CN111088209B - Recombinant clostridium butyricum for producing 1, 4-butanediol and construction method and application thereof - Google Patents

Abstract

The invention discloses a recombinant clostridium butyricum for producing 1, 4-butanediol and a construction method and application thereof, wherein the recombinant clostridium butyricum is constructed by expressing 4-hydroxybutyryl coenzyme A dehydratase in the clostridium butyricum. Compared with the prior art, the invention has the following advantages: (1) compared with wild bacteria which can not produce 1, 4-butanediol, the recombinant bacteria constructed by the invention have the yield of 66mg/L in the process of producing 1, 4-butanediol. (2) The invention further inactivates the etfA gene in the clostridium butyricum, and the yield of the 1, 4-butanediol produced by the recombinant bacterium is further improved to 182 mg/L.

Description

Recombinant clostridium butyricum for producing 1, 4-butanediol and construction method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant clostridium butyricum for producing 1, 4-butanediol, and a construction method and application thereof.

Background

1, 4-butanediol is an important basic organic chemical raw material and fine chemical raw material, and downstream derivatives thereof are many and have wide application. The derivatives are fine chemical products with high added value, are widely used for solvents, medicines, cosmetics, plasticizers, curing agents and pesticides, and are mainly used for manufacturing Tetrahydrofuran (THF), polybutylene terephthalate (PBT), gamma-butyrolactone (GBL), N-methylpyrrolidone (NMP), N-vinyl pyrrolidone (NVP) and the like. China is the fastest growing area of global demand, and the increase is estimated to be 4% -6%. Some foreign producers have avoided the excess of BDO capacity, and the German Basff company is the largest BDO producer worldwide with the annual capacity of 46.7 ten thousand tons. However, the industrial production of 1, 4-butanediol mainly uses fossil fuel, which greatly consumes resources and is environmentally responsible, so that it is necessary to develop a biological preparation method for producing 1, 4-butanediol.

The existing 1, 4-butanediol methods have two kinds, the first method is that succinic acid after TCA circulation is used as a substrate to produce 1, 4-butanediol through some genetic modification; another is the production in the xylose non-phosphorylation pathway. The first method has an energy output of 0 and a reducing power of 1, the second method has an energy output of 1 and a reducing power of 0, and both the energy output and the reducing power are lower. Therefore, it is required to develop a process route for producing 1, 4-butanediol with high energy yield and high reducing power. In addition, the clostridium butyricum is mainly used for acetone-butanol ethanol (ABE) fermentation, is also applied to the production of products such as 2, 3-butanediol, acetoin and the like at present, has high yield and wide application range, has little application to similar 1, 4-butanediol, and is developed into a clostridium model by utilizing the clostridium butyricum to construct a new biological way capable of efficiently producing the 1, 4-butanediol.

Acetoin is a prerequisite for the synthesis of 2, 3-butanediol, clostridium acetobutylicum has genes for the synthesis of acetoin, and metabolic steps flowing to the acetoin are fewer compared with 1, 4-butanediol which needs to be further synthesized by crotonyl-coenzyme A, and metabolic branches are fewer, so that the metabolic direction of the acetoin is easier to control. The production of 1, 4-butanediol has more metabolic branches and redundant metabolites, and is relatively more difficult to control the conversion of metabolic pathways and thus more difficult to produce.

Disclosure of Invention

The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a recombinant clostridium butyricum for producing 1, 4-butanediol aiming at the defects of the prior art.

The technical problem to be solved by the invention is to provide a construction method of the recombinant clostridium butyricum.

The technical problem to be finally solved by the invention is to provide the application of the recombinant clostridium butyricum.

In order to solve the technical problem, the invention discloses a recombinant clostridium butyricum for producing 1, 4-butanediol, which is constructed by expressing 4-hydroxybutyryl coenzyme A dehydratase in the clostridium butyricum. The metabolic profile is shown in FIG. 1. The metabolic process of FIG. 1 shows that the expression of 4 hydroxybutyryl-CoA dehydratase in C.butyricum and the utilization of crotonyl-CoA, is 1, 4-butanediol produced by 4HBD conversion from crotonyl-CoA.

Wherein the clostridium butyricum is CGMCC NO.5234 or ATCC 824.

Wherein the 4-hydroxybutyryl-CoA dehydratase is expressed by a gene fragment of any one of 4HBD, namely cbei _2100(4HBD-1), ckl _3020(4HBD-2), abfD (4HBD-3), lf65-02412(4HBD-4) and ctk-c07640(4 HBD-5); wherein the nucleotide sequences of cbei _2100, ckl _3020, abfD, lf65-02412 and ctk-c07640 are respectively shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.

Among them, the recombinant Clostridium butyricum is preferably Clostridium acetobutylicum having introduced therein a gene fragment of any one of cbei _2100(4HBD-1), ckl _3020(4HBD-2), abfD (4HBD-3), lf65-02412(4HBD-4) and ctk-c07640(4 HBD-5).

More preferably Clostridium butyricum having the cbei _2100(4HBD-1) gene fragment introduced thereinto.

The construction method of the recombinant clostridium butyricum for producing 1, 4-butanediol comprises the following steps:

(1) inserting any one gene fragment of cbei _2100, ckl _3020, abfD, lf65-02412 and ctk-c07640 into Nde I enzyme cutting sites of the vector plasmid to obtain a recombinant plasmid;

(2) methylating the recombinant plasmid obtained in the step (1) to obtain a methylated recombinant plasmid;

(3) transferring the methylated recombinant plasmid obtained in the step (2) into clostridium butyricum, and screening to obtain the recombinant plasmid.

In the step (1), the vector plasmid is PSY8, and the nucleotide sequence of the vector plasmid is shown as SEQ ID NO. 7.

Preferably, the etfA gene in the recombinant clostridium butyricum is inactivated so as to further improve the yield of 1, 4-butanediol, and the nucleotide sequence of the obtained recombinant clostridium butyricum is shown as SEQ ID NO. 6.

The construction method of the recombinant clostridium butyricum for producing 1, 4-butanediol comprises the step of inserting the intron sequence shown in SEQ ID NO.8 between the 639bp and 640bp of the etfA gene by using the knockout technology of the intron II gene.

Wherein the gene knockout technology of the intron type II comprises the following steps:

(1) analyzing the sequence shown in SEQ ID NO.6 to obtain the nucleotide sequence of the intron and the insertion position of the gene;

(2) constructing an intron sequence shown in SEQ ID NO.8 into a gene knockout plasmid to obtain a recombinant plasmid;

(3) and (3) transforming the recombinant plasmid obtained in the step (2) into clostridium butyricum, and screening to obtain the strain with the inactivated etfA gene.

In the step (2), the gene knockout plasmid is PMTL007C-E2, and the nucleotide sequence of the gene knockout plasmid is shown as SEQ ID NO. 9.

The application of the recombinant clostridium butyricum in preparing 1, 4-butanediol also falls within the protection scope of the invention.

Wherein, the application comprises the following steps: and (2) culturing the recombinant clostridium butyricum in a seed culture medium to obtain a seed solution, transferring the seed solution into a fermentation culture medium according to the volume ratio of 10%, and performing anaerobic fermentation for about 84 hours.

Wherein the seed culture medium is 3g/L of yeast powder, 5g/L of peptone, 10g/L of anhydrous glucose, 2g/L of ammonium acetate, 2g/L of sodium chloride, 3g/L of magnesium sulfate heptahydrate, 1g/L of potassium dihydrogen phosphate, 1g/L of dipotassium hydrogen phosphate, 0.1g/L of ferrous sulfate heptahydrate, and the balance of water.

Wherein the fermentation medium is solution I: 60g/L of anhydrous glucose and water as a solvent; solution II: 2.2g/L of ammonium acetate, 0.5g/L of monopotassium phosphate, 0.5g/L of dipotassium phosphate and water as a solvent; solution III: 0.01g/L of sodium chloride, 0.2g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate heptahydrate, 0.01g/L of manganese sulfate monohydrate and water as a solvent; solution IV: VB 11 mg/L, 1mg/L of p-amino cresol, 0.01mg/L of biotin and water as a solvent.

And adding the solution III and the solution IV which are prepared by expanding 1000 times into the 80% solution I and the 10% solution II, wherein the solution III and the solution IV are one thousandth of each (taking 50mL system as an example, fermentation liquor is 40mL of solution 1 and 5mL of solution 2; and solutions 3 and 4 are 50 mu L respectively, and the solutions 1, 2,3 and 4 are mixed and then added with 5mL of seed solution) to finally obtain a fermentation culture medium.

Has the advantages that: compared with the prior art, the invention has the following advantages:

(1) the invention takes the more cheap and common glucose as the substrate for production.

(2) The process route for preparing the 1, 4-butanediol is anaerobic fermentation, the energy and the reducing power can be furthest reserved in the metabolic process for product synthesis and thallus growth, the energy output is 2, the reducing power is 1, the product synthesis and the thallus growth are more facilitated, and the synthesis path has the maximum theoretical yield.

(3) Compared with wild bacteria which can not produce 1, 4-butanediol, the recombinant bacteria constructed by the invention have the yield of 66mg/L in the process of producing 1, 4-butanediol.

(4) The invention further inactivates the etfA gene in the clostridium butyricum, and the yield of the 1, 4-butanediol produced by the recombinant bacterium is further improved to 182 mg/L.

Drawings

FIG. 1 is a comparison of metabolic pathways for producing 2, 3-butanediol versus 1, 4-butanediol.

FIG. 2 is a plasmid map of the type II intron gene knock-out plasmid PMTL 007C-E2.

FIG. 3 is a colony PCR gel electrophoresis chart of Clostridium acetobutylicum for verifying whether eftA is knocked out.

FIG. 4 is a diagram showing the results of PCR on the 4HBD 1-3 colonies.

FIG. 5 is a diagram showing the PCR results of 4HBD 4-5 colonies.

FIG. 6 is a gas chromatogram of a 1, 4-butanediol standard.

FIG. 7 is a gas phase diagram after fermentation of wild fungi.

FIG. 8 is a gas chromatogram after fermentation of a recombinant bacterium which expresses only the 4HBD gene.

FIG. 9 is a gas chromatogram after fermentation of a recombinant bacterium which expresses 4HBD after the etfA gene is knocked out.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.

Example 1: method for constructing etfA gene inactivation mutant strain of clostridium acetobutylicum

1. Design of introns

According to the etfA gene sequence of Clostridium acetobutylicum recorded in NCBI database (shown as SEQ ID NO. 6), a suitable insertion gene site (http:// www.Clostron.Com) is designed by software, and is inserted between 640 bases of 639-.

2. Construction of etfA insertion inactivation vector:

the vector PMTL007C-E2 is subjected to double digestion by Xhol and BSrGI, and the sequence of the vector is shown as SEQ ID NO. 9. The enzyme digestion product is converted into a purification kit (Takara), and then is connected with an intron sequence S-639 through one-step cloning (Clonexpress), the recombinant plasmid connected through one-step cloning is transformed into Escherichia coli E.coli DH5a (the laboratory), the Escherichia coli E.coli DH5a is coated on an LB plate containing 25ug/mL chloramphenicol resistance, the culture is carried out at 37 ℃ for 12h, a transformant is picked up, the transformant is inoculated into a liquid LB culture medium containing 25 ug/chloramphenicol resistance, the culture is carried out at 37 ℃ and 200rpm for 12h, the recombinant plasmid is extracted, and the sequence verification is carried out to obtain an etfA insertion inactivation vector PMTL007C-E2-etfA, the nucleotide sequence of the etfA insertion inactivation vector is shown as SEQ ID No.6, and the graph of the inserted plasmid is shown as figure 2.

3. Methylation of vector PMTL 007C-E2-etfA:

since it has been demonstrated that Clostridium acetobutylicum (Clostridium acetobutylicum) ATCC824 contains restriction enzyme Cac824I, which cleaves unmethylated foreign DNA, the recombinant inactivation vector PMTL007C-E2-etfA was methylated before transformation of Clostridium acetobutylicum. The specific operation steps are to prepare the chemical competence of E.coli TOP10/pANZ (laboratory), to transform the sequencing successful inactivation vector into E.coli E.COli TOP10, because the pANZ plasmid has tetracycline resistance, to coat the plasmid on LB plate containing 25ug/mL chloramphenicol and 10ug/mL tetracycline double resistance, to culture for 12h at 37 ℃, to pick up the transformant, to receive the liquid containing 25ug/mL chloramphenicol and 10ug/mL tetracycline LB medium, to culture for 12h at 200rpm at 37 ℃, to extract the methylation deletion vector (the pANZ plasmid contains a Bacillus subtilis phage gene, which can code methyltransferase, and can realize the methylation of exogenous plasmid in E.coli).

4. Electrotransformation of the methylated knock-out plasmid to clostridium acetobutylicum:

under anaerobic conditions, 60mL of C.acetobutylicum B3 culture solution which grows to the middle logarithmic phase and is cultured by 2XYTG medium is taken, centrifuged at 4000rpm at 40 ℃ for 10min, supernatant is discarded, sufficient precooled electrotransformation buffer EPB (27omM sucrose, sfnmNaHZpO4, pH7.4) is added, washing is carried out twice, and 2.3mL of EPB is used for heavy suspension; 570uL was then added to a 0.4cm electric beaker and placed in an ice bath to cool, and 20uL of the methylated plasmid was added and placed in an ice bath for 2 min. Performing electric conversion at an O kV voltage and a 25uF capacitor; the electrotransfer solution was then added to l mL of 2XYTG medium at 37 ℃ for resuscitation and culture for 4h, and 100uL of cells were collected by centrifugation and plated on P2 plates containing 20ug/mL of validamycin A for anaerobic culture for 24 h.

5. Screening of etfA insertionally inactivated mutants

Transformants are picked, colony PCR verification is carried out on the transformants by using primers etfA-F (the sequence of which is shown in SEQ ID NO. 10) and etfA-R (the sequence of which is shown in SEQ ID NO. 11), and mutant strains with introns inserted into the genome are screened (after insertion, about 1Kbp on a gene band electrophoretogram is amplified by PCR compared with the wild type). The correctly inserted mutants were passaged three times and spread on P2 solid medium containing erythromycin and validamycin resistances, and mutants lacking the knockout plasmid (mutants that could not grow on the plate containing both erythromycin and validamycin resistances) were selected, and the colony PCR results of the results of gel nucleic acid electrophoresis were shown in FIG. 3.

Example 2:

1. designing a recombinant plasmid: searching gene sequences of cbei _2100, ckl _3020, abfD, lf65-02412 and ctk-c07640 of clostridium acetobutylicum recorded in an NCBI industrial database, respectively inserting the gene sequences into Nde I enzyme cutting sites in PSY8 serving as vector plasmids, and synthesizing the gene sequences with Jinwei. Inoculating the obtained puncturing strain to LB medium containing 25 ug/ampicillin, culturing at 37 deg.C and 200rpm for 12 hr, extracting recombinant plasmid, preserving the strain with glycerol, and storing in refrigerator at-80 deg.C.

2. Methylation of the recombinant PSY8 plasmid:

since it has been demonstrated that Clostridium acetobutylicum (Clostridium acetobutylicum) ATCC824 contains restriction enzyme Cac824, which cleaves unmethylated foreign DNA, the recombinant vector PSY8 is methylated before transformation of Clostridium acetobutylicum.

The specific operation steps are to prepare the chemical competence of E.coli Top10/pANZ (the laboratory), to transform the extracted recombinant plasmid vector into E.coli E.COli Top10, because the pANZ plasmid has tetracycline resistance, to coat the plasmid into LB plate containing 25ug/mL ampicillin resistance and 10ug/mL tetracycline double resistance, to culture for 12h at 37 ℃, to pick up the transformant, to receive the liquid containing 25ug/mL ampicillin and 10ug/mL tetracycline LB medium, to culture for 12h at 37 ℃ and 200rpm, to extract the methylation deletion vector (the pANZ plasmid contains a Bacillus subtilis phage gene, which can code methyltransferase, to realize the methylation of exogenous plasmid in E.coli).

3. Electrotransformation of the methylated recombinant plasmid to clostridium acetobutylicum and etfA knockout clostridium acetobutylicum of example 1:

under anaerobic conditions, 60mL of C.acetobutylicum B3 culture medium grown to mid-log phase in 2XYTG medium was centrifuged at 4000rpm at 4 ℃ for 10min to discard the supernatant, and sufficient precooled electroporation buffer EPB (270mM sucrose, 5mM NaHZpO) was added₄pH7.4), washing twice, re-suspending with 2.3mL of EPB, adding 570uL of the mixture into a 0.4cm electric rotating cup, placing the electric rotating cup in an ice bath for cooling, adding 20uL of the methylated plasmid, and placing the electric rotating cup in the ice bath for 30 min; 2.0kV voltage, 25uF capacitance.

Then adding the electrotransformation liquid into 10mL of 2xYTG culture medium at 37 ℃ for recovery and culture for 4h, centrifugally collecting 100uL of cells, coating the cells in a P2 plate containing 20ug/mL of validamycin for anaerobic culture for 24-36h, and verifying whether the 4HBD gene is successfully electrotransferred into clostridium acetobutylicum: after plate culture, 30 single colonies were picked to a thiamphenicol resistant P2 plate and colony PCR was performed after growth. PCR conditions were as follows: at 95 ℃ for 10 min; 38cycles X (95 ℃, 10 s; 52 ℃, 15 s; 72 ℃, 2 min).

The results of colony PCR were analyzed by nucleic acid gel electrophoresis, and the results are shown in FIG. 4 and FIG. 5 (in which 4HBD-1, 2,3, 4, 5 respectively indicate that the inserted genes are cbei _2100, ckl _3020, abfD, lf65-02412, ctk-c07640)

Example 3: the recombinant bacteria and the wild bacteria are fermented in a 50mL blue-mouth bottle respectively

P2 plate medium: 3g/L of yeast powder, 5g/L of peptone, 10g/L of anhydrous glucose, 2g/L of ammonium acetate, 2g/L of sodium chloride, 3g/L of magnesium sulfate heptahydrate, 1g/L of potassium dihydrogen phosphate, 1g/L of dipotassium hydrogen phosphate, 0.1g/L of ferrous sulfate heptahydrate, 15g/L of agar and the balance of water.

2. Seed culture medium: 3g/L of yeast powder, 5g/L of peptone, 10g/L of anhydrous glucose, 2g/L of ammonium acetate, 2g/L of sodium chloride, 3g/L of magnesium sulfate heptahydrate, 1g/L of potassium dihydrogen phosphate, 1g/L of dipotassium hydrogen phosphate, 0.1g/L of ferrous sulfate heptahydrate and the balance of water.

3. Fermentation medium:

solution I: 60g/L of anhydrous glucose and water as a solvent;

solution II: 2.2g/L of ammonium acetate, 0.5g/L of monopotassium phosphate, 0.5g/L of dipotassium phosphate and water as a solvent;

solution III: 0.01g/L of sodium chloride, 0.2g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate heptahydrate, 0.01g/L of manganese sulfate monohydrate and water as a solvent;

solution IV: VB 11 mg/L, 1mg/L of p-amino cresol, 0.01mg/L of biotin and water as a solvent.

Adding solution III and solution IV which are prepared by expanding 1000 times into 80% solution I and 10% solution II, wherein the solution III and the solution IV are respectively one thousandth, and finally obtaining the fermentation culture medium.

4. Plate culture: the recombinant bacterium constructed in example 1, the recombinant bacterium of clostridium butyricum constructed in example 2 and the wild strain are inoculated into a plate culture medium for anaerobic culture at the culture temperature of 37 ℃ for 12 h. The recombinant bacteria and wild bacteria cultured by the plate are inoculated into a seed culture medium (agar is not added, and other components are the same as those of a P2 plate culture medium) to be statically cultured for 12h at 37 ℃.

5. Liquid fermentation: adding 5mL of recombinant bacteria and wild bacteria cultured in the seed culture medium into 45mL of prepared fermentation culture medium, respectively, placing in an anaerobic box for culturing at 37 ℃ for 72h, sampling every 12h and storing in a refrigerator at-80 ℃, and stopping fermentation after 72 h.

Example 4: detecting the yield of 1, 4-butanediol by using a high-efficiency gas chromatograph

1. Chromatographic conditions are as follows: capillary column (30m × 0.32mm, 0.25 μ L); temperature programming: the initial temperature is 70 deg.C, storing for 0.5min, increasing to 180 deg.C at 20 deg.C/min, maintaining for 2min, and increasing to 235 deg.C at 25 deg.C/min, and maintaining for 2 min. The gasification chamber temperature is 240 ℃, the FID detector temperature is 250 ℃, the split ratio is 10: 1, sample size 1 microliter

2. The specific operation is as follows

(1) Preparation of gas phase vial: samples of 12, 24, 36, 48, 60, 72 hours were taken out from a-80 ℃ refrigerator, 1mL of the thawed sample was taken out of 5mL of the fermentation broth and placed in a 1.5mL centrifuge tube, marked, aspirated by a l mL syringe, and injected into a gas phase vial through a filter to prepare a sample.

(2) Preparing a standard product: 10g/L of butanol, 2g/L of ethanol, 4g/L of acetone, 2g/L of acetoin, 2g/L of acetic acid, 2g/L of butyric acid and 2g/L of 1, 4-butanediol. And (4) pumping the prepared standard solution into a gas phase small bottle through a filter membrane to prepare a standard product.

(3) Product quantification: 1.4 butanediol yield (1, 4-butanediol content in the sample/Peak area of the sample).)

FIG. 6 is a gas chromatogram of a 1, 4-butanediol sample, showing that the peak time of 1, 4-butanediol is around 11.069. FIG. 7 is a gas phase diagram of a wild-type bacterium after fermentation, and it can be found that the wild-type bacterium itself does not produce 1, 4-butanediol. FIG. 8 is a gas chromatogram after fermentation of a recombinant bacterium which expresses only the 4HBD gene. FIG. 9 is a gas chromatogram after fermentation of a recombinant bacterium which expresses 4HBD after the etfA gene is knocked out.

TABLE 1 yield of final 1, 4-butanediol in the experiment

The detection results are shown in Table 1, and it can be seen from Table 1 that the yield of 1.4 butanediol of the mutant bacteria expressing 4HBD after knockout of eftA is improved greatly compared with the yield of 1, 4-butanediol after overexpression of only 4HBD gene, wherein the yield of 1, 4-butanediol is the highest and reaches 66mg/L when Cbei-2100 is used for expressing 4-hydroxybutyryl coenzyme A dehydratase, and the yield of 1, 4-butanediol reaches 182mg/L after eftA inactivation.

The invention provides a recombinant clostridium butyricum for producing 1, 4-butanediol and a construction method and an application thought and a method thereof, and a method and a way for realizing the technical scheme are many. All the components not specified in the present embodiment can be realized by the prior art.

Sequence listing

<110> Nanjing university of industry

<120> recombinant clostridium butyricum for producing 1, 4-butanediol and construction method and application thereof

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1455

<212> DNA

<213> 4-hydroxybutyryl-CoA dehydratase (cbei _2100)

<400> 1

atgccattaa aaacaaagga acaatatatt gaaagcctta gaaagttaaa cctaaaagtg 60

tacatgtttg gaaaaccagt tgacaatgtt gttgataatc caatcataag accatctctt 120

aattcagttg ctatgactta tgaattagca caaatgccag agtatgaaga tttaatgaca 180

gctacttcta atttaacagg ggaaaaagta aatagatttg ctcatttaca tcaaagcaca 240

gatgatctta ttaaaaaagt aaagatgcaa agattacttg gtcaaaaaac cgcatcatgt 300

ttccaaaggt gcgttggtat ggatgctttt aatgcattat acagttcaac atacgaaata 360

gataaagcgt gtggaacaaa ttatcatgaa aactttaata agtttttaaa atatgttcaa 420

gaaaacgact taaccgttga tggtgcaatg actgatccta aaggggacag aggattatcg 480

ccaagtaaac aagctgatgg agatttatac ttaagggttg ttgaaagaag agaagatggg 540

gtagttgtta gaggagcaaa atgtcaccaa actggaatgt taaattctca tgaggtagta 600

gtaatgccaa caatagcatt aactccaaat gataaggatt gggcaatatc ttttgcagtg 660

cctacagatg ctgaaggaat aattcatata tatggaaggc aatcttgtga tacaagaaaa 720

ttggagccag gtgctgatat agatcttgga aatgctgaat ttggtggaca agaaacatta 780

acaatatttg ataatgtatt tgtaccaaat gaaagaattt tcttaaatgg agaaactgat 840

tttgctggaa tgatagttga aagatttgca ggatatcata gacaaagtta tggtggatgc 900

aaagtcggag ttggagatgt tctaattgga gctgctgcag tagctgctga ttataatgga 960

gctcacaaag catctcatgt taaagataaa ttaatagaaa tgactcactt aaatgaaact 1020

ttattctctt gtggtattgc atgttcagca atgggttcta aaacagaagc tggaaattat 1080

tatattgata atctgctagc aaatgtatgt aagcaaaatg ttacaagatt cccttatgaa 1140

atatgtagac ttgcagaaga tattgctgga ggaatcatgg tgactatgcc atcagaagca 1200

gattttaaag atgaaaagat aggtccatat atagataaat atttgagagg tgtaaactca 1260

gtttcaacag aaaatagaat gagaatatta agattaatag aaaatatttg tttagggact 1320

gcagctgtcg gatatagaac agaatcaatg catggagctg gatcaccaca agctcaaaga 1380

attatgatag ctagacaagg aaatttagca gctaaaaaga aaatagctaa gaaaatagct 1440

agaattaaag aataa 1455

<210> 2

<211> 1455

<212> DNA

<213> 4-hydroxybutyryl-CoA dehydratase (ckl _3020)

<400> 2

atgtctttaa tgactgggga agaatatgta gaaagtttac gtaaattaaa attaaacgtt 60

tattatctgg gagagaaaat agataatcca gtggataacc ctgtacttcg tccatcttta 120

aattctgtta agatgactta tgacttggcc caagaagcag aatatgagga tttaatgaca 180

acaacatcaa atataacagg tgaaaaaata aatagattta caaatttaca tcaaagcagt 240

gaagatttgg taaaaaaagt taaaatgcag agattgtgcg gacaaaaaac tgcagcctgt 300

ttccaaagat gcgttggtat ggactcattt aatgcagtgt acagtactac ttttgaaata 360

gataaggaat ataatacaaa ttattttgaa aatttcaaga aatttttaac ttatgttcaa 420

aagaacgatt taacagtaga tggagctatg acagatccaa aaggagacag aggattgtca 480

ccaagcaaac aggctgatcc agatttatat ttaagagttg tagaaagaag agaagacggt 540

gtagttgtaa gaggagcaaa ggctcaccag acaggtatat gtaattctca tgaagtattg 600

gttatgccaa ctattgctat gagaccggat gataaagatt atgcaatagc tttttctgtt 660

cctacagatg cagaaggaat aactatgata attggaagac agtcctgtga tactagaaaa 720

atggaaaaag atgcggacat agatgttggt aataaagaat ttggcggagt agaagcatta 780

gtagtatttg acgatgtatt tgttccaaat gacaggatat tcttaaatgg tgaaactgaa 840

tatgcaggaa tgttagtaga gagatttgca ggatatcata gacaaagtta tggtggatgc 900

aaagtaggag tgggagatgt attgataggc gctgctgcag tggctgcaga ctataatgga 960

gctgcaaagg catctcacat aaaggataaa ttaatagaaa tgatgcattt aaatgaaact 1020

ctttacgctt gcggaattgc gtgctcagca gaaggacatg caacaaaagc tggaaattac 1080

caaatagact tgcttcttgc aaatgtatgt aagcaaaata taacaagatt cccttatgaa 1140

attgtgagat tagcagaaga tatagcagga ggattaatgg ttactatgcc ttctgaaaag 1200

gattataaga gtccagaggt tggaaaatat gtagagaagt acttggtggg agttgcatcc 1260

gtacctgttg aagacagaat gaagatatta agattattag aaaatatatg tcttggaacg 1320

gctgcagtag gatatagaac tgaatccatg catggagcag gttcacctca agcacagaga 1380

ataatgatat caagacaggg aaacctagca cataagaaga aacttgcaaa gaagatagct 1440

agaatagaag attaa 1455

<210> 3

<211> 430

<212> PRT

<213> 4-hydroxybutyryl-CoA dehydratase (abfD)

<400> 3

Met Leu Met Thr Ala Glu Gln Tyr Ile Glu Ser Leu Arg Lys Leu Asn

1 5 10 15

Thr Arg Val Tyr Met Phe Gly Glu Lys Ile Glu Asn Trp Val Asp His

20 25 30

Pro Met Ile Arg Pro Ser Ile Asn Cys Val Ala Met Thr Tyr Glu Leu

35 40 45

Ala Gln Asp Pro Gln Tyr Ala Asp Leu Met Thr Thr Lys Ser Asn Leu

50 55 60

Ile Gly Lys Thr Ile Asn Arg Phe Ala Asn Leu His Gln Ser Thr Asp

65 70 75 80

Asp Leu Arg Lys Lys Val Lys Met Gln Arg Leu Leu Gly Gln Lys Thr

85 90 95

Ala Ser Cys Phe Gln Arg Cys Val Gly Met Asp Ala Phe Asn Ala Val

100 105 110

Phe Ser Thr Thr Tyr Glu Ile Asp Gln Lys Tyr Gly Thr Asn Tyr His

115 120 125

Lys Asn Phe Thr Glu Tyr Leu Lys Tyr Ile Gln Glu Asn Asp Leu Ile

130 135 140

Val Asp Gly Ala Met Thr Asp Pro Lys Gly Asp Arg Gly Leu Ala Pro

145 150 155 160

Ser Ala Gln Lys Asp Pro Asp Leu Phe Leu Arg Ile Val Glu Lys Arg

165 170 175

Glu Asp Gly Ile Glu Glu Gly Ala Asp Ile Asp Leu Gly Asn Lys Gln

180 185 190

Phe Gly Gly Gln Glu Ala Leu Val Val Phe Asp Asn Val Phe Ile Pro

195 200 205

Asn Asp Arg Ile Phe Leu Cys Gln Glu Tyr Asp Phe Ala Gly Met Met

210 215 220

Val Glu Arg Phe Ala Gly Tyr His Arg Gln Ser Tyr Gly Gly Cys Lys

225 230 235 240

Val Gly Val Gly Asp Val Val Ile Gly Ala Ala Ala Leu Ala Ala Asp

245 250 255

Tyr Asn Gly Ala Gln Lys Ala Ser His Val Lys Asp Lys Leu Ile Glu

260 265 270

Met Thr His Leu Asn Glu Thr Leu Tyr Cys Cys Gly Ile Ala Cys Ser

275 280 285

Ala Glu Gly Tyr Pro Thr Ala Ala Gly Asn Tyr Gln Ile Asp Leu Leu

290 295 300

Leu Ala Asn Val Cys Lys Gln Asn Ile Thr Arg Phe Pro Tyr Glu Ile

305 310 315 320

Val Arg Leu Ala Glu Asp Ile Ala Gly Gly Leu Met Val Thr Met Pro

325 330 335

Ser Glu Ala Asp Phe Lys Ser Glu Thr Val Val Gly Arg Asp Gly Glu

340 345 350

Thr Ile Gly Asp Phe Cys Asn Lys Phe Phe Ala Ala Ala Pro Thr Cys

355 360 365

Thr Thr Glu Glu Arg Met Arg Val Leu Arg Phe Leu Glu Asn Ile Cys

370 375 380

Leu Gly Ala Ser Ala Val Gly Tyr Arg Thr Glu Ser Met His Gly Ala

385 390 395 400

Gly Ser Pro Gln Ala Gln Arg Ile Met Ile Ala Arg Gln Gly Asn Ile

405 410 415

Asn Ala Lys Lys Glu Leu Ala Lys Ala Ile Ala Gly Ile Lys

420 425 430

<210> 4

<211> 1455

<212> DNA

<213> 4-hydroxybutyryl-CoA dehydratase (lf65-02412)

<400> 4

atgccattaa aaacaaagga acaatatatt gaaagtctta ggaagttaaa cctaaaagtg 60

tacatgtttg gaaaaccagt tgacaatgtt gttgataatc caatcataag accatctctt 120

aattcggttg ctatgactta tgaattagca caaatgccag agcatgaaga tttaatgaca 180

gctacttcta atttaacagg ggaaaaagta aatagatttg ctcatttaca tcaaagcaca 240

gatgatctta ttaaaaaagt aaagatgcaa agattacttg gtcaaaaaac cgcatcatgt 300

ttccaaaggt gcgttggtat ggatgctttt aatgcactat acagttcaac atacgaaata 360

gataaagcgt gtggaacaaa ttatcatgaa aactttaata agtttttaaa atatgttcaa 420

gaaaacgact taaccgttga tggtgcaatg actgatccta agggggacag aggattatcg 480

ccaagtaaac aagctgatgg agatttatac ttaagggttg ttgaaagaag agaagatggg 540

gtagttgtta gaggagcaaa atgtcaccaa actggaatgt taaattctca tgaggtagta 600

gtaatgccaa caatagcatt aactccaaat gataaggatt gggcaatatc ttttgcagtg 660

cctacagatg ctgaaggaat aattcatata tacggaaggc aatcttgtga tacaagaaaa 720

ctagaaccag gtgcagatat agatcttgga aacgctgaat ttggtggaca agaaacatta 780

acaatatttg ataatgtatt tgtaccaaat gaaagaattt tcttaaatgg agagactgat 840

tttgctggaa tgatcgttga aagatttgca ggatatcata gacaaagtta tggtggatgt 900

aaagttggag ttggagatgt tctaattgga gctgctgcag tagctgctga ttataacgga 960

gctcacaaag catctcatgt taaagataaa ttaatagaaa tgactcactt aaatgaaact 1020

ttattctctt gtggtattgc atgttcagca atgggttcta aaacagaagc tggaaattat 1080

tatattgata atttgctagc aaatgtatgt aagcaaaatg ttacaagatt cccttatgaa 1140

atatgtagac ttgcagaaga tattgcagga ggaatcatgg ttactatgcc atcagaagct 1200

gactttaaag atgaaaagat aggaccatat atagataaat atttgagagg tgtaaactca 1260

gtttcaacag aaaatagaat gagaatatta agattaatag aaaatatttg tttaggaact 1320

gcagctgtcg gatatagaac agaatcaatg catggagcag gatcaccaca agctcaaaga 1380

attatgatag ctagacaagg aaatttagca gctaaaaaga aaatagctaa gaaaatagct 1440

agaattgaag aataa 1455

<210> 5

<211> 1455

<212> DNA

<213> 4-hydroxybutyryl-CoA dehydratase (ctk-c07640)

<400> 5

atggctttaa tgacaggaga acaatatgta gaaagtatac gtaaattaaa tttaaatatt 60

tatatgttgg gagataaggt agacagtccg gtggacaatc ctatacttcg tccatcattg 120

aattctgtaa agatgactta tgaattggct cagcaacctg agtatgagga tttaatgaca 180

acgacttcaa acttaacagg taaaaaaata aatagattta caaatttaca tcagaattca 240

gaggatctag ttaaaaaagt aaagatgcag agattactag gacaaaaaac agctgcctgt 300

tttcaaagat gtgttggaat ggatgctttt aatgcagttt atagtactac ttatgaagtg 360

gacaaggaat ataaaacaaa ttattttgaa aatttcaaga agtttttagc atacgtacag 420

gacaatgatt taactgtaga tggagctatg actgatccga aaggtgacag aggactttca 480

ccaagtaaac aggctgatcc tgatttatac ctgagagtag tagagagaag gcctgatgga 540

attgttgtaa gaggtgccaa agctcatcaa acaggcattt gtaattctca tgaggtactt 600

gtaatgccaa cagtagccat gagaccagaa gataaagact atgctgtagc attttcagta 660

cctacagata caaagggaat aactatgata attggcagac aatcctgtga cactagaaag 720

tctgaaaaaa atgcagacat agatgttgga aacaaggtat ttggtggagt agaggcttta 780

gttgtatttg atgatgtatt tgtaccaaat gacaggatat ttttaaatgg tgaaagtgaa 840

tatgcaggga tgcttgtaga aaggtttgca ggatatcata gacagagtta tggtggatgc 900

aaagttggtg taggtgatgt cttgatagga gctgctgcat tggctgcaga ttataatgga 960

gcagcaagag catctcatat aaaggataaa ctaatagaaa tgacacattt aaatgaaact 1020

ctatatgcat gtggaattgc atgttctgca gaagggcatc caactaaggc aggaaactat 1080

gaaataaatt tacttcttgc aaatgtatgt aaacaaaatg taactagatt tccatatgaa 1140

atagtaagat tggcagaaga tatagcaggt ggattaatgg ttactcttcc atctgaaaaa 1200

gactataaca atcctgaaac taaaggatat atagagaagt atttagtagg agttaattct 1260

gtatctacag aaaatagaat gagaatattg agattaatag aaaacttaag tcttggaact 1320

gcagcagtag gctacagaac cgaatcaatg cacggagcag gttcaccaca agcccagaga 1380

ataatgatat caagacaggg taatcttcct gctaagaaga aattggcaaa agctatagct 1440

agaatagaag aatag 1455

<210> 6

<211> 1011

<212> DNA

<213> Artificial sequence (artificial)

<400> 6

atgaataaag cagattacaa gggcgtatgg gtgtttgctg aacaaagaga cggagaatta 60

caaaaggtat cattggaatt attaggtaaa ggtaaggaaa tggctgagaa attaggcgtt 120

gaattaacag ctgttttact tggacataat actgaaaaaa tgtcaaagga tttattatct 180

catggagcag ataaggtttt agcagcagat aatgaacttt tagcacattt ttcaacagat 240

ggatatgcta aagttatatg tgatttagtt aatgaaagaa agccagaaat attattcata 300

ggagctactt tcataggaag agatttagga ccaagaatag cagcaagact ttctactggt 360

ttaactgctg attgtacatc acttgacata gatgtagaaa atagagattt attggctaca 420

agaccagcgt ttggtggaaa tttgatagct acaatagttt gttcagacca cagaccacaa 480

atggctacag taagacctgg tgtgtttgaa aaattacctg ttaatgatgc aaatgtttct 540

gatgataaaa tagaaaaagt tgcaattaaa ttaacagcat cagacataag aacaaaagtt 600

tcaaaagttg ttaagcttgc taaagatatt gcagatatcg gagaagctaa ggtattagtt 660

gctggtggta gaggagttgg aagcaaagaa aactttgaaa aacttgaaga gttagcaagt 720

ttacttggtg gaacaatagc cgcttcaaga gcagcaatag aaaaagaatg ggttgataag 780

gaccttcaag taggtcaaac tggtaaaact gtaagaccaa ctctttatat tgcatgtggt 840

atatcaggag ctatccagca tttagcaggt atgcaagatt cagattacat aattgctata 900

aataaagatg tagaagcccc aataatgaag gtagcagatt tggctatagt tggtgatgta 960

aataaagttg taccagaatt aatagctcaa gttaaagctg ctaataatta a 1011

<210> 7

<211> 4040

<212> DNA

<213> vector plasmid (PSY8)

<400> 7

ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 60

ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 120

caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tgtttatgtt 180

acagtaatat tgacttttaa aaaaggattg attctaatga agaaagcaga caagtaagcc 240

tcctaaattc actttagata aaaatttagg aggcatatca aatgaacttt aataaaattg 300

atttagacaa ttggaagaga aaagagatat ttaatcatta tttgaaccaa caaacgactt 360

ttagtataac cacagaaatt gatattagtg ttttataccg aaacataaaa caagaaggat 420

ataaatttta ccctgcattt attttcttag tgacaagggt gataaactca aatacagctt 480

ttagaactgg ttacaatagc gacggagagt taggttattg ggataagtta gagccacttt 540

atacaatttt tgatggtgta tctaaaacat tctctggtat ttggactcct gtaaagaatg 600

acttcaaaga gttttatgat ttataccttt ctgatgtaga gaaatataat ggttcgggga 660

aattgtttcc caaaacacct atacctgaaa atgctttttc tctttctatt attccatgga 720

cttcatttac tgggtttaac ttaaatatca ataataatag taattacctt ctacccatta 780

ttacagcagg aaaattcatt aataaaggta attcaatata tttaccgcta tctttacagg 840

tacatcattc tgtttgtgat ggttatcatg caggattgtt tatgaactct attcaggaat 900

tgtcagatag gcctaatgac tggcttttat aaatcgatta tgtcttttgc gcattcactt 960

cttttctata taaatatgag cgaagcgaat aagcgtcgga aaagcagcaa aaagtttcct 1020

ttttgctgtt ggagcatggg ggttcagggg gtgcagtatc tgacgtcaat gccgagcgaa 1080

agcgagccga agggtagcat ttacgttaga taaccccctg atatgctccg acgctttata 1140

tagaaaagaa gattcaacta ggtaaaatct taatataggt tgagatgata aggtttataa 1200

ggaatttgtt tgttctaatt tttcactcat tttgttctaa tttcttttaa caaatgttct 1260

ttttttttta gaacagttat gatatagtta gaatagttta aaataaggag tgagaaaaag 1320

atgaaagaaa gatatggaac agtctataaa ggctctcaga ggctcataga cgaagaaagt 1380

ggagaagtca tagaggtaga caagttatac cgtaaacaaa cgtctggtaa cttcgtaaag 1440

gcatatatag tgcaattaat aagtatgtta gatatgattg gcggaaaaaa acttaaaatc 1500

gttaactata tcctagataa tgtccactta agtaacaata caatgatagc tacaacaaga 1560

gaaatagcaa aagctacagg aacaagtcta caaacagtaa taacaacact taaaatctta 1620

gaagaaggaa atattataaa aagaaaaact ggagtattaa tgttaaaccc tgaactacta 1680

atgagaggcg acgaccaaaa acaaaaatac ctcttactcg aatttgggaa ctttgagcaa 1740

gaggcaaatg aaatagattg acctcccaat aacaccacgt agttattggg aggtcaatct 1800

atgaaatgcg attaagcttg gctgcaggtc gacggatccc cgggaattct ataaaatata 1860

aataattttc taaaaaactt aacttcatgt gaaaagtttg ttaaaatata aatgagcacg 1920

ttaatcattt aacatagata attaaatagt aaaagggagt gtcgacatat ggtgcactct 1980

cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc 2040

tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 2100

ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa 2160

gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac 2220

gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat 2280

acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 2340

aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 2400

attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 2460

tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 2520

gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg 2580

cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 2640

tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 2700

agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 2760

tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 2820

tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 2880

tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact 2940

acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 3000

accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 3060

tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 3120

cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 3180

tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat 3240

actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 3300

tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 3360

cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 3420

gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 3480

tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt 3540

gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 3600

gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 3660

ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 3720

acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 3780

agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 3840

cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 3900

tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 3960

gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 4020

ttttgctcac atgttctttc 4040

<210> 8

<211> 309

<212> DNA

<213> intron (DNA)

<400> 8

ttatccttaa ttgccgatat cgtgcgccca gatagggtgt taagtcaagt agtttaaggt 60

actactctgt aagataacac agaaaacagc caacctaacc gaaaagcgaa agctgatacg 120

ggaacagagc acggttggaa agcgatgagt tacctaaaga caatcgggta cgactgagtc 180

gcaatgttaa tcagatataa ggtataagtt gtgtttactg aacgcaagtt tctaatttcg 240

gttgcaatcc gatagaggaa agtgtctgaa acctctagta caaagaaagg taagttaccg 300

atatcgact 309

<210> 9

<211> 8925

<212> DNA

<213> knock-out plasmid (PMTL007C-E2)

<400> 9

cctgcagggt gtagtagcct gtgaaataag taaggaaaaa aaagaagtaa gtgttatata 60

tgatgattat tttgtagatg tagataggat aatagaatcc atagaaaata taggttatac 120

agttatataa aaattacttt aaaaattaat aaaaacatgg taaaatataa atcgtataaa 180

gttgtgtaat ttttaagctt gagctcataa caatttcaca caggaaacag ctatgaccat 240

gattacggat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac 300

ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc 360

ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gctaataaag 420

atcttgtaca atctgtagga gaacctatgg gaacgaaacg aaagcgatgc cgagaatctg 480

aatttaccaa gacttaacac taactgggga taccctaaac aagaatgcct aatagaaagg 540

aggaaaaagg ctatagcact agagcttgaa aatcttgcaa gggtacggag tactcgtagt 600

agtctgagaa gggtaacgcc ctttacatgg caaaggggta cagttattgt gtactaaaat 660

taaaaattga ttagggagga aaacctcaaa atgaaaccaa caatggcaat tttagaaaga 720

atcagtaaaa attcacaaga aaatatagac gaagttttta caagacttta tcgttatctt 780

ttacgtccag atatttatta cgtggcgacg cgtgaagttc ctatactttc tagagaatag 840

gaacttcgcg actcatagaa ttatttcctc ccgttaaata atagataact attaaaaata 900

gacaatactt gctcataagt aacggtactt aaattgttta ctttggcgtg tttcattgct 960

tgatgaaact gatttttagt aaacagttga cgatattctc gattgaccca ttttgaaaca 1020

aagtacgtat atagcttcca atatttatct ggaacatctg tggtatggcg ggtaagtttt 1080

attaagacac tgtttacttt tggtttagga tgaaagcatt ccgctggcag cttaagcaat 1140

tgctgaatcg agacttgagt gtgcaagagc aaccctagtg ttcggtgaat atccaaggta 1200

cgcttgtaga atccttcttc aacaatcaga tagatgtcag acgcatggct ttcaaaaacc 1260

acttttttaa taatttgtgt gcttaaatgg taaggaatac tcccaacaat tttatacctc 1320

tgtttgttag ggaattgaaa ctgtagaata tcttggtgaa ttaaagtgac acgagtattc 1380

agttttaatt tttctgacga taagttgaat agatgactgt ctaattcaat agacgttacc 1440

tgtttactta ttttagccag tttcgtcgtt aaatgccctt tacctgttcc aatttcgtaa 1500

acggtatcgg tttcttttaa attcaattgt tttattattt ggttgagtac tttttcactc 1560

gttaaaaagt tttgagaata ttttatattt ttgttcatac cagcaccaga agcaccagca 1620

tctcttgggt taattgaggc ctgagtataa ggtgacttat acttgtaatc tatctaaacg 1680

gggaacctct ctagtagaca atcccgtgct aaattgtagg actgcccttt aataaatact 1740

tctatattta aagaggtatt tatgaaaagc ggaatttatc agattaaaaa tactttctct 1800

agagaaaatt tcgtctggat tagttactta tcgtgtaaaa tctgataaat ggaattggtt 1860

ctacataaat gcctaacgac tatccctttg gggagtaggg tcaagtgact cgaaacgata 1920

gacaacttgc tttaacaagt tggagatata gtctgctctg catggtgaca tgcagctgga 1980

tataattccg gggtaagatt aacgacctta tctgaacata atgccatatg aatccctcct 2040

aatttatacg ttttctctaa caacttaatt atacccacta ttattatttt tatcaatata 2100

gaagttccta tactttctag agaataggaa cttcacgcgt tgggaaatgg caatgatagc 2160

gaaacaacgt aaaactcttg ttgtatgctt tcattgtcat cgtcacgtga ttcataaaca 2220

caagtgaatg tcgacagtga atttttacga acgaacaata acagagccgt atactccgag 2280

aggggtacgt acggttcccg aagagggtgg tgcaaaccag tcacagtaat gtgaacaagg 2340

cggtacctcc ctacttcacc atatcatttt ctgcagcccc ctagaaataa ttttgtttaa 2400

ctttaagaag gagatataca tatatggcta gatcgtccat tccgacagca tcgccagtca 2460

ctatggcgtg ctgctagcgc tatatgcgtt gatgcaattt ctatgcactc gtagtagtct 2520

gagaagggta acgcccttta catggcaaag gggtacagtt attgtgtact aaaattaaaa 2580

attgattagg gaggaaaacc tcaaaatgaa accaacaatg gcaattttag aaagaatcag 2640

taaaaattca caagaaaata tagacgaagt ttttacaaga ctttatcgtt atcttttacg 2700

tccagatatt tattacgtgg cgtatcaaaa tttatattcc aataaaggag cttccacaaa 2760

aggaatatta gatgatacag cggatggctt tagtgaagaa aaaataaaaa agattattca 2820

atctttaaaa gacggaactt actatcctca acctgtacga agaatgtata ttgcaaaaaa 2880

gaattctaaa aagatgagac ctttaggaat tccaactttc acagataaat tgatccaaga 2940

agctgtgaga ataattcttg aatctatcta tgaaccggta ttcgaagatg tgtctcacgg 3000

ttttagacct caacgaagct gtcacacagc tttgaaaaca atcaaaagag agtttggcgg 3060

cgcaagatgg tttgtggagg gagatataaa aggctgcttc gataatatag accacgttac 3120

actcattgga ctcatcaatc ttaaaatcaa agatatgaaa atgagccaat tgatttataa 3180

atttctaaaa gcaggttatc tggaaaactg gcagtatcac aaaacttaca gcggaacacc 3240

tcaaggtgga attctatctc ctcttttggc caacatctat cttcatgaat tggataagtt 3300

tgttttacaa ctcaaaatga agtttgaccg agaaagtcca gaaagaataa cacctgaata 3360

tcgggagctc cacaatgaga taaaaagaat ttctcaccgt ctcaagaagt tggagggtga 3420

agaaaaagct aaagttcttt tagaatatca agaaaaacgt aaaagattac ccacactccc 3480

ctgtacctca cagacaaata aagtattgaa atacgtccgg tatgcggacg acttcattat 3540

ctctgttaaa ggaagcaaag aggactgtca atggataaaa gaacaattaa aactttttat 3600

tcataacaag ctaaaaatgg aattgagtga agaaaaaaca ctcatcacac atagcagtca 3660

acccgctcgt tttctgggat atgatatacg agtaaggaga tctggaacga taaaacgatc 3720

tggtaaagtc aaaaagagaa cactcaatgg gagtgtagaa ctccttattc ctcttcaaga 3780

caaaattcgt caatttattt ttgacaagaa aatagctatc caaaagaaag atagctcatg 3840

gtttccagtt cacaggaaat atcttattcg ttcaacagac ttagaaatca tcacaattta 3900

taattctgaa ctccgcggga tttgtaatta ctacggtcta gcaagtaatt ttaaccagct 3960

caattatttt gcttatctta tggaatacag ctgtctaaaa acgatagcct ccaaacataa 4020

gggaacactt tcaaaaacca tttccatgtt taaagatgga agtggttcgt gggggatccc 4080

gtatgagata aagcaaggta agcagcgccg ttattttgca aattttagtg aatgtaaatc 4140

cccttatcaa tttacggatg agataagtca agctcctgta ttgtatggct atgcccggaa 4200

tactcttgaa aacaggttaa aagctaaatg ttgtgaatta tgtgggacgt ctgatgaaaa 4260

tacttcctat gaaattcacc atgtcaataa ggtcaaaaat cttaaaggca aagaaaaatg 4320

ggaaatggca atgatagcga aacaacgtaa aactcttgtt gtatgctttc attgtcatcg 4380

tcacgtgatt cataaacaca agtgaatgtc gagcacccgt tctcggagca ctgtccgacc 4440

gctttggccg ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga 4500

tcatggcgac cacacccgtc ctgtggatcg ccaagccgcc gatggtagtg tggggtctcc 4560

ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact 4620

gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc 4680

cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc 4740

cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt 4800

ttctacaaac tcttcctgtc gtcatatcta caagccatcc ccccacagat acgggcgcgc 4860

cgccattatt tttttgaaca attgacaatt catttcttat tttttattaa gtgatagtca 4920

aaaggcataa cagtgctgaa tagaaagaaa tttacagaaa agaaaattat agaatttagt 4980

atgattaatt atactcattt atgaatgttt aattgaatac aaaaaaaaat acttgttatg 5040

tattcaatta cgggttaaaa tatagacaag ttgaaaaatt taataaaaaa ataagtcctc 5100

agctcttata tattaagcta ccaacttagt atataagcca aaacttaaat gtgctaccaa 5160

cacatcaagc cgttagagaa ctctatctat agcaatattt caaatgtacc gacatacaag 5220

agaaacatta actatatata ttcaatttat gagattatct taacagatat aaatgtaaat 5280

tgcaataagt aagatttaga agtttatagc ctttgtgtat tggaagcagt acgcaaaggc 5340

ttttttattt gataaaaatt agaagtatat ttattttttc ataattaatt tatgaaaatg 5400

aaagggggtg agcaaagtga cagaggaaag cagtatctta tcaaataaca aggtattagc 5460

aatatcatta ttgactttag cagtaaacat tatgactttt atagtgcttg tagctaagta 5520

gtacgaaagg gggagcttta aaaagctcct tggaatacat agaattcata aattaattta 5580

tgaaaagaag ggcgtatatg aaaacttgta aaaattgcaa agagtttatt aaagatactg 5640

aaatatgcaa aatacattcg ttgatgattc atgataaaac agtagcaacc tattgcagta 5700

aatacaatga gtcaagatgt ttacataaag ggaaagtcca atgtattaat tgttcaaaga 5760

tgaaccgata tggatggtgt gccataaaaa tgagatgttt tacagaggaa gaacagaaaa 5820

aagaacgtac atgcattaaa tattatgcaa ggagctttaa aaaagctcat gtaaagaaga 5880

gtaaaaagaa aaaataattt atttattaat ttaatattga gagtgccgac acagtatgca 5940

ctaaaaaata tatctgtggt gtagtgagcc gatacaaaag gatagtcact cgcattttca 6000

taatacatct tatgttatga ttatgtgtcg gtgggacttc acgacgaaaa cccacaataa 6060

aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac taaacaatca agctaggata 6120

tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt aatacatacg ctattaagat 6180

gtaaaaatac ggataccaat gaagggaaaa gtataatttt tggatgtagt ttgtttgttc 6240

atctatgggc aaactacgtc caaagccgtt tccaaatctg ctaaaaagta tatcctttct 6300

aaaatcaaag tcaagtatga aatcataaat aaagtttaat tttgaagtta ttatgatatt 6360

atgtttttct attaaaataa attaagtata tagaatagtt taataatagt atatacttaa 6420

tgtgataagt gtctgacagt gtcacagaaa ggatgattgt tatggattat aagcggccgg 6480

ccagtgggca agttgaaaaa ttcacaaaaa tgtggtataa tatctttgtt cattagagcg 6540

ataaacttga atttgagagg gaacttagat ggtatttgaa aaaattgata aaaatagttg 6600

gaacagaaaa gagtattttg accactactt tgcaagtgta ccttgtacct acagcatgac 6660

cgttaaagtg gatatcacac aaataaagga aaagggaatg aaactatatc ctgcaatgct 6720

ttattatatt gcaatgattg taaaccgcca ttcagagttt aggacggcaa tcaatcaaga 6780

tggtgaattg gggatatatg atgagatgat accaagctat acaatatttc acaatgatac 6840

tgaaacattt tccagccttt ggactgagtg taagtctgac tttaaatcat ttttagcaga 6900

ttatgaaagt gatacgcaac ggtatggaaa caatcataga atggaaggaa agccaaatgc 6960

tccggaaaac atttttaatg tatctatgat accgtggtca accttcgatg gctttaatct 7020

gaatttgcag aaaggatatg attatttgat tcctattttt actatgggga aatattataa 7080

agaagataac aaaattatac ttcctttggc aattcaagtt catcacgcag tatgtgacgg 7140

atttcacatt tgccgttttg taaacgaatt gcaggaattg ataaatagtt aacttcaggt 7200

ttgtctgtaa ctaaaaacaa gtatttaagc aaaaacatcg tagaaatacg gtgttttttg 7260

ttaccctaag tttaaactcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt 7320

ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 7380

ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 7440

tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 7500

gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt 7560

agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 7620

taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 7680

gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 7740

gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 7800

caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 7860

aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 7920

tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 7980

acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga 8040

ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac 8100

gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcagggcccc 8160

ctgcttcggg gtcattatag cgattttttc ggtatatcca tcctttttcg cacgatatac 8220

aggattttgc caaagggttc gtgtagactt tccttggtgt atccaacggc gtcagccggg 8280

caggataggt gaagtaggcc cacccgcgag cgggtgttcc ttcttcactg tcccttattc 8340

gcacctggcg gtgctcaacg ggaatcctgc tctgcgaggc tggccggcta ccgccggcgt 8400

aacagatgag ggcaagcgga tggctgatga aaccaagcca accaggaagg gcagcccacc 8460

tatcaaggtg tactgccttc cagacgaacg aagagcgatt gaggaaaagg cggcggcggc 8520

cggcatgagc ctgtcggcct acctgctggc cgtcggccag ggctacaaaa tcacgggcgt 8580

cgtggactat gagcacgtcc gcgagctggc ccgcatcaat ggcgacctgg gccgcctggg 8640

cggcctgctg aaactctggc tcaccgacga cccgcgcacg gcgcggttcg gtgatgccac 8700

gatcctcgcc ctgctggcga agatcgaaga gaagcaggac gagcttggca aggtcatgat 8760

gggcgtggtc cgcccgaggg cagagccatg acttttttag ccgctaaaac ggccgggggg 8820

tgcgcgtgat tgccaagcac gtccccatgc gctccatcaa gaagagcgac ttcgcggagc 8880

tggtgaagta catcaccgac gagcaaggca agaccgatcg ggccc 8925

<210> 10

<211> 19

<212> DNA

<213> upstream primer (etfA-F)

<400> 10

acagaccaca aatggctac 19

<210> 11

<211> 20

<212> DNA

<213> downstream primer (etfA-R)

<400> 11

tgaatcttgc atacctgcta 20

Claims (8)

1. The recombinant clostridium butyricum for producing 1, 4-butanediol is characterized in that the recombinant clostridium butyricum is constructed by expressing 4-hydroxybutyryl coenzyme A dehydratase in clostridium butyricum;

wherein, the clostridium butyricum is ATCC 824;

wherein the expressed 4-hydroxybutyryl-CoA dehydratase is expressed by any one gene segment of cbei _2100, ckl _3020, abfD, lf65-02412 and ctk-c 07640; wherein the nucleotide sequences of the cbei _2100, ckl _3020, abfD, lf65-02412 and ctk-c07640 gene segments are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.

2. The method of constructing recombinant clostridium butyricum producing 1, 4-butanediol of claim 1, comprising the steps of:

(1) inserting any one gene fragment of cbei _2100, ckl _3020, abfD, lf65-02412 and ctk-c07640 into the enzyme cutting site of the vector plasmid to obtain a recombinant plasmid;

(2) methylating the recombinant plasmid obtained in the step (1) to obtain a methylated recombinant plasmid;

(3) transferring the methylated recombinant plasmid obtained in the step (2) into clostridium acetobutylicum, and screening to obtain the recombinant plasmid.

3. The construction method according to claim 2, wherein in step (1), the vector plasmid is PSY8, and the nucleotide sequence thereof is shown in SEQ ID NO. 7.

4. The recombinant clostridium butyricum for producing 1, 4-butanediol according to claim 1, wherein the etfA gene in the recombinant clostridium butyricum is inactivated, and the nucleotide sequence of the etfA gene in the obtained recombinant clostridium butyricum is shown as SEQ ID No. 6.

5. The method for constructing the recombinant clostridium butyricum for producing 1, 4-butanediol of claim 4, wherein the method for constructing the recombinant clostridium butyricum with inactivated etfA gene is to insert an intron sequence shown in SEQ ID No.8 between the 639bp and 640bp of the etfA gene by using a two-type intron gene knockout technology.

6. The method of claim 5, wherein the knockout technique for the type II intron gene comprises the steps of:

(1) constructing an intron sequence shown in SEQ ID NO.8 into a gene knockout plasmid to obtain a recombinant plasmid;

(2) and (2) converting the recombinant plasmid obtained in the step (1) into clostridium acetobutylicum, and screening to obtain the strain with the inactivated etfA gene.

7. The construction method according to claim 6, wherein in the step (1), the gene knockout plasmid is PMTL007C-E2, and the nucleotide sequence is shown as SEQ ID NO. 9.

8. Use of the recombinant clostridium butyricum of claim 1 for preparing 1, 4-butanediol.

Images