CN101519673B

CN101519673B - Method for improving ratio of butanol produced from clostridium

Info

Publication number: CN101519673B
Application number: CN 200810033954
Authority: CN
Inventors: 蒋宇; 赵蒙; 杨晟; 姜卫红; 何慧琪; 陈军; 杨蕴刘
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Priority date: 2008-02-27
Filing date: 2008-02-27
Publication date: 2013-02-06
Anticipated expiration: 2028-02-27
Also published as: CN101519673A

Abstract

The invention discloses a method for improving ratio of target product (butanol) in the fermentation products of clostridium acetobutylicum. The method inhibits the generation of acetone by effectively inhibiting the metabolic pathway from acetoacetyl-CoA to acetone in the clostridium acetobutylicum. The synthesis of acetone can be blocked by knocking out an acetoacetate decarboxylase gene so as to cause the acetoacetate decarboxylase to be inactive, thereby effectively improving the ratio of butanol in the fermentation products.

Description

A kind of method that improves ratio of butanol produced from clostridium

Technical field

The invention belongs to technical field of bioengineering, specifically, is the method that improves ratio of butanol produced from clostridium about a kind of.

Background technology

Butanols is the large basic material that serves many purposes, and in the chemical fields such as dyestuff, paint, plastics, resin, rubber, can be used as the synthetic precursor of multiple organic compound; It is requisite solvent in microbiotic and the synthetic drug production process; Also be the food grade extractant of food, perfume industry simultaneously.The more important thing is, butanols or a kind of octane value (motoroctane number) are higher than high-grade fuel and the fuel dope of ethanol.Studies show that, the vapour pressure of butanols and vaporization heat are respectively 0.63psi and 141.3kcal kg ^-1, be lower than 2.25psi and the 204.1kcal kg of ethanol ^-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 occur, 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.

Butanols can be by chemical method and biological process production.During the World War I at the beginning of the method for microbiological anaerobic fermentative production butanols equal solvent can be traced back to last century.Because clostridium acetobutylicum produces three kinds of solvent acetone, butanols and ethanol (Acetone-Butanol-Ethanol, ABE) during the fermentation, also claim " ABE fermentation " therefore produce solvent fermentation.Wherein, butanols and acetone are main tunnings, and its metabolism network as shown in Figure 1.

Because the solvent of clostridium acetobutylicum production is except 60-70% is butanols, also have simultaneously the acetone of 20-30% and these low-value byproducts of ethanol of 10%, therefore reducing the by product ratio and improving ratio of butanol is the effective means that improves the biological butanol economy.The first thinking is by making up allos butanols the way of production (table 1) in the Host Strains that does not produce butanols at itself.

Table 1, propyl carbinol fermentation genetic engineering bacterium

Host Strains	Butanols output	Molar yield %	Reference
				E.coli B.subtillus S.cerevisiae E coli E.coli	1.03mM 0.19mM 1mg/L 552mg/L 9.23mM	1.9 - - - -	Dupont patent WO2007041269，Page 67 Dupont patent WO2007041269，Page 75 Dupont patent WO2007041269，Page 90 Metabolic engineering，2007，doi： 10.1016/j.ymben.2007.08.003 Nature，2008，doi：10.1038/nature06450

As shown in table 1, front four kinds of engineered butanols metabolic engineerings all are the allos pathways metabolisms (being acetyl-CoA → acetoacetyl-CoA → beta-hydroxy-butanoic acid coenzyme A → crotonyl-CoA → butyryl coenzyme A → butyraldehyde → six step of butanols biocatalytic reaction) that make up in Host Strains from the acetyl-CoA to the butanols; And last a kind of gene engineering colibacillus is produced propyl carbinol by the ketone acid approach that makes up take L-threonine as initial substrate.Owing to itself not producing the bacterial strain of butanols, very limited to the tolerance of butanols toxicity such as Host Strains listed in the table 1, the metabolic engineering that therefore utilizes these bacterium to produce propyl carbinol is shouldered heavy responsibilities.

Another kind of thinking is that the clostridium acetobutylicum of natural product butanols is transformed, and reduces the by product acetone ratio to improve ratio of butanol, and the method for having reported mainly contains following several:

1) suppresses transcribing of thiophorase (Coenzyme Atransferase, CoAT) from acetoacetyl-CoA to the acetone pathways metabolism or E.C. 4.1.1.4 gene

Tummala utilizes Antisense RNA Technique to suppress the transcribing of E.C. 4.1.1.4 (acetoacetate decarboxylase, AADC) gene adc in the clostridium acetobutylicum in reporting in 2003, but acetone output does not reduce; Then, the identical method of utilizing Tummala suppresses (the coenzyme A transferase of thiophorase in this bacterium, CoAT) gene ctfAB's transcribes, although suppressed the generation of acetone, but because this method has unavoidably suppressed transcribing of butanols and ethanol route of synthesis key gene adhE simultaneously, almost without the generation (Tummala of ethanol and butanols, S.B., Welker, N.E., Papoutsakis, E.T., Journal of bacteriology 2003 185:1923-1934); Therefore cross expression ethanol dehydrogenase E (Alcohol dehydrogenase E on the basis of transcribing that suppresses ctfAB, ADHE), but only improved the output of ethanol, the output of butanols only returns to the wild mushroom level, the ratio of butanol (Tummala that descends on the contrary, S.B., Junne, S.G., Papoutsakis, E.T., Journal of bacteriology 2003 185:3644-3653).

As seen, suppress the thiophorase activity and can't improve ratio of butanol; And that acetone can not be thoroughly blocked in the genetic transcription that suppresses E.C. 4.1.1.4 is synthetic.Therefore, block the synthetic of acetone, improve ratio of butanol, must thoroughly suppress E.C. 4.1.1.4 active.This can realize by the means that this gene is knocked out.The method of carrying out gene knockout at clostridium acetobutylicum comprises: the 1) homologous recombination of non-replicating integrative vector; 2) homologous recombination of rf integrative vector; 3) based on the Targetron gene Knockout of prokaryotic organism two class introns.The first is used nonreplication vector, and what successfully knock out has a buk, pta, and aad reaches solR gene (Green, E.M., and Bennett, G.N.Appl Biochem Biotechnol1996.57-58:213-221; Green, E.M., Boynton, Z.L., Harris, L.M., Rudolph, F.B., Papoutsakis, E.T., and Bennett, G.N.Microbiology 1996.142 (Pt 8): 2079-2086; Nair, R.V., Green, E.M., Watson, D.E., Bennett, G.N., and Papoutsakis, E.T.Journal of bacteriology 1999 181:319-330.).Second method is to adopt the shuttle plasmid that can copy in clostridium acetobutylicum to make up to knock out plasmid, because have reproducible starting point, the method has increased the chance of homologous recombination.Harris has successfully knocked out spo0A (Harris, L.M., Welker, N.E., and Papoutsakis, E.T.Journal of bacteriology 2002.184:3586-3597) by the method.The third method is the novel method that does not rely on homologous recombination that latest developments are got up.The knockout carrier that utilizes the Targetron technology of having reported has pMAL007 (Heap, J.T., et al, J.Microbiol.Methods, 2007,70:452-464), pSY6 (Shao, L., et al., Cell Res., 2007, doi:10.1038/cr.2007.91).

2) traditional mutagenesis

Chinese invention patent according to patent No. ZL 95111733.5, by the mutagenic obtained mutant strain Clostridium acetobutylicum CCTCC M 94061 of traditional separation, the ratio of butanols brings up to 70% from original 60% in its fermented liquid, butanols: acetone: ethanol is 7: 2: 1, and butanols output reaches approximately 14.5g/L during with 8% corn dextrin batch fermentation.

In sum, by synthesizing of the by products such as blocking-up acetone, improve the ratio of target product butanols for transformation efficiency and the productive rate of further raising butanols, the production cost that reduces butanols is significant.

Summary of the invention

Purpose of the present invention just is the ratio of synthesizing to improve target product butanols in the product solvent clostridium tunning by blocking-up acetone.

The method of the ratio of butanols in the solvent clostridium tunning is produced in raising of the present invention, by suppressing by the pathways metabolism of acetoacetyl-CoA to acetone, blocks the synthetic of acetone with this, thereby realizes improving the ratio of butanols in the tunning.

Particularly, described inhibition is by the pathways metabolism of acetoacetyl-CoA to acetone, realize by the activity that suppresses thiophorase (Coenzyme A transferase, CoAT) and/or E.C. 4.1.1.4 (acetoactate decarboxylase, ADC).

More specifically, described inhibition E.C. 4.1.1.4 is active realizes by interrupting E.C. 4.1.1.4 encoding gene (adc gene).

More specifically, described interruption E.C. 4.1.1.4 encoding gene (adc gene) is realized by the Targetron gene Knockout.

On the other hand, the invention provides the recombinant bacterial strain of a kind of Clostridium acetobutylicum, E.C. 4.1.1.4 encoding gene in the genome of this bacterial strain (adc gene) is knocked.

On the other hand, the present invention also provides the application of a kind of E.C. 4.1.1.4 encoding gene recombinant bacterial strain that (adc gene) knocks out for the production of propyl carbinol.

Of the present invention passing through suppresses by the pathways metabolism of acetoacetyl-CoA to acetone, blocks the synthetic method of acetone with this, can successfully block the synthetic of acetone, thus the ratio of butanols in the Effective Raise tunning.

Description of drawings

Fig. 1 is the schematic diagram of the ABE metabolism network of clostridium acetobutylicum.

Fig. 2 is the pcr amplification product electrophorogram of adc-targetron fragment, and wherein, swimming lane 1 is DNA Marker; Swimming lane 2 is adc-targetron.

Fig. 3 is that Clostridium acetobutylicum ATCC 824adc knocks out transformant colony PCR amplification product electrophorogram, wherein, swimming lane ctr is the electrophoresis result take wild-type Clostridium acetobutylicum ATCC 824 genomes as the amplified production of template, swimming lane 1-20 is respectively take the Clostridium acetobutylicum ATCC 824 transformant 1-20 numbers electrophoresis result as the amplified production of template, and swimming lane M is 1kb DNA ladder.

Fig. 4 is 6 of Clostridium acetobutylicum ATCC 824, the electrophorogram of the Genomic PCR product of 8,11, No. 18 transformants, wherein, swimming lane 1 is the electrophoresis result take wild-type Clostridium acetobutylicum ATCC 824 genomes as template; 6,8,11, No. 18 transformant genomes that swimming lane 2-5 is respectively with Clostridium acetobutylicum ATCC 824 are the electrophoresis result of template, and swimming lane 6 is Marker.

Fig. 5 is the electrophorogram that Clostridium acetobutylicum CCTCC M 94061 adc knock out transformant bacterium colony PCR checking, wherein, swimming lane ctr is the electrophoresis result take Clostridium acetobutylicum CCTCC M 94061 genomes as the amplified production of template, swimming lane 1-19 is respectively 94061 transformant 1-19 numbers electrophoresis result as the amplified production of template take Clostridium acetobutylicum CCTCC M, and swimming lane M is 1kb DNA ladder.

Fig. 6 carries out electrophorogram behind the bacterium colony PCR to the single bacterium after the line of 4,8,9,14, No. 17 transformants separates among Fig. 4, wherein, swimming lane ctr is the electrophoresis result take Clostridium acetobutylicum CCTCC M 94061 genomes as the amplified production of template, and swimming lane 1-3 is the electrophoresis result after the single bacterium after No. 4 transformants are rule among Fig. 4 carries out bacterium colony PCR; Swimming lane 4-6 is the electrophoresis result after No. 8 single bacterium after the transformant line carry out bacterium colony PCR; It is for 7-9 number the electrophoresis result after single bacterium of No. 9 transformants carries out bacterium colony PCR; 10-12 is the electrophoresis result after single bacterium of No. 14 transformants carries out bacterium colony PCR; 13-16 is the electrophoresis result after single bacterium of No. 17 transformants carries out bacterium colony PCR.

Fig. 7 is the comparative result that Clostridium acetobutylicum wild mushroom and adc knock out the acetone output after the fermentation of mutant bacteria.

Fig. 8 be Clostridium acetobutylicum CCTCC M 94061 with and the comparison of the fermentation results of adc knockout mutant strain.

Embodiment

Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples are only for explanation the present invention but not for limiting scope of the present invention.

The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, condition described in " molecular cloning: laboratory manual " (New York:Cold Spring Harbor Laboratory Press, 1989), or the condition that production firm is recommended is carried out.

The recombinant plasmid vector pSY6-adc that mentions among the present invention refers to be used to the recombinant plasmid vector that knocks out the adc gene.

The adc-targetron fragment of mentioning among the present invention refers at IBS, EBS2, and EBS1d site base is after revising, be used for knocking out the fragment of adc gene, belong to a L1.LtrB intron part, described L1.LtrB two class introns are protokaryon two class introns, comprise the ltrA gene.

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

Plasmid pSY6, sequence is the shuttle plasmid of E.coli and C.acetobutylicum shown in SEQ ID NO:1, express erythromycin (Ery) resistant gene in C.acetobutylicum.

Plasmid pANS, sequence has spectinomycin (Spc) resistance shown in SEQ ID NO:2.

Plasmid pIMP1-ptb, sequence has the NdeI restriction enzyme site shown in SEQ ID NO:5, be the shuttle plasmid of E.coliacetobutylicum.

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

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

The toolenzyme that the present invention uses: KOD archaeal dna polymerase, restriction enzyme, Taq polysaccharase are available from Fermentas, and T4 ligase enzyme and calf alkaline phosphatase (CIAP) are all available from TaKaRa company.

The prescription of the CGM liquid nutrient medium that uses among the present invention is: 2g (NH ₄) ₂SO ₄, 1g K ₂HPO ₄, 0.5gKH ₂PO ₄, 0.1g MgSO ₄7H ₂O, 0.015g FeSO ₄7H ₂O, 0.01g CaCl ₂, 0.01g MnSO ₄H ₂O, 0.002g CoCl ₂, 0.002g ZnSO ₄, 2g Tryptone, 1g Yeast Extraction, 50g Glucose is dissolved in the 1L water.CGM solid culture based formulas is for adding the agar powder of 1.5% (w/v) in the respective liquid substratum.

The ETM buffer formulation is as follows: 270mM sucrose, 0.6mM Na ₂HPO ₄, 4.4mM NaH ₂PO ₄, 10mMMgCl ₂

The ET buffer formulation is as follows: 270mM sucrose, 0.6mM Na ₂HPO ₄, 4.4mM NaH ₂PO ₄

The P2 culture medium prescription is as follows: glucose 60g; K ₂HPO ₄0.5g; KH ₂PO ₄0.5g; CH ₃COONH ₄2.2g; MgSO ₄7H ₂O 0.2g; MnSO ₄H ₂O 0.01g; NaCl 0.01g; FeSO ₄7H ₂O 0.01g; Aminobenzoic acid (ρ-aminobenzoic acid) 1mg; VITMAIN B1 (thiamine) 1mg; Vitamin H (biotin) 0.01mg is dissolved in the 1L water.

Clostridium acetobutylicum adc-among the present invention refers to the Clostridiumacetobutylicum recombinant bacterial strain of adc gene knockout.

According to acetone, butanols, ethanol (ABE) pathways metabolism of producing the solvent clostridium, the present invention is devoted to block the synthetic of acetone by suppressing by the pathways metabolism of acetoacetyl-CoA to acetone with this, thereby improves the ratio of butanols in the tunning.In order to suppress by the pathways metabolism of acetoacetyl-CoA to acetone, the present invention manages the key enzyme E.C. 4.1.1.4 in this approach of inactivation, comes the inactivation E.C. 4.1.1.4 by interrupting the E.C. 4.1.1.4 encoding gene specifically.The Targetron gene Knockout interrupts the E.C. 4.1.1.4 encoding gene more specifically.

Plasmid pSY6 (the Shao L of the present invention by utilizing this laboratory to make up, Hu S., Yang Y., Chen J., Yang Y, Jiang W, Yang S, Cell research, 2007, doi:10.1038/cr.2007.91), at Clostridium acetobutylicumATCC 824, carry out gene knockout among the Clostridium acetobutylicum CCTCC M 94061.

The present invention passes through PCR, amplify the targetron segment of adc, with it behind XhoI and BsrG I double digestion, be connected with the pSY6 carrier of cutting through same enzyme, obtain knocking out plasmid pSY6-adc, electricity turns Clostridium acetobutylicumATCC 824 or Clostridium acetobutylicum CCTCC M 94061.Identified the recombinant bacterium of Intron insertion by bacterium colony PCR.Determine that through checking order adc has inserted L1.LtrB intron fragment.

Below be described in further detail.

Embodiment 1, make up the pSY6-adc plasmid vector

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

1.1, design of primers

With reference to Targetron ^TMThe method that Gene Knockout System (TA0100) Kit provides is designed for respectively primer adc-IBS, the adc-EBS1d and the adc-EBS2 that make up the pSY6-adc plasmid vector, and its sequence is as follows respectively:

adc-IBS：3’-aaaactcgagataattatccttattagtcaggtttgtgcgcccagatagggtg-5’；

adc-EBS1d：3’-cagattgtacaaatgtggtgataacagataagtcaggtttgataacttacctttctttgt-5’；

adc-EBS2：3’-tgaacgcaagtttctaatttcggttactaatcgatagaggaaagtgtct-5’。

Simultaneously, also use the universal primer EBS that is provided by TargetronTM Gene Knockout System (TA0100) Kit.

Wherein adc-IBS2 and universal primer EBS are positioned at intron inside, and adc-IBS and adc-EBS1d are positioned at intron upstream and downstream both sides.The theoretical insertion point of the intron of test kit prediction is between the 180th and 181 base of genome adc gene, and forward inserts.

1.2, adc-targetron fragment preparation

With reference to the method that manufacturer provides, its template that provides is provided, with adc-IBS, adc-EBS1d, adc-EBS2 and EBS are primer, use Targetron ^TMGene Knockout System (TA0100) Kit carries out pcr amplification, and the PCR product is carried out gel electrophoresis, and the result as shown in Figure 2, result according to Fig. 2, at the 350bp place band is arranged, consistent with expected result, illustrate through pcr amplification to obtain the adc-targetron fragment.

Then, use the glue of Hua Shun company to reclaim test kit purifying recovery PCR product, obtain the adc-targetron fragment.

1.3, the pSY6-adc recombinant plasmid vector makes up

The adc-targetron fragment of purifying is cut through XhoI and BsrGI enzyme, with the carrier pSY6 that cuts through XhoI and BsrGI enzyme equally, use the T4 ligase enzyme to connect, with the connection product Transformed E .coli DH5 α competent cell that obtains, and in the LB solid medium that contains acillin Amp (100mg/L), cultivate, screening, acquisition has the transformant of Amp resistance.

3 transformants of random choose check order, sequencing result shows, the transformant that obtains all contains just like the sequence shown in the SEQ ID NO:3, be the intron sequences of specific recognition adc gene insertion point, this result shows that the plasmid that obtains has inserted the adc-targetron fragment that contains adc-targetron fragment upstream and downstream primer adc-IBS and adc-EBS1d between restriction enzyme site XhoI and BsrGI.

According to above-mentioned detected result, connect the positive transformant that obtains behind the product Transformed E .coli DH5 α competent cell and contain recombinant plasmid pSY6-adc.

Embodiment 2, the knocking out of Clostridium acetobutylicum ATCC824 adc gene

With the pSY6-adc plasmid through E.coli ER2275/pANS after Cac8 I site methylates, electricity turns Clostridiumacetobutylicum ATCC 824, after recovery is spent the night, get 100 μ l enchylema and coat the selectivity flat board of 40 μ g/ml Ery, in the anaerobic box in 37 ℃ cultivate 48-96 hour after, choose single bacterium, adopt bacterium colony PCR checking, concrete steps are as follows:

2.1, the methylating of pSY6-adc plasmid

With pANS Transformed E .coli ER2275, obtain E.coli ER2275/pANS.

The competent cell of preparation E.coli ER2275/pANS, then with pSY6-adc Plasmid Transformation E.coliER2275/pANS, because the pANS plasmid has the Spc resistance, therefore containing 100 μ g/ml Amp, incubated overnight on the LB culture medium flat plate of 50 μ g/mlSpc, then choose single bacterium colony and be added with 100 μ g/ml Amp to 4ml, in the LB substratum of 50 μ g/mlSpc, incubated overnight.Then, with the E.coli ER2275 that contains pANS and pSY6-adc that obtains, with the extracting of plasmid extraction test kit, thereby obtain the methylated plasmid pSY6-adc of Cac824I recognition site.

2.2, electricity turns the pSY6-adc plasmid to Clostridium acetobutylicum ATCC 824

Clostridium acetobutylicum ATCC 824 is separated single bacterium in the line of CGM flat board, in anaerobic box, cultivated 48-96 hour for 37 ℃, choose single bacterium colony of Clostridium acetobutylicum ATCC 824 to CGM nutrient solution (2g (NH ₄) ₂SO ₄, 1g K ₂HPO ₄, 0.5g KH ₂PO ₄, 0.1g MgSO ₄7H ₂O, 0.015g FeSO ₄7H ₂O, 0.01gCaCl ₂, 0.01g MnSO ₄H 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; Then, be forwarded in the 100ml CGM substratum with 1% ratio, be cultured to OD in 37 ℃ ₆₀₀Be about 0.7, get 50ml bacterium liquid, 4000rpm, 4 ℃ of centrifugal 10min, supernatant discarded; Suspend again 4000rpm, 4 ℃ of centrifugal 10min, supernatant discarded with the ETM damping fluid; Then suspend with 1.0ml ET damping fluid; Get 100 μ l suspension, add 1-3 μ g pSY6-adc plasmid, behind the mixing, add (2mm) in the electric revolving cup, use MicroPulser ^TMThe electroporation electricity turns, and the electric shock condition is voltage 2.0kV, and all the other are with reference to service manual.After the electric shock, add again CGM substratum 1ml, cultivate about 10h for 37 ℃, get 200 μ l enchylema and coat the CGM flat board that contains erythromycin (40 μ g/ml), cultivated approximately 2-3 days, obtain C.acetobutylicum ATCC 824 transformants with Ery resistance.

2.3, make up the bacterium colony PCR primer that adc knocks out

Design pair of primers adc163-177 and adc276-259, its sequence is as follows:

adc163-177：3’-gagcccttagtcagg-5’；

adc276-259：3’-ataatctcccttaactcc-5’。

Because during the adc-targetron design of primers, expectation in karyomit(e) adc gene between the 180-181nt forward insert intron, thereby the primer of choosing checking is the adc180/181nt flanking sequence, if there is the insertion of intron gene inside, then at the corresponding swimming lane of bacterium colony PCR rear electrophoresis, can the band of amplification to about the 1kb, if wild-type is then without this band.Because the amplified band size of wild-type be about 100bp, be difficult for and primer dimer is differentiated in agarose electrophoresis, so after about the 400bp of adc180/181nt downstream, design again primer adc714-699, under its sequence shown in:

adc714-699：3’-aacttcagctctaggc-5’。

Utilize primer pair adc163-177 and adc714-699 to increase, the stripe size of wild-type is about 500bp, has the positive bacterium colony amplified band size of Intron insertion to be 1.5kb.

2.4, bacterium colony PCR checking

Take C.acetobutylicum ATCC 824 transformants as template, take adc163-177 and adc276-259 as primer, adopt the identical reaction system of embodiment 1, carry out pcr amplification.

PCR reaction conditions: 95 ℃ of 5min; 95 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 1.5m, 30 circulations; 72 ℃ of 2min.

Get above-mentioned PCR product, use agarose gel electrophoresis to detect, the result as shown in Figure 3.According to the result of Fig. 3, there are 6,8,11, No. 18 transformants of obvious band that the insertion of intron may be arranged in adc gene inside at the 1.5kb place.

2.5, other checking of knocking out of adc

According to Fig. 3 result, with 1.5kb place band clearly 6,8,11, No. 18 transformants be seeded to the CGM culture medium culturing, extract genomic dna.

The genomic dna that obtains take extracting is as template, take adc163-177 and adc714-699 as primer pair, condition by embodiment 2.4 increases, pcr amplification product is carried out electrophoresis detection, detected result as shown in Figure 4, according to the result of Fig. 4,11 and No. 18 transformant is mixed with the adc wild mushroom that inserts without intron, and the adc of No. 6 and No. 8 transformants has inserted intron.

The 1.5kb band of No. 6 and No. 8 transformants is reclaimed in repurity, and this band is checked order, and sequencing result is shown in SEQID NO:4.

Sequencing result shows, the adc gene that Clostridium acetobutylicum ATCC 824 adc knock out mutant strain has inserted the intron fragment at 180/181nt site forward, therefore so that the adc gene inactivation, thereby successfully knock out the adc gene of ClostridiumacetobutylicumATCC824, i.e. No. 6 and No. 8 bacterial strain corresponding to the transformant Clostridium acetobutylicum ATCC824 recombinant bacterial strain that is the adc gene knockout.

Embodiment 3, the knocking out of Clostridium acetobutylicum CCTCC M 94061 adc genes

Adopt the method identical with embodiment 2 with the methylated plasmid pSY6-adc of Cac824 I recognition site, electricity turns C.acetobutylicum CCTCC M 94061, thereby the C.acetobutylicum CCTCC M94061 transformant that acquisition has the Ery resistance, and transformant verified, the result is as follows:

After the transformant of C.acetobutylicum CCTCC M 94061 carries out colony PCR amplification, pcr amplification product is carried out the electrophoresis checking, the result is as shown in Figure 5, according to the result of Fig. 5,3,4,5,6,8,9, the recombinant bacterium that insertion is arranged in 14, No. 17 transformants, but owing to being mixed with wild mushroom, therefore, the separation of also above 8 bacterium colonies further need being rule.

Single bacterium after line separated carries out colony PCR amplification, and the PCR product is carried out the electrophoresis checking, the result as shown in Figure 6, according to the result of Fig. 6, wherein 2,5,6, No. 11 swimming lanes have clear band at the 1.5kb place, with 2,5,6, No. 11 transformant is numbered respectively 4 (2); 8 (5); 8 (6); 14 (11).

Respectively purifying reclaims among Fig. 62,5,6, the 1.5kb band of No. 11 swimming lanes, and check order, sequencing result is shown in SEQID NO:4, according to this result, therefore the adc gene that Clostridium acetobutylicum CCTCC M 94061 adc knock out mutant strain has inserted the intron fragment at 180/181nt site forward, so that the adc gene inactivation, thereby successfully knocks out the adc gene of Clostridium acetobutylicum CCTCC M 94061.

Embodiment 4, Clostridium acetobutylicum adc-fermentation

Clostridium acetobutylicum ATCC824 and Clostridiumacetobutylicum CCTCC M 94061 recombinant bacteriums with the adc gene knockout carry out fermenting experiment in the P2 substratum respectively.Detailed process is as follows:

Clostridium acetobutylicum thalline is rule at the CGM flat board, and 37 ℃ of lower anaerobism are cultivated, and obtain single bacterium.Then single bacterium is chosen to 5ml CGM substratum, 37 ℃ of lower anaerobism overnight incubation are forwarded to 50ml CGM substratum with the nutrient solution that obtains by 5% inoculum size, are cultured to logarithmic growth mid-term, then be forwarded in the 100ml P2 substratum by 10% inoculum size, cultivate 48h in 37 ℃ of anaerobism.Then, utilize the gas Chromatographic Determination solvent production, result such as table 2 and shown in Figure 7.

The fermentation results of table 2, Clostridium acetobutylicum adc-

Bacterial strain	Acetone g/L	Ethanol g/L	Butanols g/L	Total solvent g/L	Acetone %	Ethanol %	Butanols %
									1	2.884	1.043	7.76	11.682	24.684	8.9	66.4
2	1.226	0.42	2.47	4.12	29.78	10.2	60
								3	0.23	1.14	4.2	5.57	4.13	20.5	75.4
4	0.24	0.92	3.56	4.71	4.96	19.5	71.3
								5	2.48	3.19	13.63	19.29	12.82	16.52	70.66
6	2.9	2.85	13.32	19.07	15.2	14.95	69.84
								7	0.23	1.1	6.13	7.46	3.09	14.75	82.2
8	0.16	0.31	2.79	3.26	4.9	9.5	85.6
								9	0.19	0.42	4.01	4.62	4.12	9.1	86.8

Annotate: bacterial strain corresponding to numbering 1-9 is respectively Clostridium acetobutylicum ATCC824, the recombinant C lostridium acetobutylicum ATCC824 that contains the pIMP1 carrier, No. 6 transformants of Clostridiumacetobutylicum ATCC824 mutant strain and No. 8 transformants of adc gene knockout, Clostridium acetobutylicumCCTCC M 94061, the recombinant C lostridium acetobutylicum CCTCC M94061 that contains the pIMP1-ptb carrier, No. 4 (2), Clostridium acetobutylicum CCTCC M 94061 mutant strains of adc gene knockout, 8 (6) numbers, 11 (14) numbers.

Such as table 2 and shown in Figure 7, compare with wild mushroom, the acetone output of the Clostridium acetobutylicum mutant strain of adc gene knockout is all less than 10% of wild mushroom, and acetone accounts for below the content to 5% of total solvent, but the ratio of butanols is significantly improved in the product.As seen, the synthetic of acetone successfully blocked in knocking out of adc gene, thereby improved the ratio of butanols in the tunning.

Then, to C.acetobutylicum CCTCC M 94061adc4 (2), C.acetobutylicum CCTCC M 94061, C.acetobutylicum CCTCC M 94061 (pIMP1-ptb) further ferments, sample determination OD600 to each time point, and the output of solvent, the result is as shown in Figure 8.Result according to Fig. 8, with contrast bacterium C.acetobutylicumCCTCC M 94061, C.acetobutylicum CCTCC M 94061 (pIMP1-ptb) compares, C.acetobutylicumCCTCC M 94061adc4 (2) tunning in, acetone output obviously descends, and the ratio of butanols obviously improves.As seen the acetone route of synthesis of C.acetobutylicum CCTCC M 94061adc4 (2) is blocked, thereby has improved the ratio of butanols in the tunning.

Above-described embodiment shows, C.acetobutylicum ATCC824adc-6, C.acetobutylicum ATCC824adc-8, C.acetobutylicum CCTCC M 94061adc4 (2), the acetone approach of C.acetobutylicum CCTCC M 94061adc8 (6) and C.acetobutylicum CCTCC M 94061adc11 (14) is blocked.

In sum, the present invention has successfully blocked the synthetic of acetone by suppressing by the pathways metabolism of acetoacetyl-CoA to acetone, thus the ratio of butanols in the Effective Raise tunning, thereby significant for per unit area yield butanols metabolic engineering.

Although below only be illustrated take knocking out as example of adc gene, but those skilled in the art is by of the present invention open, obviously can find out, for produce the solvent clostridium by the pathways metabolism of acetoacetyl-CoA to acetone, knock out wherein another key enzyme---thiophorase, can reach equally blocking-up acetone synthetic, improve the purpose of butanol content, thereby should belong to scope of the present invention equally.

In addition, although above embodiment only is illustrated as an example of Clostridium acetobutylicum ATCC824 and Clostridiumacetobutylicum CCTCC M 94061 example, but those skilled in the art is by of the present invention open, be easy to find out that method of the present invention is equally applicable to other product solvent clostridium, these bacterial strains include but not limited to: C.acetobutilicum, C.beijerinckii, C.saccharobutylicum, C.saccharoperbutylacetonicum, C.aurantibutyricum, C.butyricum, C.casaveris, C.pasteurianum, C.puniceum, C.sporogenes, C.tetanomorphum, C.thermosaccharolyticum, C.felsineum.Therefore these contents should belong to scope of the present invention equally.

Sequence table

Claims

1. method that improves the ratio of butanols in the natural product solvent clostridium tunning, wherein, described natural product solvent clostridium is C.acetobutylicum, C.beijerinckii, C.saccharobutylicum, or C.saccharoperbutylacetonicum; The pathways metabolism of described natural product solvent clostridium be from the acetoacetyl-CoA to the butanols, the pathways metabolism of acetone, it is characterized in that, realized by the pathways metabolism of acetoacetyl-CoA to acetone by inhibition, described inhibition is realized by the activity that suppresses E.C. 4.1.1.4 by the pathways metabolism of acetoacetyl-CoA to acetone.

2. the method for claim 1 is characterized in that, described product solvent clostridium is C.acetobutylicum.

3. method as claimed in claim 2 is characterized in that, described C.acetobutylicum bacterial strain is Clostridium acetobutylicum ATCC 824.

4. method as claimed in claim 2 is characterized in that, described C.acetobutylicum bacterial strain is Clostridium acetobutylicum CCTCC M 94061.

5. the method for claim 1 is characterized in that, described inhibition E.C. 4.1.1.4 activity realizes by interrupting the E.C. 4.1.1.4 encoding gene.

6. the method for claim 1 is characterized in that, described interruption E.C. 4.1.1.4 encoding gene is realized by the Targetron gene Knockout.

7. the recombinant bacterial strain of a Clostridium acetobutylicum, wherein, described Clostridium acetobutylicum is Clostridium acetobutylicum ATCC 824 or Clostridium acetobutylicum CCTCC M 94061, it is characterized in that, the E.C. 4.1.1.4 encoding gene is knocked in the genome of this bacterial strain.

8. the recombinant bacterial strain of E.C. 4.1.1.4 encoding gene gene knockout as claimed in claim 7 is for the production of the application of propyl carbinol.