CN101328486A - Recombinant plasmid for acetone-butanol clostridium gene disruption - Google Patents

Abstract

The invention provides a recombinant plasmid, a preparation method and application thereof, two knockout recombinant plasmid vectors pSY6-buk and pSY6-solR which are prepared by taking the recombinant plasmid as a starting plasmid, a method and application for preparing the recombiant plasmid vectors pSY6-buk and pSY6-solR.. The recombinant plasmid can be taken as a knonckout starting plasmid of an acetone and butanol, can perform the gene knockout efficiently and rapidly, and can be evolved into a novel genetic manipulation system in order to improve the imperfect condition of the prior manipulation system in the current research on the clostridium acetobutylicum, thereby providing convenience for the research on the metabolic engineering of the clostridium acetobutylicum. The recombinant plasmid vectors pSY6-buk and pSY6-solR can be directly applied to establishing knockout strains of the clostridium acetobutylicum buk and solR genes.

Description

A kind of recombinant plasmid that can be used for the acetone-butanol clostridium gene interruption

Technical field

The invention belongs to gene engineering technology field, specifically, relate to a kind of recombinant plasmid pSY6 that acetone-butanol clostridium gene interrupts that can be used for.

Background technology

Clostridium acetobutylicum (Clostridium acetobylicum) is a kind of Gram-positive, the bacterium that can produce spore, obligate anaerobic, it can utilize several kinds of carbon source, produce solvent acetone, butanols and ethanol (Abou-Zeid during the fermentation, A.A., Fouad, M., and Yassein, M.Zentralbl Bakteriol Naturwiss 1978.133:125-134).Acetone, butanols are the large basic materials that serves many purposes, and in chemical fields such as dyestuff, paint, plastics, resin, rubber, can be used as multiple organic compound synthetic precursor; It is requisite solvent in microbiotic and the synthetic drug production process; Also be the food grade extractant of food, perfume industry simultaneously.On the other hand, butanols still is that a kind of octane value (motor octanenumber) is higher than alcoholic acid high-grade fuel and fuel dope.Studies show that the vapour pressure of butanols and vaporization heat are respectively 0.63psi and 141.3kcal kg ^-1, be lower than alcoholic acid 2.25psi and 204.1kcal kg ^-1Its high boiling point (118 ℃) and low-steam pressure help the cold start-up of automobile; Because the hydrophobicity of butanols is stronger than ethanol, therefore be easier to vapour, diesel hydrocarbon class A fuel A miscible; And the perfect combustion of butanols can reduce the CO of tail gas greatly ₂Discharging, and the residual hydrocarbons pollution does not take place, very favourable to purifying air.Obviously, above-mentioned advantage might make butanols become alternate-engine novel green fuel, one of renewable energy resources of the Sustainable development of alternative mineralising fuel.

Chemical such as acetone, butanols can be by chemical method and biological process production, and the latter is energy-saving and environmental protection more.During the World War I at the beginning of the method for microbiological anaerobic fermentation production of acetone, butanols equal solvent can be traced back to last century, rubber rush of demand at that time, the natural rubber resource is the situation of relative deficiency again, has promoted asynthetic rubber's research.Facts have proved, the butanols that produces with anaerobically fermenting be starting raw material to come SBR be to produce elastomeric ideal way at that time, and achieve success.Therefore, in synthetic rubber scale operation, with produce solvent clostridium Clostridium acetobutylicum serve as produce bacterium, be that the solvent fermentation of substrate is rapidly developed with the carbohydrate, once becoming the second-biggest-in-the-world biotechnology industry that is only second to alcohol, be continuous nearly half a century.Through after the semicentennial golden period of development, solvent fermentation is because of the competition extruding that is subjected to petroleum product and the influence of fermentation raw material agricultural byproducts price ascending factor, progressively withdraw from commercial production (Zverlov in the 1970's, V.V., Berezina, O., Velikodvorskaya, G.A., and Schwarz, W.H.AppliedMicrobiology and Biotechnology 2006.71:587-597).

But oil is a kind of mineralising raw material after all, and it is non-renewable and ore reserve are limited, and back petroleum times can arrive sooner or later.Along with oil price rise steadily and to the impact of Economic development, the research and development of accelerating biomass energy and not relying on petroleum base chemicals production method have become urgent subject day by day.Technology with Production by Microorganism Fermentation acetone, butanols also causes people's attention again.In order to make clostridium acetobutylicum AB fermentation method have more advantage, people wish to utilize the metabolic engineering original strain, make it more meet the industrial production demand.

Although the research of the metabolic engineering of external acetone-butanol has more than ten years, because the shortage of clostridium acetobutylicum genetic manipulation means makes progress of research slower.At present, in clostridium acetobutylicum, cross the relatively successful (Boynton of expression alien gene, Z.L., Bennett, G.N., and Rudolph, F.B.Appl Environ Microbiol 1996 62:2758-2766), there are many laboratories all to possess such condition, then still relatively difficult for gene knockout.Usually, the plasmid that is used for gene knockout in this bacterium is mainly two kinds: non-replicating integrative vector and rf integrative vector.Use nonreplication vector, what successfully knock out has a buk, pta, and aad reaches solR gene (Green, E.M., and Bennett, G.N.ApplBiochem Biotechnol 1996.57-58:213-221; Green, E.M., Boynton, Z.L., Harris, L.M., Rudolph, F.B., Papoutsakis, E.T., and Bennett, G.N.Microbiology 1996.142 (Pt8): 2079-2086; Nair, R.V., Green, E.M., Watson, D.E., Bennett, G.N., and Papoutsakis, E.T.Journal of bacteriology 1999181:319-330.).Yet, make recombination fraction in this way very low, be lower than 1 transformant/ug DNA.And some gene can't knock out with this method, as spo0A.Another kind method, then be to adopt the shuttle plasmid that can in clostridium acetobutylicum, duplicate to make up to knock out plasmid, Harris is by this method, successfully knocked out spo0A (Harris, L.M., Welker, N.E., and Papoutsakis, E.T.Journal of bacteriology 2002.184:3586-3597).Because have reproducible starting point, this method has increased the chance of homologous recombination, yet, screen the reorganization bacterium of double exchange, still relatively waste time and energy.

Recently, Minton.N.P.et al has reported in the laboratory targetron plasmid that a kind of E.coli-Clostridia shuttles back and forth, be named as pMTL007 (structural representation of this plasmid as shown in Figure 1), can be used for multiple clostridial gene knockout [http://www.clostridia.net/Marie/Events/ClosTronWorkshopManual.p df], this plasmid contains replicon pCB102 and the promotor that derives from Clostridium butyricum, and its promotor is an induction type, induced by IPTG.PMTL007 uses and derives from the L1.LtrB intron, and has inserted key player on a team's mark (erythromycin) in the 4th structural domain of intron, is used for determining whether intron is inserted into the chromogene group.[http://hcai.nottingham.ac.uk/minton.pdf]。But because also not open about the details of pMTL007, plasmid can't obtain.

Two class intron Lactococcus lactis L1.LtrB (Klein, J.R., and G.M.Dunny.2002.Front Biosci7:d1843-56) successfully be used for multiple Gram-negative bacteria and gram-positive microorganism, as: Escherichia coli, Staphylocccus aureus, Clostridium perfringens (Chen, Y., B.A.McClane, D.J.Fisher, J.I.Rood, and P.Gupta.Appl Environ Microbiol, 2005 71:7542-7547; Frazier, C.L., J.San Filippo, A.M.Lambowitz, and D.A.Mills.Appl Environ Microbiol, 2003 69:1121-1128; Karberg, M., H.Guo, J.Zhong, R.Coon, J.Perutka, and A.M.Lambowitz.Nat Biotechnol, 2001 19:1162-1167; Yao, J., J.Zhong, Y.Fang, E.Geisinger, R.P.Novick, and A.M.Lambowitz.Rna 200612:1271-1281).

Because the imperfection of the existing genetic operating system of clostridium acetobutylicum, cause the inconvenience of basis and applied research, thereby develop a kind of new genetic operating system, improve present case, for the research of the metabolic engineering of clostridium acetobutylicum provides convenient, its importance highlights day by day.

Summary of the invention

First purpose of the present invention is to overcome incomplete defective of genetic operating system, thereby a kind of recombinant plasmid pSY6 is provided, it being developed into a kind of new genetic operating system, for the metabolic engineering research of clostridium acetobutylicum provides convenient.

Another object of the present invention is, the preparation method of recombinant plasmid pSY6 is provided.

Another object of the present invention is, the application of recombinant plasmid pSY6 is provided.

Another object of the present invention is, pSY6-buk is provided recombinant plasmid vector.

Another object of the present invention is, recombinant plasmid vector pSY6-buk is provided the preparation method.

Another object of the present invention is, the application of recombinant plasmid vector pSY6-buk is provided.

Another object of the present invention is, pSY6-solR is provided recombinant plasmid vector.

Another object of the present invention is, recombinant plasmid vector pSY6-solR is provided the preparation method.

Another object of the present invention is, the application of recombinant plasmid vector pSY6-solR is provided.

For achieving the above object, the present inventor is a template with the pJIR750ai plasmid, pcr amplification has obtained L1.LtrB two class introns, then with L1.LtrB two class introns and plasmid vector pIMP1-ptb plasmid enzyme restriction, purifying also connects, to obtain to connect product Transformed E .coli DH5a competent cell again, cultivate, make up a kind of targetron plasmid pSY6 that knows clearly.

Then, the contriver is the plasmid that sets out with this recombinant plasmid, made up the recombinant plasmid vector that two kinds of genes (buk and solR gene) knock out, difference called after pSY6-buk and pSY6-solR, also further made up the recombinant bacterial strain of buk, solR gene knockout respectively, thereby confirmed that above-mentioned plasmid can be applied to the metabolic engineering of clostridium acetobutylicum.

Recombinant plasmid pSY6 provided by the invention can be used as the plasmid that sets out that acetone-butanol clostridium gene knocks out, because pSY6 does not contain key player on a team's mark, this plasmid can need not under the antibiotic-screening, carries out gene and knocks out continuously.In addition, the 4th structural domain of intron do not insert the external source fragment, can improve the insertion efficient that knocks out plasmid, can develop into a kind of new genetic operating system, to improve incomplete situation of existing genetic operating system in the present acetone-butanol shuttle research, thereby for the metabolic engineering research of clostridium acetobutylicum provides convenient, recombinant plasmid vector pSY6-buk and pSY6-solR can be directly used in the bacterial strain that knocks out that makes up clostridium acetobutylicum buk and solR gene.

Description of drawings

Fig. 1 is the structural representation of plasmid pMTL007.

Fig. 2 is the PCR product electrophoresis result of L1.LtrB two class introns.

Fig. 3 cuts the rear electrophoresis result for pIMP1-ptb plasmid and L1.LtrB two class intron PCR product enzymes.

Fig. 4 cuts qualification result for the enzyme of pSY6 recombinant plasmid.

Fig. 5 is buk-targetron and the segmental electrophoresis result of solR-targetron.

Fig. 6 is the PCR checking result of pSY6-buk and pSY6-solR recombinant plasmid bacterium colony.

Fig. 7 is bacterium colony PCR checking Clostridium acetobutylicum ATCC 824buk knockout mutant strain qualification result.

Fig. 8 is Clostridium acetobutylicum ATCC 824buk knockout mutant strain 7,20 genome PCR qualification results.

Fig. 9 inserts the synoptic diagram of Clostridium acetobutylicum ATCC 824 for buk targetron.

Figure 10 is bacterium colony PCR checking Clostridium acetobutylicum ATCC 824 solR knockout mutant strain results.

Figure 11 is Clostridium acetobutylicum ATCC 824 solR knockout mutant strains 13,14 genome PCR qualification results.

Figure 12 inserts the synoptic diagram of Clostridium acetobutylicum ATCC 824 for solR targetron.

Embodiment

Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples only are used to the present invention is described but not are used to limit scope of the present invention.

Recombinant plasmid vector pSY6-buk that mentions among the present invention and pSY6-solR refer to the recombinant plasmid vector that buk gene and solR gene are knocked out respectively.Buk-targetron that mentions among the present invention and solR-targetron fragment refer to respectively at IBS, EBS2, EBS1d site base is after revising, be used to knock out the fragment of buk and solR gene, belong to a L1.LtrB intron part, described L1.LtrB two class introns are protokaryon two class introns, comprise the ltrA gene.

The experimental technique of unreceipted actual conditions in the following example, common " molecular cloning laboratory manual " (New York:Cold Spring Harbor Laboratory Press according to people such as normal condition such as Sambrook, 1989) condition described in, or the condition of advising according to manufacturer.

The present invention is by utilizing targetron gene knockout principle, made up a kind of new targetron plasmid pSY6 that derives, and utilizes this plasmid, can carry out gene knockout in Clostridium acetobutylicum ATCC 824.

The present invention passes through PCR, with the pJIR750ai plasmid is template, amplification L1.LtrB two class introns, cut through the NdeI enzyme, be connected into the E.coli-C.acetobutylicum shuttle vectors pIMP1-ptb that cuts through same enzyme, the recombinant plasmid vector that obtains is pSY6, and pSY6 can be used as the targetron carrier that is used for Clostridium acetobutylicum ATCC 824 gene knockouts.By PCR, amplification buk, the targetron segment of solR through XhoI and BsrG I double digestion, is connected with the pSY6 carrier that same enzyme is cut, and what obtain knocks out plasmid pSY6-buk, pSY6-solR electricity commentaries on classics Clostridium acetobutylicumATCC 824.Identify the reorganization bacterium that intron inserts by bacterium colony PCR.Determine buk through checking order, L1.LtrB intron fragment has been inserted in the solR site respectively.

Bacterial strain and plasmid that the present invention uses are respectively:

Plasmid pIMP1-ptb, sequence is the shuttle plasmid of E.coli acetobutylicum shown in SEQ ID NO:22;

Plasmid pANS, sequence has spectinomycin resistance gene and Φ 3T3 methylase gene (this gene function) shown in SEQ ID NO:23;

Plasmid pJIR750ai has L1.LtrB two class introns available from Sigma-Aldrich company;

Bacterial strain E.coli ER2275 is available from NEB company.

The PCR purifying Kit that uses among the present invention and DNA glue reclaim Kit all available from magnificent Shun's biological products company limited, Targetron ^TMGene Knockout System (TA0100) Kit gives birth to worker's genome extracting Kit and gives birth to worker's biotechnology company limited available from Shanghai available from Sigma-Aldrich.

Enzyme and reagent that the present invention uses are: the KOD archaeal dna polymerase is available from Toyobo company, and restriction enzyme, Taq polysaccharase are available from Fermentas company, and T4 ligase enzyme and calf alkaline phosphatase (CIAP) are all available from TaKaRa company; Agar is available from western Bath Bioisystech Co., Ltd.

Other conventional reagent are homemade or the import packing.

Embodiment 1, make up the pSY6 plasmid vector

With the pJIR750ai plasmid is template, the L1.LtrB two class introns that contained by this plasmid of pcr amplification and the IEP segment (length is about 3.4kb) of ltrA genes encoding, with amplified production after the NdeI enzyme is cut, be connected into through same enzyme and cut among the E.coli-C.acetobutylicum shuttle vectors pIMPl-ptb of also dephosphorization, the recombinant plasmid vector that obtains, called after pSY6, concrete experimental procedure is as follows:

1.1, design of primers and synthetic

The design primer is to In-1 (SEQ ID NO:4) and In-2 (SEQ ID NO:5), introduce the restriction enzyme site of NdeI and XhoI at the 5 ' end of primer I n-1, introduce the restriction enzyme site of NdeI at the 5 ' end of primer I n-2, wherein, NdeI is used to make up pSY6, and XhoI then can make things convenient for follow-up targetron to knock out segmental connection.

1.2, pcr amplification

With the pJIR750ai plasmid is template, and In-1 and In-2 are primer, carry out pcr amplification.

Wherein, PCR reaction system (100 μ l) is: KOD enzyme 1 μ l, KOD buffer 10 μ l, MgCl ₂(25 μ M) 10 μ l, dNTP (2.5 μ M) 8 μ l, primer I n-1 (10 μ M) 4 μ l, primer I n-2 (10 μ M) 4 μ l, plasmid template 2 μ l, ddH ₂O 61 μ l.

The PCR reaction conditions is: 95 ℃, and 5min; 95 ℃, 30s, 55 ℃, 30s, 72 ℃, 3.5min, 30 circulations; 72 ℃, 10min.

Get above-mentioned PCR product, use polyacrylamide gel electrophoresis to detect, the result as shown in Figure 2, wherein, Lane1 is L1.LtrB two class introns, about 3.4kb; Lane2 is 1kb DNA ladder.

According to the result of Fig. 2, obtain L1.LtrB two class intron fragments through pcr amplification, size is 3.4kb.

Again the product at 3.4kb place behind the electrophoresis is tapped rubber, with reference to service manual, use the glue of Hua Shun company to reclaim the Kit recovery, the L1.LtrB two class introns behind the acquisition purifying then, the L1.LtrB two class introns that obtain behind the purifying carry out electrophoresis detection, and the result only shows has a band at 3.4kb size place.

1.3, make up the pSY6 plasmid vector

L1.LtrB behind the purifying two class introns and plasmid vector pIMP1-ptb plasmid are cut with the NdeI enzyme respectively, use the glue of Hua Shun company to reclaim the recovery of Kit purifying then, plasmid after the NdeI enzyme is cut is with the calf alkaline phosphatase, handle half an hour in 37 ℃ of dephosphorizations, and then the pIMP1-ptb plasmid after the glue recovery Kit purifying recovery dephosphorization of use Hua Shun company, wherein electrophoresis result is as shown in Figure 3.

According to the result of Fig. 3, the fragment that the pIMP1-ptb plasmid is cut, obtained after the dephosphorizationization, purifying through the NdeI enzyme, its size is 4.6kb; L1.LtrB two class intron PCR products are cut also through the NdeI enzyme and are obtained fragment behind the purifying, and size is 3.4kb.

Then, segment and pIMP1-ptb plasmid vector after the enzyme that reclaims cut, use the T4 ligase enzyme, in 16 ℃, connection is spent the night, with the connection product Transformed E .coli DH5a competent cell that obtains, then, the picking recon in the LB substratum that contains 100 μ g/ml erythromycin (Amp), 37 ℃, after the overnight incubation, the extracting plasmid.

Cut the plasmid of identifying that extracting obtains with the BamHI enzyme, the result as shown in Figure 4, wherein, Lane1-9 is a recombinant plasmid 1,2,5,6,7 be reverse cloning, 3,4,8,9 is that forward is cloned; Lane10 is 1kb DNA ladder.

According to the result of Fig. 4,3,4,8,9 is the purpose recon, and L1.LtrB two class intron forwards are connected in ptb promotor downstream, the sub-called after pSY6 of the reverse cloning that is obtained plasmid vector.No. 4 forward clones of picking checks order, and the result shows that its sequence is shown in SEQ ID NO:1.

Certainly; use other suitable carriers to act on behalf of pIMP1-ptb; for example; can adopt the method for replacing replicon or promotor; can construct the suitable plasmid vector of alternative pIMP1-ptb; staff for this area is conspicuous, so this also belongs to the claimed content of the present invention.Equally, the content that discloses according to the present invention uses other protokaryon two class introns to replace L1.LtrB two class introns, and this staff for this area is conspicuous, so this also belongs to the claimed content of the present invention.

Embodiment 2, make up pSY6-buk, pSY6-solR plasmid vector

By PCR, the buk targetron segment that increases respectively and solR targetron segment through XhoI and BsrG I double digestion, are connected respectively with the pSY6 carrier that same enzyme is cut, and what obtain knocks out plasmid pSY6-buk and pSY6-solR.Wherein, amplification buk targetron, the template of solR targetron and design of primers come from the Targetron of Sigma-Aldrich company ^TMGene Knockout System (TA0100) Kit.Concrete steps are as follows:

2.1, design of primers and synthetic

With reference to Targetron ^TMThe method that Gene Knockout System (TA0100) Kit provides designs primer buk-IBS (SEQ ID NO:6), buk-EBS1d (SEQ ID NO:7) and buk-EBS2 (SEQ ID NO:8) respectively, is used to make up the pSY6-buk plasmid vector; And solR-IBS (SEQ ID NO:9), solR-EBS1d (SEQ IDNO:10) and solR-EBS2 (SEQ ID NO:11), be used to make up the pSY6-solR plasmid vector.

In addition, also need to use the universal primer EBS that provides by TargetronTM Gene Knockout System (TA0100) Kit.

2.2, pcr amplification

Method that provides with reference to manufacturer and the template that provides thereof are used the Targetron of Sigma-Aldrich ^TMGeneKnockout System (TA0100) Kit carries out pcr amplification, and the PCR product is carried out gel electrophoresis, the result as shown in Figure 5, wherein, Fig. 5 a is the fragment electrophoresis result of buk-targetron, Fig. 5 b is the fragment electrophoresis result of solR-targetron.

According to Fig. 5 result, at the 350bp place band is arranged, illustrate through pcr amplification to obtain buk-targetron and solR-targetron fragment respectively.

Then, use the glue of Hua Shun company to reclaim Kit purifying recovery PCR product, results buk-targetron and solR-targetron fragment.

2.3, make up pSY6-buk and pSY6-solR recombinant plasmid vector

Use XhoI and BsrGI to carry out enzyme the buk-targetron that obtains behind the PCR purifying and solR-targetron fragment and cut, use XhoI and BsrGI enzyme to cut carrier pSY6 simultaneously, enzyme is cut after product and is all reclaimed the Kit purifying with the glue of Hua Shun company and reclaim.

Endonuclease bamhi after the recovery is cut carrier with enzyme respectively, uses the T4 ligase enzyme, in 16 ℃ of connections of spending the night, connects product Transformed E .coli DH5 α competent cell, cultivates in the LB substratum, and the recon to obtaining adopts bacterium colony PCR checking then.Bacterium colony PCR verification method is as follows:

As primer, the bacterium colony on the picking flat board is done template with buk-IBStrop (SEQ ID NO:12) and buk-EBS1d (SEQ ID NO:13), carries out the PCR reaction, with checking pSY6-buk recombinant plasmid; As primer, the bacterium colony on the picking flat board is done template for solR-IBStrop (SEQ IDNO:14) and solR-EBS1d (SEQ ID NO:15), carries out the PCR reaction, with checking pSY6-solR recombinant plasmid.

The PCR reaction system adopts the reaction system identical with embodiment 1.

PCR reaction conditions: 95 ℃ of 5min; 95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 2min.

Get above-mentioned PCR product, carry out polyacrylamide gel electrophoresis and detect, detected result as shown in Figure 6, wherein, Fig. 6 a is the PCR checking result of pSY6-buk recombinant plasmid bacterium colony, the bacterium colony that Lane1-10 is used to verify, Lane11 are 1kb DNA ladder; The negative contrast of Lane12; Lane13 is over against photograph, is template with buk-targetron PCR product; Fig. 6 b is the PCR checking result of pSY6-solR recombinant plasmid bacterium colony, and the bacterium colony that Lane1-13 is used to verify, Lane14 are 1kb DNA ladder; The negative contrast of Lane15; Lane16 is over against photograph, is template with solR-targetron PCR product.

As seen from Figure 6, the band position of Lane1-10 is with consistent over against shining (Lane13), buk-targetron is described, the solR-targetron fragment has been connected in the pSY6 carrier, connects the recon that obtains behind the product Transformed E .coli DH5 α competent cell and contains recombinant plasmid pSY6-buk and pSY6-solR respectively.

Therefrom respectively select again 3 bacterium colonies recombinant plasmid (pSY6-buk is No.1,2,3; PSY6-solR is No.1,2,3) check order, and the result shows that the sequence of pSY6-buk and pSY6-solR is respectively shown in SEQ ID NO:2 and SEQ ID NO:3.

Then, with 20% glycerine, under-70 ℃ of conditions, preserve the E.coliDH5 α that contains recombinant plasmid pSY6-buk and pSY6-solR and be kept at-20 ℃.

Embodiment 3, make up the recombinant bacterial strain Clostridium acetobutylicum of buk gene knockout

With the pSY6-buk plasmid through E.coli ER2275/pANS after Cac8 I site methylates, electricity changes Clostridiumacetobutylicum ATCC 824, after recovery is spent the night, get 100 μ l enchylema and coat Amp (25 μ g/ml) resistant panel, in 37 ℃, cultivate after 48-96 hour in the anaerobic box, choose single bacterium and adopt bacterium colony PCR checking, concrete steps are as follows:

3.1, the methylating of pSY6-buk plasmid

PANS Transformed E .coli ER2275 obtains E.coli ER2275/pANS.

The chemoreception attitude cell of preparation E.coli ER2275/pANS, then with pSY6-buk plasmid Transformed E .coliER2275/pANS, because the pANS plasmid has the spectinomycin resistance, so containing 100 μ g/ml Amp, incubated overnight on the LB culture medium flat plate of 50 μ g/ml spectinomycins, choose single bacterium colony then and be added with 100 μ g/ml Amp to 4ml, in the LB substratum of 50 μ g/ml spectinomycins, incubated overnight.With reference to operational manual,,, obtain the methylated plasmid pSY6-buk of Cac824 I recognition site with plasmid extraction Kit extracting plasmid with the E.coli ER2275 that contains pANS and pSY6-buk that obtains.

When electricity changes Clostridium acetobutylicum, for guaranteeing the quality of foreign DNA, can use the Kit extracting, be 200-300ug/ml for controlling its concentration, and OD260/OD280 is between 1.7～1.9, and available spectrophotometer detects the plasmid quality.

3.2, electricity changes the pSY6-buk plasmid to Clostridium acetobutylicum ATCC 824

Single bacterium colony of choosing the E.coli ER2275 that contains pANS and pSY6-buk in the step 3.1 is to CGM substratum (2g (NH ₄) ₂SO ₄, 1g K ₂HPO ₄, 0.5g KH ₂PO ₄, 0.1g MgSO ₄.7H ₂O, 0.015g FeSO ₄.7H ₂O, 0.01gCaCl ₂, 0.01g MnSO ₄.H ₂O, 0.002g CoCl ₂, 0.002g ZnSO ₄, 2g Tryptone, 1g Yeast Extraction, 50g Glucose is dissolved in the 1L water), 37 ℃ of overnight incubation; Be forwarded in 100ml CGM substratum with 1% ratio next day, and being cultured to OD600 in 37 ℃ is about 0.7, gets 50ml bacterium liquid, 4000rpm, 4 ℃ of centrifugal 10min, supernatant discarded; Again with ETM damping fluid (270mM sucrose, 0.6mM Na ₂HPO ₄, 4.4mM NaH ₂PO ₄, 10mM MgCl ₂) suspend 4000rpm, 4 ℃ of centrifugal 10min, supernatant discarded; Then with 1.0ml ET damping fluid (270mM sucrose, 0.6mM Na ₂HPO ₄, 4.4mM NaH ₂PO ₄) suspend; Get 100 μ l suspension, add 1 μ g plasmid, behind the mixing, add (2mm) in the electric revolving cup, use MicroPulser ^TMThe electroporation electricity changes, and the electric shock condition is voltage 2.0kV, and all the other are with reference to service manual.After the electric shock, add CGM substratum 1ml again, 37 ℃ of overnight incubation suspend with 100 μ l enchylema, coat erythromycin (25 μ g/ml) resistant panel, cultivate about 2-3 days, and flat board grows 70 bacterium colonies, calculates electric transformation efficiency, and electric transformation efficiency adopts following method to calculate:

Electricity transformation efficiency=flat board colony number * enchylema cumulative volume/coat enchylema volume of flat board of go up growing

Calculate according to this method, obtaining electric transformation efficiency is 0.77 * 10 ³Transformant/μ g DNA.

3.3, make up the bacterium colony PCR primer that buk knocks out

The design primer is to buk-for and buk-rev, and its sequence is respectively shown in SEQ ID NO:16 and SEQ ID NO:17.

Because during the buk-targetron design of primers, expectation in karyomit(e) buk gene between the 49-50nt forward insert intron, thereby the primer of choosing checking is a buk49/50nt upstream and downstream about about 100, if there is intron to insert (as shown in Figure 9), the bacterium colony PCR band about 1kb that can increase then, if wild-type, then increase less than this big or small band.

3.4, bacterium colony PCR checking

The bacterium colony that obtains with embodiment 3.2 is a template, and the primer that embodiment 3.3 makes up carries out bacterium colony PCR to being primer, the insertion situation of intron in the bacterium colony that check embodiment 3.2 obtains.

The PCR reaction system adopts the identical reaction system of embodiment 1.

PCR reaction conditions: 95 ℃ of 5min; 95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 70s, 30 circulations; 72 ℃ of 2min.

Get above-mentioned PCR product, the use polyacrylamide gel electrophoresis detects, the result as shown in Figure 7, Lane1 is a template with wild-type Clostridium acetobutylicum ATCC 824 genomes, the negative contrast of Lane2, Lane3 is a template with the pSY6-buk plasmid, and Lane4 is 1kbDNA ladder, and Lane5-17 is template with the bacterium colony.

According to Fig. 7 result, in 26 clostridium acetobutylicums being selected, can increase band about 1kb of 16 bacterium colonies is arranged, illustrate that these 16 bacterium colonies have the insertion (as shown by arrows) of intron, it is 61.5% that intron inserts efficient.

3.5, other checkings of knocking out of buk

Positive reorganization bacterium 7 among the extracting bacterium colony PCR at random, 20 genome is a template, identify that with PCR intron inserts situation, in this PCR process, use two pairs of primers (respectively still simultaneously), wherein, a pair of primer pair with identify that the primer that bacterium colony PCR uses is identical, another to primer to be buk49/50 downstream primer buk-rev and intron inside primer EBS2.The result as shown in Figure 8, wherein, Lane1 and 6 is a template with wild-type Clostridium acetobutylicum ATCC 824 genomes, Lane2 and 7 is a template with Clostridium acetobutylicum ATCC 824buk knockout mutant strain No.7 genome, Lane3 and 8 is a template with Clostridium acetobutylicum ATCC 824buk knockout mutant strain No.20 genome, Lane4 and 9 negative contrasts, Lane5 and 10 is a template with the pSY6-buk plasmid, and Lane11 is 1kb DNAladder; The PCR primer of Lane1-5 is to being buk-for and buk-rev, and the PCR primer of Lane6-10 is to being buk-rev and buk-EBS2.

The buk gene inside of Fig. 8 presentation of results reorganization bacterium 7,20 has the intron fragment to insert.

Repurity is reclaimed by the 1kb band that colony PCR amplification obtained, and this band is checked order, and sequencing result is identical with sequence shown in the SEQ ID NO:18.

Sequencing result shows, the solR gene that Clostridium acetobutylicum ATCC 824buk knocks out mutant strain has inserted the intron fragment at 49/50 site forward, synoptic diagram as shown in Figure 9, make the buk gene inactivation, thereby successfully made up the recombinant bacterial strain Clostridium acetobutylicum of buk gene knockout.

Embodiment 4, the solR gene knockout recombinant bacterial strain Clostridium acetobutylicum make up

With the pSY6-solR plasmid through E.coli ER2275/pANS after Cac8 I site methylates, electricity changes Clostridiumacetobutylicum ATCC 824, after recovery is spent the night, get the screening of 100 μ l enchylema coating Emr (25 μ g/ml) resistant panel, 37 ℃, cultivated 48-96 hour in the anaerobic box, choose single bacterium and adopt bacterium colony PCR checking.Concrete steps are as follows:

Embodiment 4.1 and 4.2 adopts and embodiment 3.1 and 3.2 identical method and steps, the pSY6-solR plasmid and its electricity gone to Clostridium acetobutylicum ATCC 824 of methylating, and recording its electric transformation efficiency is 0.48 * 10 ³Transformant/μ g DNA.

4.3, design of primers

The bacterium colony PCR primer that design solR knocks out is to solR-for and solR-rev, and its sequence is respectively shown in SEQ ID NO:19 and SEQ ID NO:20.

During the solR-targetron design of primers, expectation 468/469nt in karyomit(e) solR gene oppositely inserts between the intron, so choose the primer of checking is solR468/469 upstream and downstream about about 150, if there is intron to insert (as shown in figure 12), the bacterium colony PCR band about 1.2kb that can increase then, if wild-type, then increase less than this big or small band, but size about 300bp.

4.4, the checking that knocks out of SolR:

Adopt the method identical with embodiment 3.4, identify the reorganization bacterium that solR knocks out with bacterium colony PCR, wherein, the bacterium colony that obtains with embodiment 4.2 is a template, carry out pcr amplification with solR468/469 upstream primer solR-for and downstream primer solR-rev as primer, insert segment to identify; The result as shown in figure 10, wherein, among Figure 10 a, it is template that Lane1-11 knocks out bacterium colony with Clostridium acetobutylicum ATCC 824 solR, Lane12 is 1kb DNA ladder, the negative contrast of Lane13, Lane14 is a template with wild-type Clostridium acetobutylicum ATCC 824 genomes, Lane15 is a template with the pSY6-solR plasmid; Among Figure 10 b, Lane1-13 is template with the bacterium colony, and Lane14 is 1kb DNAladder.

According to Figure 10 result, there are 6 to have the intron fragment to insert the solR site in 24 bacterium of institute's picking, as shown by arrows, it is 25% that intron inserts efficient.

Picking colony PCR identifies correct reorganization bacterium No.13 at random, 14, and the extracting genome is a template with the genome, identifies that with two couples of PCR whether its intron inserts respectively.

Wherein, a pair of primer is to being the inner primer EBS2 of solR468/469 upstream primer solR-for and intron; Another to primer to being the identical primer of primer with bacterium colony PCR checking, the result as shown in figure 11, wherein, Lane1 and 6 is a template with wild-type Clostridium acetobutylicum ATCC 824 genomes, Lane2 and 7 is a template with Clostridiumacetobutylicum ATCC 824solR knockout mutant strain No.13 genome, Lane3 and 8 is a template with Clostridiumacetobutylicum ATCC 824solR knockout mutant strain No.14 genome, Lane4 and 9 negative contrasts, Lane5 and 10 is a template with the pSY6-solR plasmid, and Lane11 is 1kb DNA ladder; The PCR primer is solR-for among the Lane1-5, solR-rev; The PCR primer is solR-for among the Lane6-10, solR-EBS2.

There is the segmental insertion of intron in the Clostridium acetobutylicum ATCC 824solR knockout mutant strain No.13 of the presentation of results institute picking of Figure 11 and the genome solR site of No.14.

Repurity is reclaimed by the 1.2kb band that colony PCR amplification obtained, and this band is checked order, and sequencing result is identical with sequence shown in the SEQ ID NO:21.

The sequencing result explanation, the solR gene that Clostridium acetobutylicum ATCC 824solR knocks out mutant strain has oppositely inserted the intron fragment in 468/469 site, synoptic diagram as shown in figure 12, make the solR gene inactivation, thereby successfully made up the recombinant bacterial strain Clostridium acetobutylicum of solR gene knockout.

In sum, recombinant plasmid pSY6 provided by the invention can be used as the plasmid that sets out that acetone-butanol clostridium gene knocks out, efficiently (electric transformation efficiency can reach hundreds of transformants/μ g DNA), carry out gene knockout apace, can develop into a kind of new genetic operating system, to improve incomplete situation of existing genetic operating system in the present acetone-butanol shuttle research, thereby for the metabolic engineering research of clostridium acetobutylicum provides convenient, recombinant plasmid vector pSY6-buk and pSY6-solR can be directly used in the bacterial strain that knocks out that makes up clostridium acetobutylicum buk and solR gene.

Sequence table

＜110〉Chinese Academy of Sciences Shanghai Life Science and Technology institute

＜120〉a kind of recombinant plasmid pSY6 that can be used for the acetone-butanol clostridium gene interruption

<130>P5071045

<160>23

<170>PatentIn?version?3.1

<210>1

<211>8032

<212>DNA

＜213〉clostridium acetobutylicum

<400>1

tcgagataat?tatccttacc?agcccatagg?gtgcgcccag?atagggtgtt?aagtcaagta 60

gtttaaggta?ctactctgta?agataacaca?gaaaacagcc?aacctaaccg?aaaagcgaaa 120

gctgatacgg?gaacagagca?cggttggaaa?gcgatgagtt?acctaaagac?aatcgggtac 180

gactgagtcg?caatgttaat?cagatataag?gtataagttg?tgtttactga?acgcaagttt 240

ctaatttcgg?ttgctggtcg?atagaggaaa?gtgtctgaaa?cctctagtac?aaagaaaggt 300

aagttaagcc?tatggactta?tctgttatca?ccacatttgt?acaatctgta?ggagaaccta 360

tgggaacgaa?acgaaagcga?tgccgagaat?ctgaatttac?caagacttaa?cactaactgg 420

ggatacccta?aacaagaatg?cctaatagaa?aggaggaaaa?aggctatagc?actagagctt 480

gaaaatcttg?caagggtacg?gagtactcgt?agtagtctga?gaagggtaac?gccctttaca 540

tggcaaaggg?gtacagttat?tgtgtactaa?aattaaaaat?tgattaggga?ggaaaacctc 600

aaaatgaaac?caacaatggc?aattttagaa?agaatcagta?aaaattcaca?agaaaatata 660

gacgaagttt?ttacaagact?ttatcgttat?cttttacgtc?cagatattta?ttacgtggcg 720

acgcgttggg?aaatggcaat?gatagcgaaa?caacgtaaaa?ctcttgttgt?atgctttcat 780

tgtcatcgtc?acgtgattca?taaacacaag?tgaatgtcga?cagtgaattt?ttacgaacga 840

acaataacag?agccgtatac?tccgagaggg?gtacgtacgg?ttcccgaaga?gggtggtgca 900

aaccagtcac?agtaatgtga?acaaggcggt?acctccctac?ttcaccatat?cattttctgc 960

agccccctag?aaataatttt?gtttaacttt?aagaaggaga?tatacatata?tggctagatc 1020

gtccattccg?acagcatcgc?cagtcactat?ggcgtgctgc?tagcgctata?tgcgttgatg 1080

caatttctat?gcactcgtag?tagtctgaga?agggtaacgc?cctttacatg?gcaaaggggt 1140

acagttattg?tgtactaaaa?ttaaaaattg?attagggagg?aaaacctcaa?aatgaaacca 1200

acaatggcaa?ttttagaaag?aatcagtaaa?aattcacaag?aaaatataga?cgaagttttt 1260

acaagacttt?atcgttatct?tttacgtcca?gatatttatt?acgtggcgta?tcaaaattta 1320

tattccaata?aaggagcttc?cacaaaagga?atattagatg?atacagcgga?tggctttagt 1380

gaagaaaaaa?taaaaaagat?tattcaatct?ttaaaagacg?gaacttacta?tcctcaacct 1440

gtacgaagaa?tgtatattgc?aaaaaagaat?tctaaaaaga?tgagaccttt?aggaattcca 1500

actttcacag?ataaattgat?ccaagaagct?gtgagaataa?ttcttgaatc?tatctatgaa 1560

ccggtattcg?aagatgtgtc?tcacggtttt?agacctcaac?gaagctgtca?cacagctttg 1620

aaaacaatca?aaagagagtt?tggcggcgca?agatggtttg?tggagggaga?tataaaaggc 1680

tgcttcgata?atatagacca?cgttacactc?attggactca?tcaatcttaa?aatcaaagat 1740

atgaaaatga?gccaattgat?ttataaattt?ctaaaagcag?gttatctgga?aaactggcag 1800

tatcacaaaa?cttacagcgg?aacacctcaa?ggtggaattc?tatctcctct?tttggccaac 1860

atctatcttc?atgaattgga?taagtttgtt?ttacaactca?aaatgaagtt?tgaccgagaa 1920

agtccagaaa?gaataacacc?tgaatatcgg?gagctccaca?atgagataaa?aagaatttct 1980

caccgtctca?agaagttgga?gggtgaagaa?aaagctaaag?ttcttttaga?atatcaagaa 2040

aaacgtaaaa?gattacccac?actcccctgt?acctcacaga?caaataaagt?attgaaatac 2100

gtccggtatg?cggacgactt?cattatctct?gttaaaggaa?gcaaagagga?ctgtcaatgg 2160

ataaaagaac?aattaaaact?ttttattcat?aacaagctaa?aaatggaatt?gagtgaagaa 2220

aaaacactca?tcacacatag?cagtcaaccc?gctcgttttc?tgggatatga?tatacgagta 2280

aggagatctg?gaacgataaa?acgatctggt?aaagtcaaaa?agagaacact?caatgggagt 2340

gtagaactcc?ttattcctct?tcaagacaaa?attcgtcaat?ttatttttga?caagaaaata 2400

gctatccaaa?agaaagatag?ctcatggttt?ccagttcaca?ggaaatatct?tattcgttca 2460

acagacttag?aaatcatcac?aatttataat?tctgaactcc?gcgggatttg?taattactac 2520

ggtctagcaa?gtaattttaa?ccagctcaat?tattttgctt?atcttatgga?atacagctgt 2580

ctaaaaacga?tagcctccaa?acataaggga?acactttcaa?aaaccatttc?catgtttaaa 2640

gatggaagtg?gttcgtgggg?gatcccgtat?gagataaagc?aaggtaagca?gcgccgttat 2700

tttgcaaatt?ttagtgaatg?taaatcccct?tatcaattta?cggatgagat?aagtcaagct 2760

cctgtattgt?atggctatgc?ccggaatact?cttgaaaaca?ggttaaaagc?taaatgttgt 2820

gaattatgtg?ggacgtctga?tgaaaatact?tcctatgaaa?ttcaccatgt?caataaggtc 2880

aaaaatctta?aaggcaaaga?aaaatgggaa?atggcaatga?tagcgaaaca?acgtaaaact 2940

cttgttgtat?gctttcattg?tcatcgtcac?gtgattcata?aacacaagtg?aatgtcgagc 3000

acccgttctc?ggagcactgt?ccgaccgctt?tggccgccgc?ccagtcctgc?tcgcttcgct 3060

acttggagcc?actatcgact?acgcgatcat?ggcgaccaca?cccgtcctgt?ggatcgccaa 3120

gccgccgatg?gtagtgtggg?gtctccccat?gcgagagtag?ggaactgcca?ggcatcaaat 3180

aaaacgaaag?gctcagtcga?aagactgggc?ctttcgtttt?atctgttgtt?tgtcggtgaa 3240

cgctctcctg?agtaggacaa?atccgccggg?agcggatttg?aacgttgcga?agcaacggcc 3300

cggagggtgg?cgggcaggac?gcccgccata?aactgccagg?catcaaatta?agcagaaggc 3360

catcctgacg?gatggccttt?ttgcgtttct?acaaactctt?cctgtcgtca?tatctacaag 3420

catatggtgc?actctcagta?caatctgctc?tgatgccgca?tagttaagcc?agccccgaca 3480

cccgccaaca?cccgctgacg?cgccctgacg?ggcttgtctg?ctcccggcat?ccgcttacag 3540

acaagctgtg?accgtctccg?ggagctgcat?gtgtcagagg?ttttcaccgt?catcaccgaa 3600

acgcgcgaga?cgaaagggcc?tcgtgatacg?cctattttta?taggttaatg?tcatgataat 3660

aatggtttct?tagacgtcag?gtggcacttt?tcggggaaat?gtgcgcggaa?cccctatttg 3720

tttatttttc?taaatacatt?caaatatgta?tccgctcatg?agacaataac?cctgataaat 3780

gcttcaataa?tattgaaaaa?ggaagagtat?gagtattcaa?catttccgtg?tcgcccttat 3840

tccctttttt?gcggcatttt?gccttcctgt?ttttgctcac?ccagaaacgc?tggtgaaagt 3900

aaaagatgct?gaagatcagt?tgggtgcacg?agtgggttac?atcgaactgg?atctcaacag 3960

cggtaagatc?cttgagagtt?ttcgccccga?agaacgtttt?ccaatgatga?gcacttttaa 4020

agttctgcta?tgtggcgcgg?tattatcccg?tattgacgcc?gggcaagagc?aactcggtcg 4080

ccgcatacac?tattctcaga?atgacttggt?tgagtactca?ccagtcacag?aaaagcatct 4140

tacggatggc?atgacagtaa?gagaattatg?cagtgctgcc?ataaccatga?gtgataacac 4200

tgcggccaac?ttacttctga?caacgatcgg?aggaccgaag?gagctaaccg?cttttttgca 4260

caacatgggg?gatcatgtaa?ctcgccttga?tcgttgggaa?ccggagctga?atgaagccat 4320

accaaacgac?gagcgtgaca?ccacgatgcc?tgtagcaatg?gcaacaacgt?tgcgcaaact 4380

attaactggc?gaactactta?ctctagcttc?ccggcaacaa?ttaatagact?ggatggaggc 4440

ggataaagtt?gcaggaccac?ttctgcgctc?ggcccttccg?gctggctggt?ttattgctga 4500

taaatctgga?gccggtgagc?gtgggtctcg?cggtatcatt?gcagcactgg?ggccagatgg 4560

taagccctcc?cgtatcgtag?ttatctacac?gacggggagt?caggcaacta?tggatgaacg 4620

aaatagacag?atcgctgaga?taggtgcctc?actgattaag?cattggtaac?tgtcagacca 4680

agtttactca?tatatacttt?agattgattt?aaaacttcat?ttttaattta?aaaggatcta 4740

ggtgaagatc?ctttttgata?atctcatgac?caaaatccct?taacgtgagt?tttcgttcca 4800

ctgagcgtca?gaccccgtag?aaaagatcaa?aggatcttct?tgagatcctt?tttttctgcg 4860

cgtaatctgc?tgcttgcaaa?caaaaaaacc?accgctacca?gcggtggttt?gtttgccgga 4920

tcaagagcta?ccaactcttt?ttccgaaggt?aactggcttc?agcagagcgc?agataccaaa 4980

tactgtcctt?ctagtgtagc?cgtagttagg?ccaccacttc?aagaactctg?tagcaccgcc 5040

tacatacctc?gctctgctaa?tcctgttacc?agtggctgct?gccagtggcg?ataagtcgtg 5100

tcttaccggg?ttggactcaa?gacgatagtt?accggataag?gcgcagcggt?cgggctgaac 5160

ggggggttcg?tgcacacagc?ccagcttgga?gcgaacgacc?tacaccgaac?tgagatacct 5220

acagcgtgag?ctatgagaaa?gcgccacgct?tcccgaaggg?agaaaggcgg?acaggtatcc 5280

ggtaagcggc?agggtcggaa?caggagagcg?cacgagggag?cttccagggg?gaaacgcctg 5340

gtatctttat?agtcctgtcg?ggtttcgcca?cctctgactt?gagcgtcgat?ttttgtgatg 5400

ctcgtcaggg?gggcggagcc?tatggaaaaa?cgccagcaac?gcggcctttt?tacggttcct 5460

ggccttttgc?tggccttttg?ctcacatgtt?ctttcctgcg?ttatcccctg?attctgtgga 5520

taaccgtatt?accgcctttg?agtgagctga?taccgctcgc?cgcagccgaa?cgaccgagcg 5580

cagcgagtca?gtgagcgagg?aagcggaaga?gcgcccaata?cgcaaaccgc?ctctccccgc 5640

gcgttggccg?attcattaat?gcagctggca?cgacaggttt?cccgactgga?aagcgggcag 5700

tgagcgcaac?gcaattaatg?tgagttagct?cactcattag?gcaccccagg?ctttacactt 5760

tatgcttccg?gctcgtatgt?tgtgtggaat?tgtgagcgga?taacaatttc?acacaggaaa 5820

cagctatgac?catgattacg?ccaagctttg?gctaacacac?acgccattcc?aaccaatagt 5880

tttctcggca?taaagccatg?ctctgacgct?taaatgcact?aatgccttaa?aaaaacatta 5940

aagtctaaca?cactagactt?atttacttcg?taattaagtc?gttaaaccgt?gtgctctacg 6000

accaaaagta?taaaaccttt?aagaactttc?ttttttcttg?taaaaaaaga?aactagataa 6060

atctctcata?tcttttattc?aataatcgca?tcagattgca?gtataaattt?aacgatcact 6120

catcatgttc?atatttatca?gagctcgtgc?tataattata?ctaattttat?aaggaggaaa 6180

aaataaagag?ggttataatg?aacgagaaaa?atataaaaca?cagtcaaaac?tttattactt 6240

caaaacataa?tatagataaa?ataatgacaa?atataagatt?aaatgaacat?gataatatct 6300

ttgaaatcgg?ctcaggaaaa?gggcatttta?cccttgaatt?agtacagagg?tgtaatttcg 6360

taactgccat?tgaaatagac?cataaattat?gcaaaactac?agaaaataaa?cttgttgatc 6420

acgataattt?ccaagtttta?aacaaggata?tattgcagtt?taaatttcct?aaaaaccaat 6480

cctataaaat?atttggtaat?ataccttata?acataagtac?ggatataata?cgcaaaattg 6540

tttttgatag?tatagctgat?gagatttatt?taatcgtgga?atacgggttt?gctaaaagat 6600

tattaaatac?aaaacgctca?ttggcattat?ttttaatggc?agaagttgat?atttctatat 6660

taagtatggt?tccaagagaa?tattttcatc?ctaaacctaa?agtgaatagc?tcacttatca 6720

gattaaatag?aaaaaaatca?agaatatcac?acaaagataa?acagaagtat?aattatttcg 6780

ttatgaaatg?ggttaacaaa?gaatacaaga?aaatatttac?aaaaaatcaa?tttaacaatt 6840

ccttaaaaca?tgcaggaatt?gacgatttaa?acaatattag?ctttgaacaa?ttcttatctc 6900

ttttcaatag?ctataaatta?tttaataagt?aagttaaggg?atgcataaac?tgcatccctt 6960

aacttgtttt?tcgtgtacct?attttttgtg?aatcgattat?gtcttttgcg?cattcacttc 7020

ttttctatat?aaatatgagc?gaagcgaata?agcgtcggaa?aagcagcaaa?aagtttcctt 7080

tttgctgttg?gagcatgggg?gttcaggggg?tgcagtatct?gacgtcaatg?ccgagcgaaa 7140

gcgagccgaa?gggtagcatt?tacgttagat?aaccccctga?tatgctccga?cgctttatat 7200

agaaaagaag?attcaactag?gtaaaatctt?aatataggtt?gagatgataa?ggtttataag 7260

gaatttgttt?gttctaattt?ttcactcatt?ttgttctaat?ttcttttaac?aaatgttctt 7320

ttttttttag?aacagttatg?atatagttag?aatagtttaa?aataaggagt?gagaaaaaga 7380

tgaaagaaag?atatggaaca?gtctataaag?gctctcagag?gctcatagac?gaagaaagtg 7440

gagaagtcat?agaggtagac?aagttatacc?gtaaacaaac?gtctggtaac?ttcgtaaagg 7500

catatatagt?gcaattaata?agtatgttag?atatgattgg?cggaaaaaaa?cttaaaatcg 7560

ttaactatat?cctagataat?gtccacttaa?gtaacaatac?aatgatagct?acaacaagag 7620

aaatagcaaa?agctacagga?acaagtctac?aaacagtaat?aacaacactt?aaaatcttag 7680

aagaaggaaa?tattataaaa?agaaaaactg?gagtattaat?gttaaaccct?gaactactaa 7740

tgagaggcga?cgaccaaaaa?caaaaatacc?tcttactcga?atttgggaac?tttgagcaag 7800

aggcaaatga?aatagattga?cctcccaata?acaccacgta?gttattggga?ggtcaatcta 7860

tgaaaatgcg?attaagcttg?gctgcaggtc?gacggatccc?cgggaattct?ataaaatata 7920

aataattttc?taaaaaactt?aacttcatgt?gaaaagtttg?ttaaaatata?aatgagcacg 7980

ttaatcattt?aacatagata?attaaatagt?aaaagggagt?gtcgacatat?gc 8032

<210>2

<211>8032

<212>DNA

＜213〉clostridium acetobutylicum

<400>2

tcgagataat?tatccttaca?actcaaattg?gtgcgcccag?atagggtgtt?aagtcaagta 60

gtttaaggta?ctactctgta?agataacaca?gaaaacagcc?aacctaaccg?aaaagcgaaa 120

gctgatacgg?gaacagagca?cggttggaaa?gcgatgagtt?acctaaagac?aatcgggtac 180

gactgagtcg?caatgttaat?cagatataag?gtataagttg?tgtttactga?acgcaagttt 240

ctaatttcgg?ttagttgtcg?atagaggaaa?gtgtctgaaa?cctctagtac?aaagaaaggt 300

aagttaacca?atttgactta?tctgttatca?ccacatttgt?acaatctgta?ggagaaccta 360

tgggaacgaa?acgaaagcga?tgccgagaat?ctgaatttac?caagacttaa?cactaactgg 420

ggatacccta?aacaagaatg?cctaatagaa?aggaggaaaa?aggctatagc?actagagctt 480

gaaaatcttg?caagggtacg?gagtactcgt?agtagtctga?gaagggtaac?gccctttaca 540

tggcaaaggg?gtacagttat?tgtgtactaa?aattaaaaat?tgattaggga?ggaaaacctc 600

aaaatgaaac?caacaatggc?aattttagaa?agaatcagta?aaaattcaca?agaaaatata 660

gacgaagttt?ttacaagact?ttatcgttat?cttttacgtc?cagatattta?ttacgtggcg 720

acgcgttggg?aaatggcaat?gatagcgaaa?caacgtaaaa?ctcttgttgt?atgctttcat 780

tgtcatcgtc?acgtgattca?taaacacaag?tgaatgtcga?cagtgaattt?ttacgaacga 840

acaataacag?agccgtatac?tccgagaggg?gtacgtacgg?ttcccgaaga?gggtggtgca 900

aaccagtcac?agtaatgtga?acaaggcggt?acctccctac?ttcaccatat?cattttctgc 960

agccccctag?aaataatttt?gtttaacttt?aagaaggaga?tatacatata?tggctagatc 1020

gtccattccg?acagcatcgc?cagtcactat?ggcgtgctgc?tagcgctata?tgcgttgatg 1080

caatttctat?gcactcgtag?tagtctgaga?agggtaacgc?cctttacatg?gcaaaggggt 1140

acagttattg?tgtactaaaa?ttaaaaattg?attagggagg?aaaacctcaa?aatgaaacca 1200

acaatggcaa?ttttagaaag?aatcagtaaa?aattcacaag?aaaatataga?cgaagttttt 1260

acaagacttt?atcgttatct?tttacgtcca?gatatttatt?acgtggcgta?tcaaaattta 1320

tattccaata?aaggagcttc?cacaaaagga?atattagatg?atacagcgga?tggctttagt 1380

gaagaaaaaa?taaaaaagat?tattcaatct?ttaaaagacg?gaacttacta?tcctcaacct 1440

gtacgaagaa?tgtatattgc?aaaaaagaat?tctaaaaaga?tgagaccttt?aggaattcca 1500

actttcacag?ataaattgat?ccaagaagct?gtgagaataa?ttcttgaatc?tatctatgaa 1560

ccggtattcg?aagatgtgtc?tcacggtttt?agacctcaac?gaagctgtca?cacagctttg 1620

aaaacaatca?aaagagagtt?tggcggcgca?agatggtttg?tggagggaga?tataaaaggc 1680

tgcttcgata?atatagacca?cgttacactc?attggactca?tcaatcttaa?aatcaaagat 1740

atgaaaatga?gccaattgat?ttataaattt?ctaaaagcag?gttatctgga?aaactggcag 1800

tatcacaaaa?cttacagcgg?aacacctcaa?ggtggaattc?tatctcctct?tttggccaac 1860

atctatcttc?atgaattgga?taagtttgtt?ttacaactca?aaatgaagtt?tgaccgagaa 1920

agtccagaaa?gaataacacc?tgaatatcgg?gagctccaca?atgagataaa?aagaatttct 1980

caccgtctca?agaagttgga?gggtgaagaa?aaagctaaag?ttcttttaga?atatcaagaa 2040

aaacgtaaaa?gattacccac?actcccctgt?acctcacaga?caaataaagt?attgaaatac 2100

gtccggtatg?cggacgactt?cattatctct?gttaaaggaa?gcaaagagga?ctgtcaatgg 2160

ataaaagaac?aattaaaact?ttttattcat?aacaagctaa?aaatggaatt?gagtgaagaa 2220

aaaacactca?tcacacatag?cagtcaaccc?gctcgttttc?tgggatatga?tatacgagta 2280

aggagatctg?gaacgataaa?acgatctggt?aaagtcaaaa?agagaacact?caatgggagt 2340

gtagaactcc?ttattcctct?tcaagacaaa?attcgtcaat?ttatttttga?caagaaaata 2400

gctatccaaa?agaaagatag?ctcatggttt?ccagttcaca?ggaaatatct?tattcgttca 2460

acagacttag?aaatcatcac?aatttataat?tctgaactcc?gcgggatttg?taattactac 2520

ggtctagcaa?gtaattttaa?ccagctcaat?tattttgctt?atcttatgga?atacagctgt 2580

ctaaaaacga?tagcctccaa?acataaggga?acactttcaa?aaaccatttc?catgtttaaa 2640

gatggaagtg?gttcgtgggg?gatcccgtat?gagataaagc?aaggtaagca?gcgccgttat 2700

tttgcaaatt?ttagtgaatg?taaatcccct?tatcaattta?cggatgagat?aagtcaagct 2760

cctgtattgt?atggctatgc?ccggaatact?cttgaaaaca?ggttaaaagc?taaatgttgt 2820

gaattatgtg?ggacgtctga?tgaaaatact?tcctatgaaa?ttcaccatgt?caataaggtc 2880

aaaaatctta?aaggcaaaga?aaaatgggaa?atggcaatga?tagcgaaaca?acgtaaaact 2940

cttgttgtat?gctttcattg?tcatcgtcac?gtgattcata?aacacaagtg?aatgtcgagc 3000

acccgttctc?ggagcactgt?ccgaccgctt?tggccgccgc?ccagtcctgc?tcgcttcgct 3060

acttggagcc?actatcgact?acgcgatcat?ggcgaccaca?cccgtcctgt?ggatcgccaa 3120

gccgccgatg?gtagtgtggg?gtctccccat?gcgagagtag?ggaactgcca?ggcatcaaat 3180

aaaacgaaag?gctcagtcga?aagactgggc?ctttcgtttt?atctgttgtt?tgtcggtgaa 3240

cgctctcctg?agtaggacaa?atccgccggg?agcggatttg?aacgttgcga?agcaacggcc 3300

cggagggtgg?cgggcaggac?gcccgccata?aactgccagg?catcaaatta?agcagaaggc 3360

catcctgacg?gatggccttt?ttgcgtttct?acaaactctt?cctgtcgtca?tatctacaag 3420

catatggtgc?actctcagta?caatctgctc?tgatgccgca?tagttaagcc?agccccgaca 3480

cccgccaaca?cccgctgacg?cgccctgacg?ggcttgtctg?ctcccggcat?ccgcttacag 3540

acaagctgtg?accgtctccg?ggagctgcat?gtgtcagagg?ttttcaccgt?catcaccgaa 3600

acgcgcgaga?cgaaagggcc?tcgtgatacg?cctattttta?taggttaatg?tcatgataat 3660

aatggtttct?tagacgtcag?gtggcacttt?tcggggaaat?gtgcgcggaa?cccctatttg 3720

tttatttttc?taaatacatt?caaatatgta?tccgctcatg?agacaataac?cctgataaat 3780

gcttcaataa?tattgaaaaa?ggaagagtat?gagtattcaa?catttccgtg?tcgcccttat 3840

tccctttttt?gcggcatttt?gccttcctgt?ttttgctcac?ccagaaacgc?tggtgaaagt 3900

aaaagatgct?gaagatcagt?tgggtgcacg?agtgggttac?atcgaactgg?atctcaacag 3960

cggtaagatc?cttgagagtt?ttcgccccga?agaacgtttt?ccaatgatga?gcacttttaa 4020

agttctgcta?tgtggcgcgg?tattatcccg?tattgacgcc?gggcaagagc?aactcggtcg 4080

ccgcatacac?tattctcaga?atgacttggt?tgagtactca?ccagtcacag?aaaagcatct 4140

tacggatggc?atgacagtaa?gagaattatg?cagtgctgcc?ataaccatga?gtgataacac 4200

tgcggccaac?ttacttctga?caacgatcgg?aggaccgaag?gagctaaccg?cttttttgca 4260

caacatgggg?gatcatgtaa?ctcgccttga?tcgttgggaa?ccggagctga?atgaagccat 4320

accaaacgac?gagcgtgaca?ccacgatgcc?tgtagcaatg?gcaacaacgt?tgcgcaaact 4380

attaactggc?gaactactta?ctctagcttc?ccggcaacaa?ttaatagact?ggatggaggc 4440

ggataaagtt?gcaggaccac?ttctgcgctc?ggcccttccg?gctggctggt?ttattgctga 4500

taaatctgga?gccggtgagc?gtgggtctcg?cggtatcatt?gcagcactgg?ggccagatgg 4560

taagccctcc?cgtatcgtag?ttatctacac?gacggggagt?caggcaacta?tggatgaacg 4620

aaatagacag?atcgctgaga?taggtgcctc?actgattaag?cattggtaac?tgtcagacca 4680

agtttactca?tatatacttt?agattgattt?aaaacttcat?ttttaattta?aaaggatcta 4740

ggtgaagatc?ctttttgata?atctcatgac?caaaatccct?taacgtgagt?tttcgttcca 4800

ctgagcgtca?gaccccgtag?aaaagatcaa?aggatcttct?tgagatcctt?tttttctgcg 4860

cgtaatctgc?tgcttgcaaa?caaaaaaacc?accgctacca?gcggtggttt?gtttgccgga 4920

tcaagagcta?ccaactcttt?ttccgaaggt?aactggcttc?agcagagcgc?agataccaaa 4980

tactgtcctt?ctagtgtagc?cgtagttagg?ccaccacttc?aagaactctg?tagcaccgcc 5040

tacatacctc?gctctgctaa?tcctgttacc?agtggctgct?gccagtggcg?ataagtcgtg 5100

tcttaccggg?ttggactcaa?gacgatagtt?accggataag?gcgcagcggt?cgggctgaac 5160

ggggggttcg?tgcacacagc?ccagcttgga?gcgaacgacc?tacaccgaac?tgagatacct 5220

acagcgtgag?ctatgagaaa?gcgccacgct?tcccgaaggg?agaaaggcgg?acaggtatcc 5280

ggtaagcggc?agggtcggaa?caggagagcg?cacgagggag?cttccagggg?gaaacgcctg 5340

gtatctttat?agtcctgtcg?ggtttcgcca?cctctgactt?gagcgtcgat?ttttgtgatg 5400

ctcgtcaggg?gggcggagcc?tatggaaaaa?cgccagcaac?gcggcctttt?tacggttcct 5460

ggccttttgc?tggccttttg?ctcacatgtt?ctttcctgcg?ttatcccctg?attctgtgga 5520

taaccgtatt?accgcctttg?agtgagctga?taccgctcgc?cgcagccgaa?cgaccgagcg 5580

cagcgagtca?gtgagcgagg?aagcggaaga?gcgcccaata?cgcaaaccgc?ctctccccgc 5640

gcgttggccg?attcattaat?gcagctggca?cgacaggttt?cccgactgga?aagcgggcag 5700

tgagcgcaac?gcaattaatg?tgagttagct?cactcattag?gcaccccagg?ctttacactt 5760

tatgcttccg?gctcgtatgt?tgtgtggaat?tgtgagcgga?taacaatttc?acacaggaaa 5820

cagctatgac?catgattacg?ccaagctttg?gctaacacac?acgccattcc?aaccaatagt 5880

tttctcggca?taaagccatg?ctctgacgct?taaatgcact?aatgccttaa?aaaaacatta 5940

aagtctaaca?cactagactt?atttacttcg?taattaagtc?gttaaaccgt?gtgctctacg 6000

accaaaagta?taaaaccttt?aagaactttc?ttttttcttg?taaaaaaaga?aactagataa 6060

atctctcata?tcttttattc?aataatcgca?tcagattgca?gtataaattt?aacgatcact 6120

catcatgttc?atatttatca?gagctcgtgc?tataattata?ctaattttat?aaggaggaaa 6180

aaataaagag?ggttataatg?aacgagaaaa?atataaaaca?cagtcaaaac?tttattactt 6240

caaaacataa?tatagataaa?ataatgacaa?atataagatt?aaatgaacat?gataatatct 6300

ttgaaatcgg?ctcaggaaaa?gggcatttta?cccttgaatt?agtacagagg?tgtaatttcg 6360

taactgccat?tgaaatagac?cataaattat?gcaaaactac?agaaaataaa?cttgttgatc 6420

acgataattt?ccaagtttta?aacaaggata?tattgcagtt?taaatttcct?aaaaaccaat 6480

cctataaaat?atttggtaat?ataccttata?acataagtac?ggatataata?cgcaaaattg 6540

tttttgatag?tatagctgat?gagatttatt?taatcgtgga?atacgggttt?gctaaaagat 6600

tattaaatac?aaaacgctca?ttggcattat?ttttaatggc?agaagttgat?atttctatat 6660

taagtatggt?tccaagagaa?tattttcatc?ctaaacctaa?agtgaatagc?tcacttatca 6720

gattaaatag?aaaaaaatca?agaatatcac?acaaagataa?acagaagtat?aattatttcg 6780

ttatgaaatg?ggttaacaaa?gaatacaaga?aaatatttac?aaaaaatcaa?tttaacaatt 6840

ccttaaaaca?tgcaggaatt?gacgatttaa?acaatattag?ctttgaacaa?ttcttatctc 6900

ttttcaatag?ctataaatta?tttaataagt?aagttaaggg?atgcataaac?tgcatccctt 6960

aacttgtttt?tcgtgtacct?attttttgtg?aatcgattat?gtcttttgcg?cattcacttc 7020

ttttctatat?aaatatgagc?gaagcgaata?agcgtcggaa?aagcagcaaa?aagtttcctt 7080

tttgctgttg?gagcatgggg?gttcaggggg?tgcagtatct?gacgtcaatg?ccgagcgaaa 7140

gcgagccgaa?gggtagcatt?tacgttagat?aaccccctga?tatgctccga?cgctttatat 7200

agaaaagaag?attcaactag?gtaaaatctt?aatataggtt?gagatgataa?ggtttataag 7260

gaatttgttt?gttctaattt?ttcactcatt?ttgttctaat?ttcttttaac?aaatgttctt 7320

ttttttttag?aacagttatg?atatagttag?aatagtttaa?aataaggagt?gagaaaaaga 7380

tgaaagaaag?atatggaaca?gtctataaag?gctctcagag?gctcatagac?gaagaaagtg 7440

gagaagtcat?agaggtagac?aagttatacc?gtaaacaaac?gtctggtaac?ttcgtaaagg 7500

catatatagt?gcaattaata?agtatgttag?atatgattgg?cggaaaaaaa?cttaaaatcg 7560

ttaactatat?cctagataat?gtccacttaa?gtaacaatac?aatgatagct?acaacaagag 7620

aaatagcaaa?agctacagga?acaagtctac?aaacagtaat?aacaacactt?aaaatcttag 7680

aagaaggaaa?tattataaaa?agaaaaactg?gagtattaat?gttaaaccct?gaactactaa 7740

tgagaggcga?cgaccaaaaa?caaaaatacc?tcttactcga?atttgggaac?tttgagcaag 7800

aggcaaatga?aatagattga?cctcccaata?acaccacgta?gttattggga?ggtcaatcta 7860

tgaaaatgcg?attaagcttg?gctgcaggtc?gacggatccc?cgggaattct?ataaaatata 7920

aataattttc?taaaaaactt?aacttcatgt?gaaaagtttg?ttaaaatata?aatgagcacg 7980

ttaatcattt?aacatagata?attaaatagt?aaaagggagt?gtcgacatat?gc 8032

<210>3

<211>8032

<212>DNA

＜213〉clostridium acetobutylicum

<400>3

tcgagataat?tatccttaaa?tagctttttc?gtgcgcccag?atagggtgtt?aagtcaagta 60

gtttaaggta?ctactctgta?agataacaca?gaaaacagcc?aacctaaccg?aaaagcgaaa 120

gctgatacgg?gaacagagca?cggttggaaa?gcgatgagtt?acctaaagac?aatcgggtac 180

gactgagtcg?caatgttaat?cagatataag?gtataagttg?tgtttactga?acgcaagttt 240

ctaatttcgg?ttctattccg?atagaggaaa?gtgtctgaaa?cctctagtac?aaagaaaggt 300

aagttattga?aaaagactta?tctgttatca?ccacatttgt?acaatctgta?ggagaaccta 360

tgggaacgaa?acgaaagcga?tgccgagaat?ctgaatttac?caagacttaa?cactaactgg 420

ggatacccta?aacaagaatg?cctaatagaa?aggaggaaaa?aggctatagc?actagagctt 480

gaaaatcttg?caagggtacg?gagtactcgt?agtagtctga?gaagggtaac?gccctttaca 540

tggcaaaggg?gtacagttat?tgtgtactaa?aattaaaaat?tgattaggga?ggaaaacctc 600

aaaatgaaac?caacaatggc?aattttagaa?agaatcagta?aaaattcaca?agaaaatata 660

gacgaagttt?ttacaagact?ttatcgttat?cttttacgtc?cagatattta?ttacgtggcg 720

acgcgttggg?aaatggcaat?gatagcgaaa?caacgtaaaa?ctcttgttgt?atgctttcat 780

tgtcatcgtc?acgtgattca?taaacacaag?tgaatgtcga?cagtgaattt?ttacgaacga 840

acaataacag?agccgtatac?tccgagaggg?gtacgtacgg?ttcccgaaga?gggtggtgca 900

aaccagtcac?agtaatgtga?acaaggcggt?acctccctac?ttcaccatat?cattttctgc 960

agccccctag?aaataatttt?gtttaacttt?aagaaggaga?tatacatata?tggctagatc 1020

gtccattccg?acagcatcgc?cagtcactat?ggcgtgctgc?tagcgctata?tgcgttgatg 1080

caatttctat?gcactcgtag?tagtctgaga?agggtaacgc?cctttacatg?gcaaaggggt 1140

acagttattg?tgtactaaaa?ttaaaaattg?attagggagg?aaaacctcaa?aatgaaacca 1200

acaatggcaa?ttttagaaag?aatcagtaaa?aattcacaag?aaaatataga?cgaagttttt 1260

acaagacttt?atcgttatct?tttacgtcca?gatatttatt?acgtggcgta?tcaaaattta 1320

tattccaata?aaggagcttc?cacaaaagga?atattagatg?atacagcgga?tggctttagt 1380

gaagaaaaaa?taaaaaagat?tattcaatct?ttaaaagacg?gaacttacta?tcctcaacct 1440

gtacgaagaa?tgtatattgc?aaaaaagaat?tctaaaaaga?tgagaccttt?aggaattcca 1500

actttcacag?ataaattgat?ccaagaagct?gtgagaataa?ttcttgaatc?tatctatgaa 1560

ccggtattcg?aagatgtgtc?tcacggtttt?agacctcaac?gaagctgtca?cacagctttg 1620

aaaacaatca?aaagagagtt?tggcggcgca?agatggtttg?tggagggaga?tataaaaggc 1680

tgcttcgata?atatagacca?cgttacactc?attggactca?tcaatcttaa?aatcaaagat 1740

atgaaaatga?gccaattgat?ttataaattt?ctaaaagcag?gttatctgga?aaactggcag 1800

tatcacaaaa?cttacagcgg?aacacctcaa?ggtggaattc?tatctcctct?tttggccaac 1860

atctatcttc?atgaattgga?taagtttgtt?ttacaactca?aaatgaagtt?tgaccgagaa 1920

agtccagaaa?gaataacacc?tgaatatcgg?gagctccaca?atgagataaa?aagaatttct 1980

caccgtctca?agaagttgga?gggtgaagaa?aaagctaaag?ttcttttaga?atatcaagaa 2040

aaacgtaaaa?gattacccac?actcccctgt?acctcacaga?caaataaagt?attgaaatac 2100

gtccggtatg?cggacgactt?cattatctct?gttaaaggaa?gcaaagagga?ctgtcaatgg 2160

ataaaagaac?aattaaaact?ttttattcat?aacaagctaa?aaatggaatt?gagtgaagaa 2220

aaaacactca?tcacacatag?cagtcaaccc?gctcgttttc?tgggatatga?tatacgagta 2280

aggagatctg?gaacgataaa?acgatctggt?aaagtcaaaa?agagaacact?caatgggagt 2340

gtagaactcc?ttattcctct?tcaagacaaa?attcgtcaat?ttatttttga?caagaaaata 2400

gctatccaaa?agaaagatag?ctcatggttt?ccagttcaca?ggaaatatct?tattcgttca 2460

acagacttag?aaatcatcac?aatttataat?tctgaactcc?gcgggatttg?taattactac 2520

ggtctagcaa?gtaattttaa?ccagctcaat?tattttgctt?atcttatgga?atacagctgt 2580

ctaaaaacga?tagcctccaa?acataaggga?acactttcaa?aaaccatttc?catgtttaaa 2640

gatggaagtg?gttcgtgggg?gatcccgtat?gagataaagc?aaggtaagca?gcgccgttat 2700

tttgcaaatt?ttagtgaatg?taaatcccct?tatcaattta?cggatgagat?aagtcaagct 2760

cctgtattgt?atggctatgc?ccggaatact?cttgaaaaca?ggttaaaagc?taaatgttgt 2820

gaattatgtg?ggacgtctga?tgaaaatact?tcctatgaaa?ttcaccatgt?caataaggtc 2880

aaaaatctta?aaggcaaaga?aaaatgggaa?atggcaatga?tagcgaaaca?acgtaaaact 2940

cttgttgtat?gctttcattg?tcatcgtcac?gtgattcata?aacacaagtg?aatgtcgagc 3000

acccgttctc?ggagcactgt?ccgaccgctt?tggccgccgc?ccagtcctgc?tcgcttcgct 3060

acttggagcc?actatcgact?acgcgatcat?ggcgaccaca?cccgtcctgt?ggatcgccaa 3120

gccgccgatg?gtagtgtggg?gtctccccat?gcgagagtag?ggaactgcca?ggcatcaaat 3180

aaaacgaaag?gctcagtcga?aagactgggc?ctttcgtttt?atctgttgtt?tgtcggtgaa 3240

cgctctcctg?agtaggacaa?atccgccggg?agcggatttg?aacgttgcga?agcaacggcc 3300

cggagggtgg?cgggcaggac?gcccgccata?aactgccagg?catcaaatta?agcagaaggc 3360

catcctgacg?gatggccttt?ttgcgtttct?acaaactctt?cctgtcgtca?tatctacaag 3420

catatggtgc?actctcagta?caatctgctc?tgatgccgca?tagttaagcc?agccccgaca 3480

cccgccaaca?cccgctgacg?cgccctgacg?ggcttgtctg?ctcccggcat?ccgcttacag 3540

acaagctgtg?accgtctccg?ggagctgcat?gtgtcagagg?ttttcaccgt?catcaccgaa 3600

acgcgcgaga?cgaaagggcc?tcgtgatacg?cctattttta?taggttaatg?tcatgataat 3660

aatggtttct?tagacgtcag?gtggcacttt?tcggggaaat?gtgcgcggaa?cccctatttg 3720

tttatttttc?taaatacatt?caaatatgta?tccgctcatg?agacaataac?cctgataaat 3780

gcttcaataa?tattgaaaaa?ggaagagtat?gagtattcaa?catttccgtg?tcgcccttat 3840

tccctttttt?gcggcatttt?gccttcctgt?ttttgctcac?ccagaaacgc?tggtgaaagt 3900

aaaagatgct?gaagatcagt?tgggtgcacg?agtgggttac?atcgaactgg?atctcaacag 3960

cggtaagatc?cttgagagtt?ttcgccccga?agaacgtttt?ccaatgatga?gcacttttaa 4020

agttctgcta?tgtggcgcgg?tattatcccg?tattgacgcc?gggcaagagc?aactcggtcg 4080

ccgcatacac?tattctcaga?atgacttggt?tgagtactca?ccagtcacag?aaaagcatct 4140

tacggatggc?atgacagtaa?gagaattatg?cagtgctgcc?ataaccatga?gtgataacac 4200

tgcggccaac?ttacttctga?caacgatcgg?aggaccgaag?gagctaaccg?cttttttgca 4260

caacatgggg?gatcatgtaa?ctcgccttga?tcgttgggaa?ccggagctga?atgaagccat 4320

accaaacgac?gagcgtgaca?ccacgatgcc?tgtagcaatg?gcaacaacgt?tgcgcaaact 4380

attaactggc?gaactactta?ctctagcttc?ccggcaacaa?ttaatagact?ggatggaggc 4440

ggataaagtt?gcaggaccac?ttctgcgctc?ggcccttccg?gctggctggt?ttattgctga 4500

taaatctgga?gccggtgagc?gtgggtctcg?cggtatcatt?gcagcactgg?ggccagatgg 4560

taagccctcc?cgtatcgtag?ttatctacac?gacggggagt?caggcaacta?tggatgaacg 4620

aaatagacag?atcgctgaga?taggtgcctc?actgattaag?cattggtaac?tgtcagacca 4680

agtttactca?tatatacttt?agattgattt?aaaacttcat?ttttaattta?aaaggatcta 4740

ggtgaagatc?ctttttgata?atctcatgac?caaaatccct?taacgtgagt?tttcgttcca 4800

ctgagcgtca?gaccccgtag?aaaagatcaa?aggatcttct?tgagatcctt?tttttctgcg 4860

cgtaatctgc?tgcttgcaaa?caaaaaaacc?accgctacca?gcggtggttt?gtttgccgga 4920

tcaagagcta?ccaactcttt?ttccgaaggt?aactggcttc?agcagagcgc?agataccaaa 4980

tactgtcctt?ctagtgtagc?cgtagttagg?ccaccacttc?aagaactctg?tagcaccgcc 5040

tacatacctc?gctctgctaa?tcctgttacc?agtggctgct?gccagtggcg?ataagtcgtg 5100

tcttaccggg?ttggactcaa?gacgatagtt?accggataag?gcgcagcggt?cgggctgaac 5160

ggggggttcg?tgcacacagc?ccagcttgga?gcgaacgacc?tacaccgaac?tgagatacct 5220

acagcgtgag?ctatgagaaa?gcgccacgct?tcccgaaggg?agaaaggcgg?acaggtatcc 5280

ggtaagcggc?agggtcggaa?caggagagcg?cacgagggag?cttccagggg?gaaacgcctg 5340

gtatctttat?agtcctgtcg?ggtttcgcca?cctctgactt?gagcgtcgat?ttttgtgatg 5400

ctcgtcaggg?gggcggagcc?tatggaaaaa?cgccagcaac?gcggcctttt?tacggttcct 5460

ggccttttgc?tggccttttg?ctcacatgtt?ctttcctgcg?ttatcccctg?attctgtgga 5520

taaccgtatt?accgcctttg?agtgagctga?taccgctcgc?cgcagccgaa?cgaccgagcg 5580

cagcgagtca?gtgagcgagg?aagcggaaga?gcgcccaata?cgcaaaccgc?ctctccccgc 5640

gcgttggccg?attcattaat?gcagctggca?cgacaggttt?cccgactgga?aagcgggcag 5700

tgagcgcaac?gcaattaatg?tgagttagct?cactcattag?gcaccccagg?ctttacactt 5760

tatgcttccg?gctcgtatgt?tgtgtggaat?tgtgagcgga?taacaatttc?acacaggaaa 5820

cagctatgac?catgattacg?ccaagctttg?gctaacacac?acgccattcc?aaccaatagt 5880

tttctcggca?taaagccatg?ctctgacgct?taaatgcact?aatgccttaa?aaaaacatta 5940

aagtctaaca?cactagactt?atttacttcg?taattaagtc?gttaaaccgt?gtgctctacg 6000

accaaaagta?taaaaccttt?aagaactttc?ttttttcttg?taaaaaaaga?aactagataa 6060

atctctcata?tcttttattc?aataatcgca?tcagattgca?gtataaattt?aacgatcact 6120

catcatgttc?atatttatca?gagctcgtgc?tataattata?ctaattttat?aaggaggaaa 6180

aaataaagag?ggttataatg?aacgagaaaa?atataaaaca?cagtcaaaac?tttattactt 6240

caaaacataa?tatagataaa?ataatgacaa?atataagatt?aaatgaacat?gataatatct 6300

ttgaaatcgg?ctcaggaaaa?gggcatttta?cccttgaatt?agtacagagg?tgtaatttcg 6360

taactgccat?tgaaatagac?cataaattat?gcaaaactac?agaaaataaa?cttgttgatc 6420

acgataattt?ccaagtttta?aacaaggata?tattgcagtt?taaatttcct?aaaaaccaat 6480

cctataaaat?atttggtaat?ataccttata?acataagtac?ggatataata?cgcaaaattg 6540

tttttgatag?tatagctgat?gagatttatt?taatcgtgga?atacgggttt?gctaaaagat 6600

tattaaatac?aaaacgctca?ttggcattat?ttttaatggc?agaagttgat?atttctatat 6660

taagtatggt?tccaagagaa?tattttcatc?ctaaacctaa?agtgaatagc?tcacttatca 6720

gattaaatag?aaaaaaatca?agaatatcac?acaaagataa?acagaagtat?aattatttcg 6780

ttatgaaatg?ggttaacaaa?gaatacaaga?aaatatttac?aaaaaatcaa?tttaacaatt 6840

ccttaaaaca?tgcaggaatt?gacgatttaa?acaatattag?ctttgaacaa?ttcttatctc 6900

ttttcaatag?ctataaatta?tttaataagt?aagttaaggg?atgcataaac?tgcatccctt 6960

aacttgtttt?tcgtgtacct?attttttgtg?aatcgattat?gtcttttgcg?cattcacttc 7020

ttttctatat?aaatatgagc?gaagcgaata?agcgtcggaa?aagcagcaaa?aagtttcctt 7080

tttgctgttg?gagcatgggg?gttcaggggg?tgcagtatct?gacgtcaatg?ccgagcgaaa 7140

gcgagccgaa?gggtagcatt?tacgttagat?aaccccctga?tatgctccga?cgctttatat 7200

agaaaagaag?attcaactag?gtaaaatctt?aatataggtt?gagatgataa?ggtttataag 7260

gaatttgttt?gttctaattt?ttcactcatt?ttgttctaat?ttcttttaac?aaatgttctt 7320

ttttttttag?aacagttatg?atatagttag?aatagtttaa?aataaggagt?gagaaaaaga 7380

tgaaagaaag?atatggaaca?gtctataaag?gctctcagag?gctcatagac?gaagaaagtg 7440

gagaagtcat?agaggtagac?aagttatacc?gtaaacaaac?gtctggtaac?ttcgtaaagg 7500

catatatagt?gcaattaata?agtatgttag?atatgattgg?cggaaaaaaa?cttaaaatcg 7560

ttaactatat?cctagataat?gtccacttaa?gtaacaatac?aatgatagct?acaacaagag 7620

aaatagcaaa?agctacagga?acaagtctac?aaacagtaat?aacaacactt?aaaatcttag 7680

aagaaggaaa?tattataaaa?agaaaaactg?gagtattaat?gttaaaccct?gaactactaa 7740

tgagaggcga?cgaccaaaaa?caaaaatacc?tcttactcga?atttgggaac?tttgagcaag 7800

aggcaaatga?aatagattga?cctcccaata?acaccacgta?gttattggga?ggtcaatcta 7860

tgaaaatgcg?attaagcttg?gctgcaggtc?gacggatccc?cgggaattct?ataaaatata 7920

aataattttc?taaaaaactt?aacttcatgt?gaaaagtttg?ttaaaatata?aatgagcacg 7980

ttaatcattt?aacatagata?attaaatagt?aaaagggagt?gtcgacatat?gc 8032

<210>4

<211>40

<212>DNA

＜213〉primer

<400>4

ggaattccat?atgctcgaga?taattatcct?taccagccca 40

<210>5

<211>35

<212>DNA

＜213〉primer

<400>5

ggaattccat?atgcttgtag?atatgacgac?aggaagag 38

<210>6

<211>54

<212>DNA

＜213〉primer

<400>6

aaaaactcga?gataattatc?cttacaactc?aaattggtgc?gcccagatag?ggtg 54

<210>7

<211>60

<212>DNA

＜213〉primer

<400>7

cagattgtac?aaatgtggtg?ataacagata?agtcaaattg?gttaacttac?ctttctttgt 60

<210>8

<211>49

<212>DNA

＜213〉primer

<400>8

tgaacgcaag?tttctaattt?cggttagttg?tcgatagagg?aaagtgtct 49

<210>9

<211>52

<212>DNA

＜213〉primer

<400>9

ccgctcgaga?taattatcct?taaatagctt?tttcgtgcgc?ccagataggg?tg 52

<210>10

<211>60

<212>DNA

＜213〉primer

<400>10

cagattgtac?aaatgtggtg?ataacagata?agtctttttc?aataacttac?ctttctttgt 60

<210>11

<211>49

<212>DNA

＜213〉primer

<400>11

tgaacgcaag?tttctaattt?cggttctatt?ccgatagagg?aaagtgtct 49

<210>12

<211>20

<212>DNA

＜213〉primer

<400>12

ttatccttac?aactcaaatt 20

<210>13

<211>20

<212>DNA

＜213〉primer

<400>13

taacagataa?gtcaaattgg 20

<210>14

<211>27

<212>DNA

＜213〉primer

<400>14

cccaagctta?taattatcct?taaatag 27

<210>15

<211>40

<212>DNA

＜213〉primer

<400>15

cagattgtac?aaatgtggtg?ataacagata?agtctttttc 40

<210>16

<211>20

<212>DNA

＜213〉primer

<400>16

tactaataat?caatcctggc 20

<210>17

<211>24

<212>DNA

＜213〉primer

<400>17

agtgttatat?ttttctatct?cttc 24

<210>18

<211>991

<212>DNA

＜213〉clostridium acetobutylicum

<400>18

gtgcgccaga?tagggtgtta?agtcaagtag?tttaaggtac?tactctgtaa?gataacacag 60

aaaacagcca?acctaaccga?aaagcgaaag?ctgatacggg?aacagagcac?ggttggaaag 120

cgatgagtta?cctaaagaca?atcgggtacg?actgagtcgc?aatgttaatc?agatataagg 180

tataagttgt?gtttactgaa?cgcaagtttc?taatttcggt?tagttgtcga?tagaggaaag 240

tgtctgaaac?ctctagtaca?aagaaaggta?agttaaccaa?tttgacttat?ctgttatcac 300

cacatttgta?caatctgtag?gagaacctat?gggaacgaaa?cgaaagcgat?gccgagaatc 360

tgaatttacc?aagacttaac?actaactggg?gataccctaa?acaagaatgc?ctaatagaaa 420

ggaggaaaaa?ggctatagca?ctagagcttg?aaaatcttgc?aagggtacgg?agtactcgta 480

gtagtctgag?aagggtaacg?ccctttacat?ggcaaagggg?tacagttatt?gtgtactaaa 540

attaaaaatt?gattagggag?gaaaacctca?aaatgaaacc?aacaatggca?attttagaaa 600

gaatcagtaa?aaattcacaa?gaaaatatag?acgaagtttt?tacaagactt?tatcgttatc 660

ttttacgtcc?agatatttat?tacgtggcga?cgcgttggga?aatggcaatg?atagcgaaac 720

aacgtaaaac?tcttgttgta?tgctttcatt?gtcatcgtca?cgtgattcat?aaacacaagt 780

gaatgtcgac?agtgaatttt?tacgaacgaa?caataacaga?gccgtatact?ccgagagggg 840

tacgtacggt?tcccgaagag?ggtggtgcaa?accagtcaca?gtaatgtgaa?caaggcggta 900

cctccctact?tcacgtattt?atgacgatga?aaaagagata?tttgagaaga?ctttaagaca 960

ttcagctgaa?gagatagaaa?aatataacac?t 991

<210>19

<211>22

<212>DNA

＜213〉primer

<400>19

attcttggag?agcctgaaag?ag 22

<210>20

<211>21

<212>DNA

＜213〉primer

<400>20

acgaaaagta?tatatgagtt?g 21

<210>21

<211>1111

<212>DNA

＜213〉clostridium acetobutylicum

<400>21

aactccagcc?taatgtaggt?atatcctacg?agaacttagc?ctggttttac?tacttaacag 60

gcaaatacga?taaggcaata?gaaaactttg?tgaagtaggg?aggtaccgcc?ttgttcacat 120

tactgtgact?ggtttgcacc?accctcttcg?ggaaccgtac?gtacccctct?cggagtatac 180

ggctctgtta?ttgttcgttc?gtaaaaattc?actgtcgaca?ttcacttgtg?tttatgaatc 240

acgtgacgat?gacaatgaaa?gcatacaaca?agagttttac?gttgtttcgc?tatcattgcc 300

atttcccaac?gcgtcgccac?gtaataaata?tctggacgta?aaagataacg?ataaagtctt 360

gtaaaaactt?cgtctatatt?ttcttgtgaa?tttttactga?ttctttctaa?aattgccatt 420

gttggtttca?ttttgaggtt?ttcctcccta?atcaattttt?aattttagta?cacaataact 480

gtaccccttt?gccatgtaaa?gggcgttacc?cttctcagac?tactacgagt?actccgtacc 540

cttgcaagat?tttcaagctc?tagtgctata?gcctttttcc?tcctttctat?taggcattct 600

tgtttagggt?atccccagtt?agtgttaagt?cttggtaaat?tcagattctc?ggcatcgctt 660

tcgtttcgtt?cccataggtt?ctcctacaga?ttgtacaaat?gtggtgataa?cagataagtc 720

tttttcaata?acttaccttt?ctttgtacta?gaggtttcag?acactttcct?ctatcggaat 780

agaaccgaaa?ttagaaactt?gcgttcagta?aacacaactt?ataccttata?tctgattaac 840

attgcgactc?agtcgtaccc?gattgtcttt?aggtaactca?tcgctttcca?accgtgctct 900

gttcccgtat?cagctttcgc?ttttcggtta?ggttggctgt?tttctgtgtt?atcttacaga 960

gtagtacctt?aaactacttg?acttaacacc?ctatctgggc?gcacgaaaaa?gctatttcca?1020

tgggcagtac?aaactctgtc?tatagaagtt?taggaattac?ctatgccaaa?ataggagatt?1080

ataaaaaatc?cgaagaatat?cttaagaaag?c 1111

<210>22

<211>4605

<212>DNA

＜213〉clostridium acetobutylicum

<400>1

agcttggctg?caggtcgacg?gatccccggg?aattctataa?aatataaata?attttctaaa 60

aaacttaact?tcatgtgaaa?agtttgttaa?aatataaatg?agcacgttaa?tcatttaaca 120

tagataatta?aatagtaaaa?gggagtgtcg?acatatggtg?cactctcagt?acaatctgct 180

ctgatgccgc?atagttaagc?cagccccgac?acccgccaac?acccgctgac?gcgccctgac 240

gggcttgtct?gctcccggca?tccgcttaca?gacaagctgt?gaccgtctcc?gggagctgca 300

tgtgtcagag?gttttcaccg?tcatcaccga?aacgcgcgag?acgaaagggc?ctcgtgatac 360

gcctattttt?ataggttaat?gtcatgataa?taatggtttc?ttagacgtca?ggtggcactt 420

ttcggggaaa?tgtgcgcgga?acccctattt?gtttattttt?ctaaatacat?tcaaatatgt 480

atccgctcat?gagacaataa?ccctgataaa?tgcttcaata?atattgaaaa?aggaagagta 540

tgagtattca?acatttccgt?gtcgccctta?ttcccttttt?tgcggcattt?tgccttcctg 600

tttttgctca?cccagaaacg?ctggtgaaag?taaaagatgc?tgaagatcag?ttgggtgcac 660

gagtgggtta?catcgaactg?gatctcaaca?gcggtaagat?ccttgagagt?tttcgccccg 720

aagaacgttt?tccaatgatg?agcactttta?aagttctgct?atgtggcgcg?gtattatccc 780

gtattgacgc?cgggcaagag?caactcggtc?gccgcataca?ctattctcag?aatgacttgg 840

ttgagtactc?accagtcaca?gaaaagcatc?ttacggatgg?catgacagta?agagaattat 900

gcagtgctgc?cataaccatg?agtgataaca?ctgcggccaa?cttacttctg?acaacgatcg 960

gaggaccgaa?ggagctaacc?gcttttttgc?acaacatggg?ggatcatgta?actcgccttg 1020

atcgttggga?accggagctg?aatgaagcca?taccaaacga?cgagcgtgac?accacgatgc 1080

ctgtagcaat?ggcaacaacg?ttgcgcaaac?tattaactgg?cgaactactt?actctagctt 1140

cccggcaaca?attaatagac?tggatggagg?cggataaagt?tgcaggacca?cttctgcgct 1200

cggcccttcc?ggctggctgg?tttattgctg?ataaatctgg?agccggtgag?cgtgggtctc 1260

gcggtatcat?tgcagcactg?gggccagatg?gtaagccctc?ccgtatcgta?gttatctaca 1320

cgacggggag?tcaggcaact?atggatgaac?gaaatagaca?gatcgctgag?ataggtgcct 1380

cactgattaa?gcattggtaa?ctgtcagacc?aagtttactc?atatatactt?tagattgatt 1440

taaaacttca?tttttaattt?aaaaggatct?aggtgaagat?cctttttgat?aatctcatga 1500

ccaaaatccc?ttaacgtgag?ttttcgttcc?actgagcgtc?agaccccgta?gaaaagatca 1560

aaggatcttc?ttgagatcct?ttttttctgc?gcgtaatctg?ctgcttgcaa?acaaaaaaac 1620

caccgctacc?agcggtggtt?tgtttgccgg?atcaagagct?accaactctt?tttccgaagg 1680

taactggctt?cagcagagcg?cagataccaa?atactgtcct?tctagtgtag?ccgtagttag 1740

gccaccactt?caagaactct?gtagcaccgc?ctacatacct?cgctctgcta?atcctgttac 1800

cagtggctgc?tgccagtggc?gataagtcgt?gtcttaccgg?gttggactca?agacgatagt 1860

taccggataa?ggcgcagcgg?tcgggctgaa?cggggggttc?gtgcacacag?cccagcttgg 1920

agcgaacgac?ctacaccgaa?ctgagatacc?tacagcgtga?gctatgagaa?agcgccacgc 1980

ttcccgaagg?gagaaaggcg?gacaggtatc?cggtaagcgg?cagggtcgga?acaggagagc 2040

gcacgaggga?gcttccaggg?ggaaacgcct?ggtatcttta?tagtcctgtc?gggtttcgcc 2100

acctctgact?tgagcgtcga?tttttgtgat?gctcgtcagg?ggggcggagc?ctatggaaaa 2160

acgccagcaa?cgcggccttt?ttacggttcc?tggccttttg?ctggcctttt?gctcacatgt 2220

tctttcctgc?gttatcccct?gattctgtgg?ataaccgtat?taccgccttt?gagtgagctg 2280

ataccgctcg?ccgcagccga?acgaccgagc?gcagcgagtc?agtgagcgag?gaagcggaag 2340

agcgcccaat?acgcaaaccg?cctctccccg?cgcgttggcc?gattcattaa?tgcagctggc 2400

acgacaggtt?tcccgactgg?aaagcgggca?gtgagcgcaa?cgcaattaat?gtgagttagc 2460

tcactcatta?ggcaccccag?gctttacact?ttatgcttcc?ggctcgtatg?ttgtgtggaa 2520

ttgtgagcgg?ataacaattt?cacacaggaa?acagctatga?ccatgattac?gccaagcttt 2580

ggctaacaca?cacgccattc?caaccaatag?ttttctcggc?ataaagccat?gctctgacgc 2640

ttaaatgcac?taatgcctta?aaaaaacatt?aaagtctaac?acactagact?tatttacttc 2700

gtaattaagt?cgttaaaccg?tgtgctctac?gaccaaaagt?ataaaacctt?taagaacttt 2760

cttttttctt?gtaaaaaaag?aaactagata?aatctctcat?atcttttatt?caataatcgc 2820

atcagattgc?agtataaatt?taacgatcac?tcatcatgtt?catatttatc?agagctcgtg 2880

ctataattat?actaatttta?taaggaggaa?aaaataaaga?gggttataat?gaacgagaaa 2940

aatataaaac?acagtcaaaa?ctttattact?tcaaaacata?atatagataa?aataatgaca 3000

aatataagat?taaatgaaca?tgataatatc?tttgaaatcg?gctcaggaaa?agggcatttt 3060

acccttgaat?tagtacagag?gtgtaatttc?gtaactgcca?ttgaaataga?ccataaatta 3120

tgcaaaacta?cagaaaataa?acttgttgat?cacgataatt?tccaagtttt?aaacaaggat 3180

atattgcagt?ttaaatttcc?taaaaaccaa?tcctataaaa?tatttggtaa?tataccttat 3240

aacataagta?cggatataat?acgcaaaatt?gtttttgata?gtatagctga?tgagatttat 3300

ttaatcgtgg?aatacgggtt?tgctaaaaga?ttattaaata?caaaacgctc?attggcatta 3360

tttttaatgg?cagaagttga?tatttctata?ttaagtatgg?ttccaagaga?atattttcat 3420

cctaaaccta?aagtgaatag?ctcacttatc?agattaaata?gaaaaaaatc?aagaatatca 3480

cacaaagata?aacagaagta?taattatttc?gttatgaaat?gggttaacaa?agaatacaag 3540

aaaatattta?caaaaaatca?atttaacaat?tccttaaaac?atgcaggaat?tgacgattta 3600

aacaatatta?gctttgaaca?attcttatct?cttttcaata?gctataaatt?atttaataag 3660

taagttaagg?gatgcataaa?ctgcatccct?taacttgttt?ttcgtgtacc?tattttttgt 3720

gaatcgatta?tgtcttttgc?gcattcactt?cttttctata?taaatatgag?cgaagcgaat 3780

aagcgtcgga?aaagcagcaa?aaagtttcct?ttttgctgtt?ggagcatggg?ggttcagggg 3840

gtgcagtatc?tgacgtcaat?gccgagcgaa?agcgagccga?agggtagcat?ttacgttaga 3900

taaccccctg?atatgctccg?acgctttata?tagaaaagaa?gattcaacta?ggtaaaatct 3960

taatataggt?tgagatgata?aggtttataa?ggaatttgtt?tgttctaatt?tttcactcat 4020

tttgttctaa?tttcttttaa?caaatgttct?ttttttttta?gaacagttat?gatatagtta 4080

gaatagttta?aaataaggag?tgagaaaaag?atgaaagaaa?gatatggaac?agtctataaa 4140

ggctctcaga?ggctcataga?cgaagaaagt?ggagaagtca?tagaggtaga?caagttatac 4200

cgtaaacaaa?cgtctggtaa?cttcgtaaag?gcatatatag?tgcaattaat?aagtatgtta 4260

gatatgattg?gcggaaaaaa?acttaaaatc?gttaactata?tcctagataa?tgtccactta 4320

agtaacaata?caatgatagc?tacaacaaga?gaaatagcaa?aagctacagg?aacaagtcta 4380

caaacagtaa?taacaacact?taaaatctta?gaagaaggaa?atattataaa?aagaaaaact 4440

ggagtattaa?tgttaaaccc?tgaactacta?atgagaggcg?acgaccaaaa?acaaaaatac 4500

ctcttactcg?aatttgggaa?ctttgagcaa?gaggcaaatg?aaatagattg?acctcccaat 4560

aacaccacgt?agttattggg?aggtcaatct?atgaaaatgc?gatta 4605

<210>23

<211>6196

<212>DNA

＜213〉acetone Clostridium butylicum

<400>1

gtcgactcta?gaggatcccc?catatgcaag?ggtttattgt?tttctaaaat?ctgattacca 60

attagaatga?atatttccca?aatattaaat?aataaaacaa?aaaaattgaa?aaaagtgttt 120

ccaccatttt?ttcaattttt?ttataatttt?tttaatctgt?tatttaaata?gtttatagtt 180

aaatttacat?tttcattagt?ccattcaata?ttctctccaa?gataactacg?aactgctaac 240

aaaattctct?ccctatgttc?taatggagaa?gattcagcca?ctgcatttcc?cgcaatatct 300

tttggtatga?ttttacccgt?gtccatagtt?aaaatcatac?ggcataaagt?taatatagag 360

ttggtttcat?catcctgata?attatctatt?aattcctctg?acgaatccat?aatggctctt 420

ctcacatcag?aaaatggaat?atcaggtagt?aattcctcta?agtcataatt?tccgtatatt 480

cttttatttt?ttcgttttgc?ttggtaaagc?attatggtta?aatctgaatt?taattccttc 540

tgaggaatgt?atccttgttc?ataaagctct?tgtaaccatt?ctccataaat?aaattcttgt 600

ttgggaggat?gattccacgg?taccatttct?tgctgaataa?taattgttaa?ttcaatatat 660

cgtaagttgc?ttttatctcc?tatttttttt?gaaataggtc?taattttttg?tataagtatt 720

tctttacttt?gatctgtcaa?tggttcagat?acgacgacta?aaaagtcaag?atcactattt 780

ggttttagtc?cactctcaac?tcctgatcca?aacatgtaag?taccaataag?gttatttttt 840

aaatgtttcc?gaagtatttt?tttcacttta?ttaatttgtt?cgtatgtatt?caaatatatc 900

ctcctcacta?ttttgattag?tacctatttt?atatccatag?ttgttaatta?aataaactta 960

atttagttta?tttatagatt?tcattggctt?ctaaattttt?tatctagata?ataattattt 1020

tagttaattt?tattctagat?tatatatgat?atgatctttc?atttccataa?aactaaagta 1080

agtgtaaacc?tattcattgt?tttaaaaata?tctcttgcca?gtcacgttac?gttattagtt 1140

atagttatta?taacatgtat?tcacgaacga?aaatcgatgg?gctgcaggaa?ttcgatctga 1200

acggtctggt?tataggtaca?ttgagcaact?gactgaaatg?cctcaaaatg?ttctttacga 1260

tgccattggg?atatatcaac?ggtggtatat?ccagtgattt?ttttctccat?tttagcttcc 1320

ttagctcctg?aaaatctcga?taactcaaaa?aatacgcccg?gtagtgatct?tatttcatta 1380

tggtgaaagt?tggaacctct?tacgtgccga?tcaacgtctc?attttcgcca?aaagttggcc 1440

cagggcttcc?cggtatcaac?agggacacca?ggatttattt?attctgcgaa?gtgatcttcc 1500

gtcacaggta?tttattcggc?gcaaagtgcg?tcgggtgatg?ctgccaactt?actgatttag 1560

tgtatgatgg?tgtttttgag?gtgctccagt?ggcttctgtt?tctatcagct?gtccctcctg 1620

ttcagctact?gacggggtgg?tgcgtaacgg?caaaagcacc?gccggacatc?agcgctagcg 1680

gagtgtatac?tggcttacta?tgttggcact?gatgagggtg?tcagtgaagt?gcttcatgtg 1740

gcaggagaaa?aaaggctgca?ccggtgcgtc?agcagaatat?gtgatacagg?atatattccg 1800

cttcctcgct?cactgactcg?ctacgctcgg?tcgttcgact?gcggcgagcg?gaaatggctt 1860

acgaacgggg?cggagatttc?ctggaagatg?ccaggaagat?acttaacagg?gaagtgagag 1920

ggccgcggca?aagccgtttt?tccataggct?ccgcccccct?gacaagcatc?acgaaatctg 1980

acgctcaaat?cagtggtggc?gaaacccgac?aggactataa?agataccagg?cgtttccccc 2040

tggcggctcc?ctcgtgcgct?ctcctgttcc?tgcctttcgg?tttaccggtg?tcattccgct 2100

gttatggccg?cgtttgtctc?attccacgcc?tgacactcag?ttccgggtag?gcagttcgct 2160

ccaagctgga?ctgtatgcac?gaaccccccg?ttcagtccga?ccgctgcgcc?ttatccggta 2220

actatcgtct?tgagtccaac?ccggaaagac?atgcaaaagc?accactggca?gcagccactg 2280

gtaattgatt?tagaggagtt?agtcttgaag?tcatgcgccg?gttaaggcta?aactgaaagg 2340

acaagttttg?gtgactgcgc?tcctccaagc?cagttacctc?ggttcaaaga?gttggtagct 2400

cagagaacct?tcgaaaaacc?gccctgcaag?gcggtttttt?cgttttcaga?gcaagagatt 2460

acgcgcagac?caaaacgatc?tcaagaagat?catcttatta?atcagataaa?atatttctag 2520

atttcagtgc?aatttatctc?ttcaaatgta?gcacctgaag?tcagccccat?acgatataag 2580

ttgtaattct?catgtttgac?agcttatcat?cgataagctt?tcgtatttga?aaacgtaaaa 2640

gggttgataa?cgaagcgtca?ccgtcctaca?ttcgacgcac?ttatcgagaa?gtttaatgaa 2700

ataggttacg?agattagttg?ggaagtagta?aacgcgtggg?attacggagt?agcgcagaaa 2760

cgtgagcgag?tgtttatagt?tggtgtgcgg?aatgacctcg?gttttaagtt?cgattttccg 2820

aatccgttag?aaggcgatta?tcagacgcga?gtattgcggg?atgtgatcgg?agatctgcca 2880

gaggtcggag?aatacttcta?cgttaatcgg?gggggttggg?gtaagtgttt?ggtgaaaagt 2940

gttttcggac?tcgaagatgt?atcgccgact?ataaaaagtc?aaaacgcaaa?tcttcctcct 3000

ggttatcccg?gacaccctaa?agacgccgat?caaatctgca?accgaccgat?tagccaacaa 3060

aagcgccccg?ccgtttcacc?gttcgcgaat?gtctccgaat?ccaatctgcg?ccagacactt 3120

acgtcctgcc?agacgatatt?tccttaacgg?cgcagtatcg?gattagtcgg?taacggaatt 3180

gcttcgcgtg?ttgcgtggta?tatcgggcgg?gcgttgcgga?tcaactttca?tctaactgta 3240

agtaattatg?atagataaga?tggttgaaat?attacataaa?agatcaattt?tatttagagt 3300

aaaaataaaa?tatatggagg?ttgtttgttg?agtaaactac?gtgtaatgag?cctttttagt 3360

ggaattggtg?catttgaagc?tgcattaaga?aacataggcg?ttgattatga?actgattggc 3420

tttagtgaaa?tagataaata?tgcaatcaaa?tcttattgtg?caattcataa?tgtaagtgaa 3480

acattgaatg?ttggagacat?aagtaaagct?aagaaagata?atattccata?ttttgatttg 3540

ttaacaagtg?gatttccttg?tcccactttt?tcagtagccg?gcggccgtga?tggtatggaa 3600

tataagtgta?gcaattgttc?gcacgagcat?ttaattactt?atgaggatta?taagaaggga 3660

gtcaaatgcc?cgaagtgtga?agctgtttct?aaggctaaag?atgaacgggg?aacgctcttt 3720

tttgaaacag?ctttgttagc?agaagagaag?aagccgaaat?ttgtgatatt?agaaaatgta 3780

aaagggctaa?ttaatagtgg?caatggacaa?gtgttaagaa?taattagtga?gactatgaac 3840

aatataggct?ataggattga?cctagagcta?cttaattcaa?agtttttcaa?tgttccacag 3900

aatcgtgaac?gggtatacat?aattggcatt?agagaagatt?tggttgaaaa?tgaacaatgg 3960

gttgtaggtc?agaagcgaaa?cgatgttctg?agtaaaggta?aaaagagatt?gcaagaaata 4020

aatataaaga?gttttaattt?taaatggcct?ttacaagaca?ctgtcacaaa?gaggttgaga 4080

gaaattcttg?aggattttgt?tgatgagaag?tattacttaa?atgaagaaaa?gacgaagaaa 4140

cttgttgagc?aactaggaac?cgcaccgcta?caaaaacaag?aagtaaggga?gccgttaatg 4200

gtgggacacg?tggatttaaa?aggtcacgat?gccatcaaaa?gagtttactc?gcctgaagga 4260

ctttcaccga?cattaactac?aatgggcggc?ggtcacagag?aacctaaaat?cgcagaaaag 4320

cagaaagaag?taagggcagt?gttaacgcca?gaaagagaag?agaaaagaca?gaatggaaga 4380

cgttttaaag?aaaacggaga?accggctttc?acggttaata?caattgatcg?acatggtgta 4440

gcaatcggag?aatacccaaa?atataaaatt?agaaagctct?ctccattaga?atgttggaga 4500

ctccaagcat?ttgatgacga?agattttgaa?aaagcttttg?cagcaggaat?aagtaactca 4560

caattataca?agcaagccgg?taattcaatt?acagttagtg?tgcttgagtc?tatatttcaa 4620

gaattaatac?atacatatgt?taataaagaa?tctgaataaa?atttgtcttt?taaacaaatg 4680

caaaataaga?gggattgaga?ggtgaggaag?atggatagtt?acccagagtc?tttaaaaaga 4740

gagacagagg?agattaaaga?gcgtgttagg?aatggaaata?tcaaagaaga?caggattaaa 4800

gaaattgcag?aaacgacagt?tgagtttttg?aaatcagagg?agaaaagaca?taaatacttt 4860

tctgaagttg?ctgcagctat?ggcgtataac?ttaagtgagt?ttttcaaatc?ttatttaaaa 4920

ggagagtgaa?tatgctaact?gatcagaaaa?aattgacttg?gtaaacgctc?ttagaaaaaa 4980

aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa 5040

aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa 5100

aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa 5160

aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa 5220

aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa 5280

aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa 5340

aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa 5400

aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa 5460

aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa 5520

aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaagctt 5580

taatgcggta?gtttatcaca?gttaaattgc?taacgcagtc?aggcaccgtg?tatgaaatct 5640

aacaatgcgc?tcatcgtcat?cctcggcacc?gtcaccctgg?atgctgtagg?cataggcttg 5700

gttatgccgg?tactgccggg?cctcttgcgg?gatatcgtcc?attccgacag?catcgccagt 5760

cactatggcg?tgctgctagc?gctatatgcg?ttgatgcaat?ttctatgcgc?acccgttctc 5820

ggagcactgt?ccgaccgctt?tggccgccgc?ccagtcctgc?tcgcttcgct?acttggagcc 5880

actatcgact?acgcgatcat?ggcgaccaca?cccgtcctgt?ggatcctcta?cgccggacgc 5940

atcgtggccg?gcatcaccgg?cgccacaggt?gcggttgctg?gcgcctatat?cgccgacatc 6000

accgatgggg?aagatcgggc?tcgccacttc?gggctcatga?gcgcttgttt?cggcgtgggt 6060

atggtggcag?gccccgtggc?cgggggactg?ttgggcgcca?tctccttgca?tgcaccattc 6120

cttgcggcgg?cggtgctcaa?cggcctcaac?ctactactgg?gctgcttcct?aatgcaggag 6180

tcgcataagg?gagagc 6196

Claims (35)

1, a kind of recombinant plasmid is characterized in that, described plasmid comprises protokaryon two class intron fragments and intestinal bacteria-clostridium acetobutylicum shuttle plasmid fragment, and described protokaryon two class introns are through deriving from the clostridium acetobutylicum promoter regulation.

2, recombinant plasmid as claimed in claim 1 is characterized in that, described protokaryon two class introns are L1.LtrB two class introns.

3, recombinant plasmid as claimed in claim 1 is characterized in that, described shuttle plasmid is pIMP1-ptb.

4, recombinant plasmid as claimed in claim 1 is characterized in that, described promotor is the ptb promotor.

As each described recombinant plasmid among the claim 1-4, it is characterized in that 5, described recombinant plasmid is pSY6, its sequence is shown in SEQ ID NO:1.

6, construction of recombinant plasmid method as claimed in claim 1 is characterized in that, said method comprising the steps of:

A) pcr amplification, purifying prepare protokaryon two class introns;

B) enzyme is cut intron and intestinal bacteria-clostridium acetobutylicum shuttle plasmid, connects, and makes up shuttle plasmid.

7, construction process as claimed in claim 6 is characterized in that, described protokaryon two class introns are L1.LtrB two class introns.

8, construction process as claimed in claim 6 is characterized in that, described plasmid is pIMP1-ptb.

As claim 7 or 8 described construction processs, it is characterized in that 9, the promotor of described intron is the ptb promotor.

10, construction process as claimed in claim 6 is characterized in that, described recombinant plasmid is pSY6, and its sequence is shown in SEQ ID NO:1.

11, construction process as claimed in claim 6 is characterized in that, comprises also after the digested plasmid step that purifying reclaims the step of plasmid.

12, construction process as claimed in claim 6 is characterized in that, also comprises plasmid dephosphorization step after the digested plasmid step.

13, construction process as claimed in claim 12 is characterized in that, comprises also after the plasmid dephosphorization step that purifying reclaims the step of dephosphorization plasmid.

14, construction process as claimed in claim 6 is characterized in that, makes up the shuttle plasmid step and also comprises and will connect the step of product transformed competence colibacillus cell and cultivation and extracting plasmid.

15, the application of recombinant plasmid as claimed in claim 1 is characterized in that, described shuttle plasmid is used to make up the gene knockout plasmid of clostridium acetobutylicum.

16, application as claimed in claim 15 is characterized in that, described recombinant plasmid is pSY6, and its sequence is shown in SEQID NO:1.

17, application as claimed in claim 15 is characterized in that, described gene knockout plasmid vector comprises the plasmid of buk gene knockout and the plasmid of solR gene knockout.

18, application as claimed in claim 17 is characterized in that, the plasmid of described buk gene knockout is pSY6-buk, and its sequence is shown in SEQ ID NO:2.

19, application as claimed in claim 17 is characterized in that, the plasmid of described solR gene knockout is pSY6-solR, and its sequence is shown in SEQ ID NO:3.

20, a kind of plasmid of buk gene knockout is characterized in that, described plasmid comprises that the buk-targetron enzyme is cut the back fragment and the described recombinant plasmid enzyme of claim 1 is cut the back fragment.

21, plasmid as claimed in claim 20 is characterized in that, described plasmid is pSY6-buk, and its sequence is shown in SEQID NO:2.

22, the construction process of plasmid as claimed in claim 20 is characterized in that, may further comprise the steps:

A) pcr amplification, purifying obtains the buk-targetron fragment;

B) enzyme is cut buk-targetron fragment and plasmid, and connects, and makes up the plasmid of buk gene knockout.

23, construction process as claimed in claim 22 is characterized in that, described structure plasmid step comprises that also purifying reclaims enzyme and cuts back buk-targetron fragment and plasmid.

24, construction process as claimed in claim 23 is characterized in that, described structure plasmid step comprises that also connecting product transformed competence colibacillus cell also cultivates and plasmid extraction.

25, construction process as claimed in claim 22 is characterized in that, described plasmid is pSY6-buk, and its sequence is shown in SEQ ID NO:2.

26, the application of the plasmid of buk gene knockout as claimed in claim 20 is characterized in that, described plasmid is used to make up the recombinant bacterial strain of buk gene knockout.

27, application as claimed in claim 26 is characterized in that, the plasmid of described buk gene knockout is pSY6-buk, and its sequence is shown in SEQ ID NO:2.

28, a kind of plasmid of solR gene knockout is characterized in that, described plasmid comprises that the solR-targetron enzyme is cut the back fragment and the described recombinant plasmid enzyme of claim 1 is cut the back fragment.

29, plasmid as claimed in claim 28 is characterized in that, described plasmid is pSY6-solR, and its sequence is shown in SEQID NO:3.

30, the construction process of plasmid as claimed in claim 28 is characterized in that, may further comprise the steps:

A) pcr amplification, purifying obtains the solR-targetron fragment;

B) enzyme is cut solR-targetron fragment and plasmid, and connects, and makes up the plasmid of solR gene knockout.

31, construction process as claimed in claim 30 is characterized in that, described structure plasmid step comprises that also purifying reclaims enzyme and cuts back solR-targetron fragment and plasmid.

32, construction process as claimed in claim 31 is characterized in that, described structure plasmid step comprises that also connecting product transformed competence colibacillus cell also cultivates and plasmid extraction.

33, construction process as claimed in claim 30 is characterized in that, described plasmid is pSY6-solR, and its sequence is shown in SEQ ID NO:3.

34, the application of the plasmid of solR gene knockout as claimed in claim 28 is characterized in that, described plasmid vector is used to make up the recombinant bacterial strain of buk gene knockout.

35, application as claimed in claim 34 is characterized in that, the plasmid of described solR gene knockout is pSY6-solR, and its sequence is shown in SEQ ID NO:3.

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