CN102618569B - Construction of butanol producing genetic engineering bacteria, strain and application thereof - Google Patents
Construction of butanol producing genetic engineering bacteria, strain and application thereof Download PDFInfo
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- CN102618569B CN102618569B CN201210067927.8A CN201210067927A CN102618569B CN 102618569 B CN102618569 B CN 102618569B CN 201210067927 A CN201210067927 A CN 201210067927A CN 102618569 B CN102618569 B CN 102618569B
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- 241000894006 Bacteria Species 0.000 title claims abstract description 44
- 238000010353 genetic engineering Methods 0.000 title claims abstract description 24
- 238000010276 construction Methods 0.000 title claims abstract description 13
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- 238000000034 method Methods 0.000 claims abstract description 19
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- 230000008569 process Effects 0.000 claims abstract description 13
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- 239000013612 plasmid Substances 0.000 claims description 43
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- 101150013191 E gene Proteins 0.000 claims description 3
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- 101100326920 Caenorhabditis elegans ctl-1 gene Proteins 0.000 abstract description 2
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- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 2
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Abstract
The invention belongs to the technical field of biological engineering, and relates to construction of a butanol-producing genetic engineering bacterium, a strain and application thereof. The construction process mainly adopts a molecular biology method to synthesize key genes in the butanol synthesis pathway of clostridium klonicat1、sucD、4hbd、cat2、abfDAnd genes in Clostridium acetobutylicumbcd -etfB-etfA、adhE, carrying out recombinant expression in Escherichia coli, and constructing a metabolic pathway from the succinic acid to the butanol. Using constructed recombinant strainsEscherichiacoliLP-99e is fermented to enable the production of butanol.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to structure, bacterial strain and the application thereof of producing the butanols genetic engineering bacterium, specifically refer to produce the strain bacterial strain that butanols construction of genetic engineering and structure thereof obtain, also relate to the method for utilizing this strain fermentation to produce butanols.
Background technology
Butanols is a kind of important organic solvent and fine chemical material, also is a kind of renewable energy source of cleaning, and butanols has reached annual 1300 ten thousand tons in the market of the U.S. at present.Because the raw material of the chemical synthesis of butanols is the propylene based on oil, along with the rapid growth of oil price, petrochemical complex is produced the butanols cost and is also increased thereupon, so biological fermentation process production butanols is subjected to extensive concern.
Biological fermentation process (ABE fermentation method) is produced butanols mainly by clostridium acetobutylicum genus fermentation generation at present, comprises
Clostridium acetobutylicum,
C. beijerinckii,
C.pasteurianum,
C. saccharoperbutylacetonicum,
C. tetanomorphum,
C. saccharoacetobutylicumDeng.Develop rapidly along with biotechnology, metabolic engineering, genetic engineering technique, adopt molecular biology method, the genetic engineering bacterium that makes up inheritance stability is produced the focus that butanols becomes research, except carrying out the genetic modification producing Clostridium acetobutylicum, reconstruct butanols pathways metabolism in other microfloras also is as intestinal bacteria etc.In the prior art, utilize genetic engineering technique that colibacillary pathways metabolism is carried out process and remould, the colibacillus engineering of structure can produce acetone, butanols.People such as Bermejo have made up one and have comprised gene
Cet4
(adhe, etfAB and
Thil) plasmid pACT, and change intestinal bacteria over to, made up the colibacillus engineering that produces acetone first, can produce the acetone of 2.32 g/ L after the fermentation; Inui etc. will derive from the key gene of synthetic butanols approach among the clostridium acetobutylicum ATCC 824
Thil,
Hbd,
Crt,
Bcd-etfB-etfA,
AdhE, the thiolase of encoding respectively, 3-maloyl group-CoA desaturase, enoyl-CoA hydratase, butyryl-CoA desaturase and aldehyde/alcoholdehydrogenase are at expression in escherichia coli, and the activity of enzyme changes to some extent, and genetic engineering bacterium fermentation back produces 1.18 g/ L butanols; Nielsen etc. exist
E.coliIn made up the butanols metabolic pathway of clostridium acetobutylicum, directly express polycistron and can access 34 mg/L butanols, express respectively separately along anti-gene, butanols output can reach 200 mg/L, if express yeast ammonium formiate dehydrogenase gene simultaneously, reached and express colibacillary 3-phosphate dehydrogenase, butanols output can be brought up to 580 mg/L; People such as Shen have made up the clostridium acetobutylicum pathways metabolism through modifying in intestinal bacteria; having obtained Ter(acyl-CoA transferring enzyme) the irreversible reaction of catalysis produced NADH and acetyl-CoA motivating force, and used the air lift means to make the recombinant bacterial strain anaerobically fermenting can produce the butanols of 30 g/ L.Above-mentioned prior art shows the gene of the synthetic middle coding key enzyme of clone's butanols, further expresses in intestinal bacteria, and the colibacillus engineering of structure can produce butanols.
Summary of the invention
Technical purpose of the present invention has been to provide a kind of construction process and bacterial strain of new product butanols genetic engineering bacterium, makes that this construction process is that a kind of structure contains key gene in the Ke Shi clostridium butanols route of synthesis
Cat1,
SucD, 4
Hbd,
Cat2,
AbfDWith the gene among the clostridium acetobutylicum ATCC 824
Bcd-etfB-etfA,
AdhThe pathways metabolism method from the Succinic Acid to the butanols of E, and bacterial strain of the present invention can lay the foundation for producing the colibacillary genetically engineered research of acetone-butanol.
For realizing technical purpose of the present invention, the present invention by the following technical solutions.
One, the construction process of product butanols genetic engineering bacterium of the present invention.
The present invention with lack lactate dehydrogenase gene (
LdhA), the pyruvate formate-lyase gene (
PflB) active bacterial strain is the bacterium that sets out, recombinant expressed gene
Cat1,
SucD, 4
Hbd,
Cat2,
Abfd, bcd-etfB-etfA,
AdhE obtains to produce the butanols genetic engineering bacterium, and the pathways metabolism of recombinant bacterial strain as shown in Figure 1.
Concrete steps are as follows:
1) be template with Ke Shi clostridium gene group DNA, purifying amplifies respectively
Cat1,
SucD, 4
Hbd,
Cat2,
AbfdGene, be inserted between the corresponding restriction enzyme site of expression plasmid pTrc99a, connect obtain in the middle of recombinant plasmid pTrcCKL-5;
2) be template with acetone-butanol clostridium gene group DNA, purifying amplifies
Trc-adhBehind the E gene, be inserted between the corresponding restriction enzyme site of expression plasmid pGEM-T Easy, connect obtain in the middle of recombinant plasmid T-A;
3) be template with acetone-butanol clostridium gene group DNA, purifying amplifies
Bcd-etfB-etfABehind the gene, in the middle of being inserted between the recombinant plasmid T-A corresponding restriction enzyme site, connect obtain in the middle of recombinant plasmid T-A-B;
4) be template with middle recombinant plasmid T-A-B, purifying amplifies
Trc-adhE-
Bcd-etfB-etfABehind the gene, in the insertion between the corresponding restriction enzyme site of interstitial granules pTrc99CKL-5, connect and obtain final recombinant plasmid pTrc99e;
5) construction recombination plasmid pTrc99e import is lacked lactate dehydrogenase gene (
LdhA), the pyruvate formate-lyase gene (
PflB) competence of the active bacterium that sets out, the positive transformant of acquisition is and produces the butanols genetic engineering bacterium.
Two, butanols genetically engineered bacteria strain is produced in a strain of adopting construction process of the present invention to obtain, its classification called after intestinal bacteria (
Escherichia coli) LP-99e, its preserving number is numbered: CCTCC NO:M 2011403.
Three, the application of product butanols genetic engineering bacterium of the present invention comprises actication of culture, seed culture, fermentation culture product butanols three steps.
(1) actication of culture: the bacterium liquid of the product butanols genetic engineering bacterium of line preservation is to the flat board that contains paraxin and penbritin, and the single bacterium colony that grows on the picking flat board is to the test tube of LB substratum;
(2) seed culture: the inoculum size according to volume ratio 1% inserts in the seed culture medium, and aerobic is cultivated thalline OD
600IPTG with 0.3mM during to 0.8-1.0 is induced to OD
600=3;
(3) fermentation culture is produced butanols: be forwarded to anaerobically fermenting in the fermention medium by inoculum size volume ratio 10%; Wherein, fermention medium is: LB, glucose 15 g/L, kantlex 30 μ g/mL, penbritin 50 μ g/mL, paraxin 25 μ g/mL, 0.3mM IPTG.
Beneficial effect of the present invention is:
Adopt genetic engineering means with key gene in the Ke Shi clostridium butanols route of synthesis
Cat1,
SucD, 4
Hbd,
Cat2,
AbfDWith the gene in the clostridium acetobutylicum
Bcd-etfB-etfA,
AdhE carries out recombinant expressed in intestinal bacteria, and the method that makes recombination bacillus coli can produce butanols and production butanols thereof is not seen open, and this application has promoted the development that genetic engineering bacterium produces butanols.Though utilize the output of colibacillus engineering production butanols not high at present, this haves laid a good foundation for producing the colibacillary genetically engineered research of acetone-butanol.In intestinal bacteria, make up new production of butanol system, as a kind of approach of novel production butanols, will become one of developing direction of studying the high yield butanols from now on.
Description of drawings
Butanols pathways metabolism figure in the product butanols genetic engineering bacterium that Fig. 1 makes up.
Recombinant plasmid pTrcCKL-5 makes up collection of illustrative plates in the middle of Fig. 2.
Recombinant plasmid T-A makes up collection of illustrative plates in the middle of Fig. 3.
Recombinant plasmid T-A-B makes up collection of illustrative plates in the middle of Fig. 4.
The structure collection of illustrative plates of Fig. 5 recombinant plasmid recombinant plasmid pTrc99e.
The middle recombinant plasmid pTrcCKL-5's of Fig. 6
EcoR V single endonuclease digestion is identified figure.
Fig. 7 recombinant plasmid pTrc99e's
SmaThe I single endonuclease digestion is identified figure.
Fig. 8 recombinant plasmid pTrc99e's
KpnThe I single endonuclease digestion is identified figure.
Product butanols genetically engineered bacteria strain of the present invention, its classification called after intestinal bacteria (
Escherichia coli) LP-99e; Its preservation mechanism full name is Chinese typical culture collection center, is called for short CCTCC, and the address is China. Wuhan. and Wuhan University; Preservation date is on November 21st, 2011, and preserving number is numbered: CCTCC NO:M 2011403.
Embodiment
The following examples elaborate to the present invention, but to the present invention without limits.
The explanation in the source of biomaterial of the present invention:
1, plasmid source:
(1) pTrc99a: available from Introvegen company;
(2) pGEM-T Easy: available from Promega company.
2, genomic templates source:
(1) Ke Shi clostridium gene group DNA:
C.
KluyveriDSM 555 is available from DSMZ;
(2) acetone-butanol clostridium gene group DNA:
C.
AcetobutylicumATCC 824 buys from ATCC.
3, starting strain:
E.coliThere are two places in the source of the competence bacterial strain of AFP111:
（1）Biotechnol?Bioeng,?2001,74:89～95。The applicant is at first by finding the above-mentioned document source of this biomaterial, and to have contacted the utterer be the David P. Clark professor of Univ Chicago USA, and its this biomaterial of gifting of mail requests, and freely obtained this biomaterial; And the applicant guaranteed to provide this biomaterial to the public in 20 years the application's days;
(2) this biomaterial also discloses in the patent documentation of Chinese patent (application number 96198547.X, applying date 1996.10.31 authorize day on January 1st, 2003, Granted publication CN1097632C) and obtains the authorization.
4, primer design and synthetic: design and outer Si Rui covered with gold leaf biotech company are synthetic voluntarily.
Embodiment 1
The present embodiment explanation makes up and comprises expressing gene
Cat1,
SucD, 4
Hbd,
Cat2,
AbfdMiddle interstitial granules pTrcCKL-5.Its process comprises:
1, design is synthetic has
NcoThe upstream primer of I restriction enzyme site and having
KpnThe downstream primer of I restriction enzyme site,
Primer1 upstream primer: 5 '-ACGT CCATGGATGAGTAAAGGGATAAAG-3 ';
Primer2 downstream primer: 5 '-ACGTGGTACCATAAAGTGTAACTAAAAATCA-3 '.
2, be template with Ke Shi clostridium gene group DNA, the pcr amplification target gene fragment, reaction conditions is: 95 ℃, 10 min; (95 ℃ of 30 s, 50 ℃ of 30 s, 72 ℃ of 8min, 35 circulations); 72 ℃, 10 min.Purifying amplifies
Trc-cat1-
SucD-4
Hbd-
Cat2-
AbfdBehind the gene, expression plasmid pTrc99a uses respectively
NcoI and
KpnI double digestion, connection obtain recombinant plasmid pTrcCKL-5.Use
EcoR V single endonuclease digestion is identified recombinant plasmid pTrcCKL-5, and obtaining four stripe size is respectively 5805bp and 4163bp, 881 bp and 514 bp, the results are shown in Figure 6, and enzyme is cut the result and shown, recombinant plasmid pTrcCKL-5 successfully constructs.
Specify: described at embodiment 1
Cat1, sucD, 4hbd, cat2, abfdThese 5 gene fragments, the present invention has adopted same primer, can clone these 5 gene fragments respectively after, be inserted into respectively between the corresponding restriction enzyme site of expression plasmid pTrc99a, obtain recombinant plasmid pTrcCKL-5; Also can disposablely clone these 5 gene fragments, disposable being inserted between the corresponding restriction enzyme site of expression plasmid pTrc99a obtains recombinant plasmid pTrcCKL-5.
Embodiment 2
The present embodiment explanation makes up and comprises expressing gene
Bcd-etfB-etfA,
AdheMiddle recombinant plasmid T-A-B.Its process comprises:
1, comprises gene
AdheThe structure of middle interstitial granules T-A,
(1) synthetic having
SmaThe upstream primer of I restriction enzyme site and not with the downstream primer of restriction enzyme site:
The Primer3 upstream primer:
5’-ATCGCCCGGGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACACCATGGATGAAAGTTACAAATCAAAA-3’；
The Primer4 downstream primer:
5’-AGCTTTAAAATGATTTTATATAGAT-3’。
(2) be template with acetone-butanol clostridium gene group DNA, the pcr amplification target gene fragment, reaction conditions is: 95 ℃, 10 min; (95 ℃ of 30 s, 48 ℃ of 30 s, 72 ℃ of 3min, 35 circulations); 72 ℃, 10 min.Purifying amplifies
Trc-adhBehind the E gene, expression plasmid pGEM-T Easy with
Trc-adhThe E fragment connects the middle recombinant plasmid T-A of acquisition.
2, comprise gene
Adhe,
Bcd-etfB-etfAThe structure of middle interstitial granules T-A-B,
(1) synthetic having
SacThe upstream primer of I restriction enzyme site and having
SacI and
SmaThe downstream primer of I restriction enzyme site restriction enzyme site:
Primer5 upstream primer: 5 '-ATCGGAGCTTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGG ATAACAATTTCACACAGGAAACAAGATCTATGGAT TTTAATTTAACAAG-3 ';
The Primer6 downstream primer:
5’-AGCTGAGCTCCCCGGGTTAATTATTAGCAGCTTTAA-3’。
(2) be template with acetone-butanol clostridium gene group DNA, the pcr amplification target gene fragment, reaction conditions is: 95 ℃, 10 min; (95 ℃ of 30 s, 48 ℃ of 30 s, 72 ℃ of 3min, 30 circulations); 72 ℃, 10 min.Purifying amplifies
Trc-bcd-etfB-etfABehind the gene, middle interstitial granules T-A uses
SacThe new middle recombinant plasmid T-A-B of acquisition is cut, connected to the I enzyme.
Embodiment 3
The present embodiment explanation makes up recombinant expressed gene
Cat1,
SucD, 4
Hbd,
Cat2,
Abfd, bcd-etfB-etfA,
AdhThe final recombinant plasmid pTrc99e of E namely connects gene on middle recombinant plasmid pTrcCKL-5 basis
AdhE,
Bcd-etfB-etfA, obtain final recombinant plasmid pTrc99e.
1, makes up recombinant expressed gene
Cat1,
SucD, 4
Hbd,
Cat2, abfd, bcd-etfB-etfA,
AdhThe final recombinant plasmid pTrc99e of E, its process comprises:
Be template with middle recombinant plasmid T-A-B, pcr amplification target gene fragment (be upstream and downstream primer with aforementioned Primer3 and Primer6), reaction conditions is: 95 ℃, 10 min; (95 ℃ of 30 s, 48 ℃ of 30 s, 72 ℃ of 6min, 35 circulations); 72 ℃, 10 min.Purifying amplifies
Trc-adhE-
Bcd-etfB-etfABehind the gene, middle interstitial granules pTrc99CKL-5 is used
SmaThe final recombinant plasmid pTrc99e of acquisition is cut, connected to the I enzyme.Use
SmaI and
KpnI single endonuclease digestion respectively identifies recombinant plasmid pTrc99e,
SmaThe band that I cuts and obtains two sizes is respectively 11313bp and 5800bp the results are shown in Figure 7;
KpnThe band that the I enzyme is cut and obtained three sizes is respectively 13914bp, 1878bp and 1358bp the results are shown in Figure 8, and enzyme is cut the result and shown, recombinant plasmid pTrc99e successfully constructs.
2, plasmid pTrc99e is imported starting strain
E.coliThe competence of AFP111 (namely lack lactate dehydrogenase gene (
LdhA), the pyruvate formate-lyase gene (
PflB) active bacterial strain), the positive transformant of acquisition is new structure bacterial strain of the present invention
Escherichia coliLP-99e.
Of particular note, described seven gene fragments of embodiment of the invention 1-3 (wherein
Bcd-etfB-etfABe considered as a gene fragment) be the approximate size that links to each other at genome in the long segment of bp more than 8000, so the recombinant plasmid pTrc99e that embodiment 3 obtains can be considered a total long gene fragment.
Embodiment 4
New intestinal bacteria LP-99e and the starting strain that makes up of present embodiment explanation overexpression
E.coliProduce the contrast of butanols ability in both fermenting processs of AFP111.
(1) actication of culture: the bacterium liquid of the intestinal bacteria LP-99e of-80 ℃ of frozen pipe preservations of line is to the flat board that contains paraxin and penbritin, and the single bacterium colony that grows on the picking flat board is to the test tube of 5mL LB substratum.
Wherein, the described prescription that contains the flat board of paraxin and penbritin is: LB+Amp (penbritin 50 μ g/mL)+Chl (paraxin 25 μ g/mL)+15% agar.
(2) seed culture: the inoculum size according to volume ratio 1% inserts in the seed culture medium, and aerobic is cultivated thalline OD
600IPTG with 0.3mM during to the 0.8-1.0 left and right sides is induced to OD
600About=3.
The prescription of described seed culture medium is: yeast powder 3 g/L, peptone 5 g/L, Zulkovsky starch 10 g/L, ammonium acetate 2 g/L, NaCl 2 g/L, MgSO
43 g/L, KH
2PO
41g/L, K
2HPO
41 g/L, FeSO
47H
2O 0.1 g/L, solid add agar 20 g/L, pH6.0.
(3) fermentation culture is produced butanols: adopt the fermentation of anaerobism serum bottle, be forwarded to anaerobically fermenting 48 h in the fermention medium by inoculum size volume ratio 10%.
Anaerobism serum bottle fermention medium is: LB+ glucose (15g/L)+Kan (kantlex 30 μ g/mL)+Amp (penbritin 50 μ g/mL)+Chl (paraxin 25 μ g/mL)+0.3mM IPTG.
The measurement result that the anaerobism serum bottle is cultivated the various parameters in back sees Table 1.
Experiment showed, the construction process by genetic engineering bacterium of the present invention, make its recombination bacillus coli that obtains to produce butanols.Though the output of the intestinal bacteria LP-99e that obtains at present production butanols is not high, this haves laid a good foundation for producing the colibacillary genetically engineered research of acetone-butanol.
Claims (3)
1. a construction process that produces the butanols genetic engineering bacterium is characterized in that: to lack lactate dehydrogenase gene, the bacterial strain of pyruvate formate-lyase gene activity
E.coliAFP111 is starting strain, recombinant expressed gene
Cat1,
SucD, 4
Hbd,
Cat2,
Abfd,
Bcd-etfB-etfA,
AdhE obtains to produce the butanols genetic engineering bacterium; It is characterized in that concrete steps are as follows:
1) be template with Ke Shi clostridium gene group DNA, purifying amplifies respectively
Cat1,
SucD, 4
Hbd,
Cat2,
AbfdGene, be inserted between the corresponding restriction enzyme site of expression plasmid pTrc99a, connect obtain in the middle of recombinant plasmid pTrcCKL-5;
2) be template with acetone-butanol clostridium gene group DNA, purifying amplifies
Trc-adhBehind the E gene, be inserted between the corresponding restriction enzyme site of expression plasmid pGEM-T Easy, connect obtain in the middle of recombinant plasmid T-A;
3) be template with acetone-butanol clostridium gene group DNA, purifying amplifies
Bcd-etfB-etfABehind the gene, in the middle of being inserted between the recombinant plasmid T-A corresponding restriction enzyme site, connect obtain in the middle of recombinant plasmid T-A-B;
4) be template with middle recombinant plasmid T-A-B, purifying amplifies
Trc-adhE-
Bcd-etfB-etfABehind the gene, in the middle of inserting between the corresponding restriction enzyme site of recombinant plasmid pTrc99CKL-5, connect and obtain final recombinant plasmid pTrc99e;
5) construction recombination plasmid pTrc99e is imported shortage lactate dehydrogenase gene, the bacterium that sets out of pyruvate formate-lyase gene activity
E.coliThe competence of AFP111, the positive transformant of acquisition are produces the butanols genetic engineering bacterium.
2. butanols genetically engineered bacteria strain is produced in a strain of adopting the described construction process of claim 1 to obtain, its classification called after intestinal bacteria (
Escherichia coli) LP-99e, its preserving number is numbered: CCTCC NO:M 2011403.
3. the application of the described product butanols of claim 1 genetic engineering bacterium is characterized in that: comprise actication of culture, seed culture, fermentation culture product butanols three steps,
(1) actication of culture: the bacterium liquid of the product butanols genetic engineering bacterium of line preservation is to the flat board that contains paraxin and penbritin, and the single bacterium colony that grows on the picking flat board is to the test tube of LB substratum;
(2) seed culture: the inoculum size according to volume ratio 1% inserts in the seed culture medium, and aerobic is cultivated thalline OD
600IPTG with 0.3mM during to 0.8-1.0 is induced to OD
600=3;
(3) fermentation culture is produced butanols: be forwarded to anaerobically fermenting in the fermention medium by inoculum size volume ratio 10%; Wherein, fermention medium is: LB, glucose 15 g/L, kantlex 30 μ g/mL, penbritin 50 μ g/mL, paraxin 25 μ g/mL, 0.3mM IPTG.
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