgcctgggta tcgatgtttc cttcgtggaa 420

aaccccgacg accctgagtc ctggcaggca gccgttcagc caaacaccaa agcattcttc 480

ggcgagactt tcgccaaccc acaggcagac gtcctggata ttcctgcggt ggctgaagtt 540

gcgcaccgca acagcgttcc actgatcatc gacaacacca tcgctaccgc agcgctcgtg 600

cgcccgctct ccctcgttgt tcacccagca accaccaccc attcacagtc cgacgaagct 660

ggcctggcac gcgcgggcgt tacccagtcc accgtccgcc tgtccgttgg catcgagacc 720

attgatgata tcatcgctga cctcgaaggc ggctttgctg caatctagct ttaaatagac 780

tcaccccagt gcttaaagcg ctgggttttt ctttttcaga ctcgtgagaa tgcaaactag 840

actagacaga gctgtccata tacactggac gaagttttag tcttgtccac ccagaacagg 900

cggttatttt catg 914

<210>12

<211>184

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic plasmid

<400>12

ggtcgagcgg cttaaagttt ggctgccatg tgaattttta gcaccctcaa cagttgagtg 60

ctggcactct cgggggtaga gtgccaaata ggttgtttga cacacagttg ttcacccgcg 120

acgacggctg tgctggaaac ccacaaccgg cacacacaaa atttttctca tggagggatt 180

catc 184

<210>13

<211>8554

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic plasmid

<400>13

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>14

<211>6583

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic plasmid

<400>14

gtaccacgcg tcatatgcgg cttaaagttt ggctgccatg tgaattttta gcaccctcaa 60

cagttgagtg ctggcactct cgggggtaga gtgccaaata ggttgtttga cacacagttg 120

ttcacccgcg acgacggctg tgctggaaac ccacaaccgg cacacacaaa atttttctca 180

tggagggatt catcatgcca aagtacgaca attccaatgc tgaccagtgg ggctttgaaa 240

cccgctccat tcacgcaggc cagtcagtag acgcacagac cagcgcacga aaccttccga 300

tctaccaatc caccgctttc gtgttcgact ccgctgagca cgccaagcag cgtttcgcac 360

ttgaggatct aggccctgtt tactcccgcc tcaccaaccc aaccgttgag gctttggaaa 420

accgcatcgc ttccctcgaa ggtggcgtcc acgctgtagc gttctcctcc ggacaggccg 480

caaccaccaa cgccattttg aacctggcag gagcgggcga ccacatcgtc acctccccac 540

gcctctacgg tggcaccgag actctattcc ttatcactct taaccgcctg ggtatcgatg 600

tttccttcgt ggaaaacccc gacgaccctg agtcctggca ggcagccgtt cagccaaaca 660

ccaaagcatt cttcggcgag actttcgcca acccacaggc agacgtcctg gatattcctg 720

cggtggctga agttgcgcac cgcaacagcg ttccactgat catcgacaac accatcgcta 780

ccgcagcgct cgtgcgcccg ctcgagctcg gcgcagacgt tgtcgtcgct tccctcacca 840

agttctacac cggcaacggc tccggactgg gcggcgtgct tatcgacggc ggaaagttcg 900

attggactgt cgaaaaggat ggaaagccag tattccccta cttcgtcact ccagatgctg 960

cttaccacgg attgaagtac gcagaccttg gtgcaccagc cttcggcctc aaggttcgcg 1020

ttggccttct acgcgacacc ggctccaccc tctccgcatt caacgcatgg gctgcagtcc 1080

agggcatcga caccctttcc ctgcgcctgg agcgccacaa cgaaaacgcc atcaaggttg 1140

cagaattcct caacaaccac gagaaggtgg aaaaggttaa cttcgcaggc ctgaaggatt 1200

ccccttggta cgcaaccaag gaaaagcttg gcctgaagta caccggctcc gttctcacct 1260

tcgagatcaa gggcggcaag gatgaggctt gggcatttat cgacgccctg aagctacact 1320

ccaaccttgc aaacatcggc gatgttcgct ccctcgttgt tcacccagca accaccaccc 1380

attcacagtc cgacgaagct ggcctggcac gcgcgggcgt tacccagtcc accgtccgcc 1440

tgtccgttgg catcgagacc attgatgata tcatcgctga cctcgaaggc ggctttgctg 1500

caatctagca ctagttcgga cctagggata tcgtcgacat cgatgctctt ctgcgttaat 1560

taacaattgg gatcctctag acccgggatt taaatcgcta gcgggctgct aaaggaagcg 1620

gaacacgtag aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg tcagctactg 1680

ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt gcagtgggct 1740

tacatggcga tagctagact gggcggtttt atggacagca agcgaaccgg aattgccagc 1800

tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc 1860

gccaaggatc tgatggcgca ggggatcaag atctgatcaa gagacaggat gaggatcgtt 1920

tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct 1980

attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct 2040

gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga 2100

actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc 2160

tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg 2220

gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc 2280

aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca 2340

tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga 2400

cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc 2460

cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga 2520

aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca 2580

ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg 2640

cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct 2700

tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc 2760

aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga 2820

atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc 2880

ttcgcccacg ctagcggcgc gccggccggc ccggtgtgaa ataccgcaca gatgcgtaag 2940

gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 3000

cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 3060

atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 3120

taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 3180

aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 3240

tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 3300

gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 3360

cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 3420

cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 3480

atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 3540

tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 3600

ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 3660

acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 3720

aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 3780

aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 3840

tttaaaggcc ggccgcggcc gcgcaaagtc ccgcttcgtg aaaattttcg tgccgcgtga 3900

ttttccgcca aaaactttaa cgaacgttcg ttataatggt gtcatgacct tcacgacgaa 3960

gtactaaaat tggcccgaat catcagctat ggatctctct gatgtcgcgc tggagtccga 4020

cgcgctcgat gctgccgtcg atttaaaaac ggtgatcgga tttttccgag ctctcgatac 4080

gacggacgcg ccagcatcac gagactgggc cagtgccgcg agcgacctag aaactctcgt 4140

ggcggatctt gaggagctgg ctgacgagct gcgtgctcgg ccagcgccag gaggacgcac 4200

agtagtggag gatgcaatca gttgcgccta ctgcggtggc ctgattcctc cccggcctga 4260

cccgcgagga cggcgcgcaa aatattgctc agatgcgtgt cgtgccgcag ccagccgcga 4320

gcgcgccaac aaacgccacg ccgaggagct ggaggcggct aggtcgcaaa tggcgctgga 4380

agtgcgtccc ccgagcgaaa ttttggccat ggtcgtcaca gagctggaag cggcagcgag 4440

aattatcgcg atcgtggcgg tgcccgcagg catgacaaac atcgtaaatg ccgcgtttcg 4500

tgtgccgtgg ccgcccagga cgtgtcagcg ccgccaccac ctgcaccgaa tcggcagcag 4560

cgtcgcgcgt cgaaaaagcg cacaggcggc aagaagcgat aagctgcacg aatacctgaa 4620

aaatgttgaa cgccccgtga gcggtaactc acagggcgtc ggctaacccc cagtccaaac 4680

ctgggagaaa gcgctcaaaa atgactctag cggattcacg agacattgac acaccggcct 4740

ggaaattttc cgctgatctg ttcgacaccc atcccgagct cgcgctgcga tcacgtggct 4800

ggacgagcga agaccgccgc gaattcctcg ctcacctggg cagagaaaat ttccagggca 4860

gcaagacccg cgacttcgcc agcgcttgga tcaaagaccc ggacacggag aaacacagcc 4920

gaagttatac cgagttggtt caaaatcgct tgcccggtgc cagtatgttg ctctgacgca 4980

cgcgcagcac gcagccgtgc ttgtcctgga cattgatgtg ccgagccacc aggccggcgg 5040

gaaaatcgag cacgtaaacc ccgaggtcta cgcgattttg gagcgctggg cacgcctgga 5100

aaaagcgcca gcttggatcg gcgtgaatcc actgagcggg aaatgccagc tcatctggct 5160

cattgatccg gtgtatgccg cagcaggcat gagcagcccg aatatgcgcc tgctggctgc 5220

aacgaccgag gaaatgaccc gcgttttcgg cgctgaccag gctttttcac ataggctgag 5280

ccgtggccac tgcactctcc gacgatccca gccgtaccgc tggcatgccc agcacaatcg 5340

cgtggatcgc ctagctgatc ttatggaggt tgctcgcatg atctcaggca cagaaaaacc 5400

taaaaaacgc tatgagcagg agttttctag cggacgggca cgtatcgaag cggcaagaaa 5460

agccactgcg gaagcaaaag cacttgccac gcttgaagca agcctgccga gcgccgctga 5520

agcgtctgga gagctgatcg acggcgtccg tgtcctctgg actgctccag ggcgtgccgc 5580

ccgtgatgag acggcttttc gccacgcttt gactgtggga taccagttaa aagcggctgg 5640

tgagcgccta aaagacacca agggtcatcg agcctacgag cgtgcctaca ccgtcgctca 5700

ggcggtcgga ggaggccgtg agcctgatct gccgccggac tgtgaccgcc agacggattg 5760

gccgcgacgt gtgcgcggct acgtcgctaa aggccagcca gtcgtccctg ctcgtcagac 5820

agagacgcag agccagccga ggcgaaaagc tctggccact atgggaagac gtggcggtaa 5880

aaaggccgca gaacgctgga aagacccaaa cagtgagtac gcccgagcac agcgagaaaa 5940

actagctaag tccagtcaac gacaagctag gaaagctaaa ggaaatcgct tgaccattgc 6000

aggttggttt atgactgttg agggagagac tggctcgtgg ccgacaatca atgaagctat 6060

gtctgaattt agcgtgtcac gtcagaccgt gaatagagca cttaaggtct gcgggcattg 6120

aacttccacg aggacgccga aagcttccca gtaaatgtgc catctcgtag gcagaaaacg 6180

gttcccccgt agggtctctc tcttggcctc ctttctaggt cgggctgatt gctcttgaag 6240

ctctctaggg gggctcacac cataggcaga taacgttccc caccggctcg cctcgtaagc 6300

gcacaaggac tgctcccaaa gatcttcaaa gccactgccg cgactgcctt cgcgaagcct 6360

tgccccgcgg aaatttcctc caccgagttc gtgcacaccc ctatgccaag cttctttcac 6420

cctaaattcg agagattgga ttcttaccgt ggaaattctt cgcaaaaatc gtcccctgat 6480

cgcccttgcg acgttggcgt cggtgccgct ggttgcgctt ggcttgaccg acttgatcag 6540

cggccgctcg atttaaatct cgagaggcct gacgtcgggc ccg 6583

<210>15

<211>5091

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic plasmid

<400>15

tcgatttaaa tctcgagagg cctgacgtcg ggcccggtac cacgcgtcat atgactagtt 60

cggacctagg gatatcgtcg acatcgatgc tcttctgcgt taattaacaa ttgggatcct 120

ctagacccgg gatttaaatc gctagcgggc tgctaaagga agcggaacac gtagaaagcc 180

agtccgcaga aacggtgctg accccggatg aatgtcagct actgggctat ctggacaagg 240

gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg gcgatagcta 300

gactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc gccctctggt 360

aaggttggga agccctgcaa agtaaactgg atggctttct tgccgccaag gatctgatgg 420

cgcaggggat caagatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa 480

gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg 540

gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc 600

ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca 660

gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc 720

actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca 780

tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat 840

acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca 900

cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg 960

ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc 1020

gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct 1080

ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct 1140

acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac 1200

ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc 1260

tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag 1320

atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg 1380

ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc cacgctagcg 1440

gcgcgccggc cggcccggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 1500

aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 1560

gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 1620

ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 1680

ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 1740

cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 1800

ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 1860

tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 1920

gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 1980

tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 2040

gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 2100

tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 2160

ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 2220

agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 2280

gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 2340

attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ggccggccgc 2400

ggccgcgcaa agtcccgctt cgtgaaaatt ttcgtgccgc gtgattttcc gccaaaaact 2460

ttaacgaacg ttcgttataa tggtgtcatg accttcacga cgaagtacta aaattggccc 2520

gaatcatcag ctatggatct ctctgatgtc gcgctggagt ccgacgcgct cgatgctgcc 2580

gtcgatttaa aaacggtgat cggatttttc cgagctctcg atacgacgga cgcgccagca 2640

tcacgagact gggccagtgc cgcgagcgac ctagaaactc tcgtggcgga tcttgaggag 2700

ctggctgacg agctgcgtgc tcggccagcg ccaggaggac gcacagtagt ggaggatgca 2760

atcagttgcg cctactgcgg tggcctgatt cctccccggc ctgacccgcg aggacggcgc 2820

gcaaaatatt gctcagatgc gtgtcgtgcc gcagccagcc gcgagcgcgc caacaaacgc 2880

cacgccgagg agctggaggc ggctaggtcg caaatggcgc tggaagtgcg tcccccgagc 2940

gaaattttgg ccatggtcgt cacagagctg gaagcggcag cgagaattat cgcgatcgtg 3000

gcggtgcccg caggcatgac aaacatcgta aatgccgcgt ttcgtgtgcc gtggccgccc 3060

aggacgtgtc agcgccgcca ccacctgcac cgaatcggca gcagcgtcgc gcgtcgaaaa 3120

agcgcacagg cggcaagaag cgataagctg cacgaatacc tgaaaaatgt tgaacgcccc 3180

gtgagcggta actcacaggg cgtcggctaa cccccagtcc aaacctggga gaaagcgctc 3240

aaaaatgact ctagcggatt cacgagacat tgacacaccg gcctggaaat tttccgctga 3300

tctgttcgac acccatcccg agctcgcgct gcgatcacgt ggctggacga gcgaagaccg 3360

ccgcgaattc ctcgctcacc tgggcagaga aaatttccag ggcagcaaga cccgcgactt 3420

cgccagcgct tggatcaaag acccggacac ggagaaacac agccgaagtt ataccgagtt 3480

ggttcaaaat cgcttgcccg gtgccagtat gttgctctga cgcacgcgca gcacgcagcc 3540

gtgcttgtcc tggacattga tgtgccgagc caccaggccg gcgggaaaat cgagcacgta 3600

aaccccgagg tctacgcgat tttggagcgc tgggcacgcc tggaaaaagc gccagcttgg 3660

atcggcgtga atccactgag cgggaaatgc cagctcatct ggctcattga tccggtgtat 3720

gccgcagcag gcatgagcag cccgaatatg cgcctgctgg ctgcaacgac cgaggaaatg 3780

acccgcgttt tcggcgctga ccaggctttt tcacataggc tgagccgtgg ccactgcact 3840

ctccgacgat cccagccgta ccgctggcat gcccagcaca atcgcgtgga tcgcctagct 3900

gatcttatgg aggttgctcg catgatctca ggcacagaaa aacctaaaaa acgctatgag 3960

caggagtttt ctagcggacg ggcacgtatc gaagcggcaa gaaaagccac tgcggaagca 4020

aaagcacttg ccacgcttga agcaagcctg ccgagcgccg ctgaagcgtc tggagagctg 4080

atcgacggcg tccgtgtcct ctggactgct ccagggcgtg ccgcccgtga tgagacggct 4140

tttcgccacg ctttgactgt gggataccag ttaaaagcgg ctggtgagcg cctaaaagac 4200

accaagggtc atcgagccta cgagcgtgcc tacaccgtcg ctcaggcggt cggaggaggc 4260

cgtgagcctg atctgccgcc ggactgtgac cgccagacgg attggccgcg acgtgtgcgc 4320

ggctacgtcg ctaaaggcca gccagtcgtc cctgctcgtc agacagagac gcagagccag 4380

ccgaggcgaa aagctctggc cactatggga agacgtggcg gtaaaaaggc cgcagaacgc 4440

tggaaagacc caaacagtga gtacgcccga gcacagcgag aaaaactagc taagtccagt 4500

caacgacaag ctaggaaagc taaaggaaat cgcttgacca ttgcaggttg gtttatgact 4560

gttgagggag agactggctc gtggccgaca atcaatgaag ctatgtctga atttagcgtg 4620

tcacgtcaga ccgtgaatag agcacttaag gtctgcgggc attgaacttc cacgaggacg 4680

ccgaaagctt cccagtaaat gtgccatctc gtaggcagaa aacggttccc ccgtagggtc 4740

tctctcttgg cctcctttct aggtcgggct gattgctctt gaagctctct aggggggctc 4800

acaccatagg cagataacgt tccccaccgg ctcgcctcgt aagcgcacaa ggactgctcc 4860

caaagatctt caaagccact gccgcgactg ccttcgcgaa gccttgcccc gcggaaattt 4920

cctccaccga gttcgtgcac acccctatgc caagcttctt tcaccctaaa ttcgagagat 4980

tggattctta ccgtggaaat tcttcgcaaa aatcgtcccc tgatcgccct tgcgacgttg 5040

gcgtcggtgc cgctggttgc gcttggcttg accgacttga tcagcggccg c 5091

Claims

1. produce the method for methionine(Met), there is the lower microorganism that produces methionine(Met) of cultivating in its sulfide compound that is included in methyl blocking, thereby produces methionine(Met), wherein

A) sulfide compound of described methyl blocking is dimethyl disulfide (DMDS);

B) microorganism of generation methionine(Met) is Corynebacterium glutamicum or intestinal bacteria,

C) at least a methionine(Met) biosynthetic enzyme that produces the microorganism of methionine(Met) is lacked of proper care, and described enzyme is selected from

C1) upborne O-ethanoyl homoserine sulfhydrylase or OSHS sulfhydrylase,

C2) upborne homoserine acetyltransferase or homoserine succinyltransferase, and upborne homoserine dehydrogenase,

C3) upborne O-ethanoyl homoserine sulfhydrylase and upborne homoserine acetyltransferase; Perhaps

C4) upborne OSHS sulfhydrylase and upborne homoserine succinyltransferase.

2. the process of claim 1 wherein that described microorganism is Corynebacterium glutamicum.

3. the method for claim 1, it also comprises the step of separating methionine(Met).

4. the method for the generation methionine(Met) of claim 1-3 any one, it is included in slowly-releasing dimethyl disulfide (DMDS) delivery system and has the lower microorganism that produces methionine(Met) of cultivating, thereby produces methionine(Met).

5. the method for claim 4, wherein said slowly-releasing DMDS delivery system is Amberlite ^TMXAD4.

6. the method for claim 4, wherein said slowly-releasing DMDS delivery system in culture with 0.1% or higher and 0.5% the level of being no more than discharge DMDS.

7. the method for claim 4, wherein said slowly-releasing DMDS delivery system in culture with 0.3% or higher and 0.5% the level of being no more than discharge DMDS.

8. but the method for claim 4, wherein said slowly-releasing DMDS delivery system comprise with water immiscibility dissolve the liquid of DMDS.

9. the method for claim 8, wherein said slowly-releasing DMDS delivery system comprise and are selected from: the liquid of animal oil, vegetables oil, organic solvent or its combination.

10. the method for claim 9, wherein said organic solvent comprise chemistry oil, chlorocarbon, fluorocarbon and Chlorofluorocarbons (CFCs).

11. the method for claim 10, wherein chemical oil comprises mineral oil and synthetic oil.

12. the method for claim 4, wherein said slowly-releasing DMDS delivery system are slowly controlled DMDS chargings.

13. the method for claim 4, wherein said slowly-releasing DMDS delivery system are that DMDS passes through the permeable membrane flow of DMDS or diffusion.

14. comprising with gaseous state, the method for claim 4, wherein said slowly-releasing DMDS delivery system carry DMDS.

15. the method for claim 14 wherein produces gaseous state DMDS by evaporation or boiling liquid DMDS.

16. the method for claim 14 is wherein by producing gaseous state DMDS via liquid D MDS drum air or oxygen bubbles.