CN106222188B - Method for producing biofuel - Google Patents
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- CN106222188B CN106222188B CN201610698228.1A CN201610698228A CN106222188B CN 106222188 B CN106222188 B CN 106222188B CN 201610698228 A CN201610698228 A CN 201610698228A CN 106222188 B CN106222188 B CN 106222188B
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- 238000004519 manufacturing process Methods 0.000 title abstract description 15
- 239000002551 biofuel Substances 0.000 title abstract description 14
- 241000588724 Escherichia coli Species 0.000 claims abstract description 19
- 101710088194 Dehydrogenase Proteins 0.000 claims abstract description 13
- 241000193755 Bacillus cereus Species 0.000 claims abstract description 10
- 101150016526 fadE gene Proteins 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 22
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 12
- 210000001072 colon Anatomy 0.000 claims description 12
- 108090000790 Enzymes Proteins 0.000 claims description 11
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 230000029087 digestion Effects 0.000 claims description 7
- 239000012634 fragment Substances 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- 238000003752 polymerase chain reaction Methods 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 230000035939 shock Effects 0.000 claims description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 4
- 239000003292 glue Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 102220013477 rs117560775 Human genes 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 238000012163 sequencing technique Methods 0.000 claims description 4
- 108090000489 Carboxy-Lyases Proteins 0.000 claims description 3
- 102220559225 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9, mitochondrial_N70S_mutation Human genes 0.000 claims description 3
- 150000002192 fatty aldehydes Chemical class 0.000 claims description 3
- 108090001018 hexadecanal dehydrogenase (acylating) Proteins 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 3
- 102220146158 rs886059141 Human genes 0.000 claims description 3
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- 230000035772 mutation Effects 0.000 claims 2
- 150000001335 aliphatic alkanes Chemical class 0.000 abstract description 12
- 238000002360 preparation method Methods 0.000 abstract description 7
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- 238000011161 development Methods 0.000 description 11
- 239000002028 Biomass Substances 0.000 description 10
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 9
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- 239000004215 Carbon black (E152) Substances 0.000 description 7
- 239000003502 gasoline Substances 0.000 description 7
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- 125000003147 glycosyl group Chemical group 0.000 description 4
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- 238000006467 substitution reaction Methods 0.000 description 3
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 235000016068 Berberis vulgaris Nutrition 0.000 description 2
- 241000335053 Beta vulgaris Species 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000305071 Enterobacterales Species 0.000 description 2
- -1 S1010 is then added Substances 0.000 description 2
- 240000000111 Saccharum officinarum Species 0.000 description 2
- 235000007201 Saccharum officinarum Nutrition 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
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- 238000003912 environmental pollution Methods 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
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- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
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- 239000002250 absorbent Substances 0.000 description 1
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- 239000003513 alkali Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000003225 biodiesel Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
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- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
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- 230000014509 gene expression Effects 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000011133 lead Substances 0.000 description 1
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- 239000000618 nitrogen fertilizer Substances 0.000 description 1
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- 235000009566 rice Nutrition 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 238000004162 soil erosion Methods 0.000 description 1
- 238000003900 soil pollution Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 235000019386 wax ester Nutrition 0.000 description 1
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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Abstract
The invention belongs to the field of biofuels, and particularly relates to a method for producing biofuels, which comprises the steps of introducing bacillus cereus dehydrogenase into escherichia coli which is transformed with accABCD/acr1/BTE/CER1 genes and whose fadE gene is knocked out, and expressing to obtain corresponding alkane. The invention has the advantages of greatly improved alkane production efficiency, simple preparation, convenient alkane secretion separation and purification, and suitability for industrial production.
Description
Technical field
The invention belongs to bio-fuel fields, and in particular to a method of produce bio-fuel.
Background technique
With international community's growing interest big to Gloal Climate Change Impact, the development and application of new energy becomes again
The hot topic of global concern, countries in the world must reinforce construction low-carbon economy with the common recognition for coping with global warming is also continuous,
The main trend for having become development of world economy to low-carbon economy transition, the maximum development of high speed development is in as economy
Middle country, China similarly face the double challenge of economic development Yu environment environmental protection.In the fossil energy of China, basic characteristics
It is rich coal, oil-poor, few gas, i.e., with oil and natural gas comparatively, the reserves of the coal in China are relatively abundant, accounts for the world
The 11.6% of reserves, this just determines critical role of the coal as non-renewable energy in our economic construction, however, with me
The sustainable development of state's economic construction, the coal in fossil energy are become increasingly conspicuous using brought problem of environmental pollution, so that solution
The certainly problem of environmental pollution in energy shortage problem and using energy source will be key point that human kind sustainable development is faced.
For this purpose, being examined closely from long-term energy development strategy angle, the development and utilization of new and renewable energy, should be arranged from " zero "
Put or the technique direction of " low " discharge development, and protect environment, the road for moving towards low-carbon economy social sustainable development it is basic
Guarantee.
Bio-fuel is the fuel using the raw material production from biomass.Biomass has light in such as plant like that
The grease or carbohydrate generated in the case where closing ability to function from luminous energy and carbon dioxide at bio-fuel raw material, because
This is able to produce the fuel small to environmental pressure.Bio-fuel, which has, is saccharified carbohydrate, the biology generated through alcoholic fermentation
Ethyl alcohol, the triglycerides of principal component as vegetable oil, the biodiesel and the combustion of biological jet that are generated by neutral lipids such as wax esters
Material etc..Generally bio-fuel is defined as by directly burning, the energy that alcohol fermentation and methane fermentation etc. are obtained from biomass.
It is (beautiful can be classified as glycosyl (sugarcane, beet etc.), starch base especially for bio-alcohol for biomass, the raw material of bio-fuel
Rice, potato, sweet potato etc.) He Muji (culled wood, straw, waste paper etc.).Glycosyl biomass can be in fairly simple pretreatment
Easily and bio-ethanol is translated directly into after process.And starch-or wood-based biomass need preprocessing process appropriate
Bio-ethanol is generated with saccharifying.The forest by-product dispersed around culled wood, a kind of municipal waste or forest, can also
For use as the wooden based biomass.In addition, can steadily guarantee raw materials requirement because they do not have the availability as food.
However, for the wooden based biomass wait be used as bio-fuel, it is necessary to eliminate the pretreatment of lignin, which increase be produced into
This, and due to the crystallization hydrogen bonding structure of cellulose, saccharification efficiency becomes very low.
For transporting upper economically viable replaceable fuel, the price of bio-fuel must mutually be competed with gasoline.In general,
The type of raw material and the cost of processing than being heavily dependent on biomass used.For example, in glycosyl, such as sugarcane and beet
In situation, the cost of raw material and processing is than being about 75:25.Meanwhile in starch-base, such as the case where corn, potato and cassava
In, ratio is about 50:50, and in the case where the wooden base, ratio is about 25:75.
The most frequently used biomass so far for bio-ethanol production is glycosyl and starch base.However, they may be used also
For use as food, therefore, if food needs rapid growth, these raw materials requirements should will receive influence, so that production cost
It is not economically feasible.It moreover has been found that the cultivation of crop such as corn needs a large amount of pesticides and nitrogenous fertilizer, lead to environmental problem,
Such as the soil erosion and pollution.
A kind of biogasoline and its preparation method are disclosed in CN 102277211A.By potato starch, modifier,
Modifying agent, hydrocarbon alcohol strain are mixed, percent mass ratio shared by each component are as follows: potato starch 90-98%, modifier
1-5%, modifying agent 1-5%, hydrocarbon alcohol strain 0.01-1%.By above-mentioned potato starch, with wine with dregs → gelatinization → sterilizing → hydrocarbon alcohol A6,
A8, B11 bacterium Tube propagation → culture bottle culture → kind of female tank culture → kind of female wine with dregs → tank ferment → karusen → mash pretreatment
Device processing → prefractionator → total solvent → destilling tower → again by hydrocarbon alcohol A6, hydrocarbon alcohol A8, hydrocarbon alcohol B11 → mixing → denaturant, modifying agent
After synthesize.Product of the present invention energy conservation and environmental protection, use cost are low, extend the service life of engine.But this method preparation is complicated,
Process is longer, is not appropriate for industrialized large-scale promotion.
Disclosed in CN 101165188A it is a kind of using cassava as the method for waste fuel oil biological butanol, it be use
Cassava crushing or tapioca add water to size mixing, then acid adding or add alkali impregnate, remove impurity, S1010 is then added, catalyst is anti-
It answers, accesses CLa-b Np-1 strain fermentation after cooling again, the mixed liquor obtained is fractionated and obtains crude product fuel oil biological butanol, slightly
Product fuel oil biological butanol obtains fine work fuel oil biological butanol after the processing of QR-W absorbent.It is needed by purposes, QR-E modifying agent is added,
Obtain qualified fuel oil biological butanol.But the fermentation efficiency of bacterium is not also high in this method, yield is lower, is equally also not suitable for extensive
Industrialized production.
CN 101899412A discloses a kind of engineering colon bacillus for being used to prepare biogasoline, and this method passes through big
Overexpression thioester enzyme gene in enterobacteria, acyl-CoA reductase gene and fatty aldehyde decarboxylase gene etc. make Escherichia coli
The ability of production biogasoline is obtained, this method production is rapid, but the yield of biogasoline need further to improve.
Summary of the invention
The purpose of the present invention is to provide a kind of engineering colon bacillus for being used to prepare biogasoline of transformation.
It is a kind of i.e. biological using short chain alkanes in the preparation of above-mentioned engineering colon bacillus another object of the present invention is to provide
The method of gasoline.
Another object of the present invention is to provide a kind of bacillus cereus dehydrogenase, the amino acid sequence of the enzyme such as:
Shown in SEQ ID No:1.The enzyme enables to Escherichia coli biology flow path to turn to fatty acid synthesis pathway, so that short
The synthetic quantity of alkane is greatly enhanced.
The present invention additionally provides a kind of methods for enhancing bacillus cereus dehydrogenase activity, and the method includes being directed to ammonia
The active site of base acid sequence carries out substitution verifying, is tested by more than 200 kinds of substitutions, finally screen to obtain G63, N70, K84,
I93、S110、R119、D128、L137、I 145、K146、L181、S182、T196、V243、A252、S263、F303、Y362、
This 21 kinds of sites E379, L404, K422 are enzyme activity critical sites, can enhance corresponding enzyme activity after being substituted, and remaining is
Enzyme activity cannot be caused to improve some to even result in enzyme activity reduction or disappear.
The present invention additionally provides a kind of method that site replaces, the having after optimizing that be substituted by improves Enzyme activity
Substitution mode, specially G63A, N70S, K84T, I93D, S110G, R119F, D128S, L137D, I145S, K146K,
L181G,S182H,T196Q,V243I,A252S,S263P,F303G,Y362T,E379M,L404W,K422E.Wherein G63A table
Show that the G of the 63rd position in the amino acid sequence of SEQ ID NO:1 is substituted by A.
The present invention provides a kind of preparation method of engineering colon bacillus for being used to prepare biogasoline, is obtained by following methods
It arrives:
Acetyl-CoA carboxylase gene GeneID (NCBI) is expanded by PCR (polymerase chain reaction): 2878573, sulphur
Lipase gene GI (NCBI): 170555, acyl-CoA reductase gene GI (NCBI): 1684885 and fatty aldehyde decarboxylase gene
GI (NCBI): 145334982 and bacillus cereus dehydrogenase GI (NCBI): KZD31264 is recycled with plastic recovery kit
Target fragment, then double digestion target fragment and prokaryotic expression carrier pET-30a (+) or pACYCDuet-1 respectively, carrier segment
After glue recycling, by carrier: genetic fragment is 1: 4 ratio mixing in molar ratio, and 4 DEG C of connections 20 are small after T4DNA ligase is added
When, 38-42 DEG C of thermal shock Transformed E .coli DH5 α competent cell of connection product, spread plate overnight incubation, the PCR screening positive
Clone；Positive colony extracts Plasmid DNA, and after digestion and sequencing identification, thermal shock Transformed E .coliBL21 (DE3), finally knocking out should
Up to purpose project Escherichia coli after the fadE gene of engineering colon bacillus.
The present invention provide it is a kind of using above-mentioned engineering colon bacillus preparation in the short chain alkanes i.e. method of biogasoline, be by
The engineering colon bacillus built includes the M9 liquid medium of kanamycins and chloramphenicol with 1-2: 100-130 ratio inoculation
In, 35-37 DEG C, culture is to OD under the conditions of 225rpm600nmWhen for 0.6-0.8, inducer IPTG to final concentration of 0.1- is added
0.2mmol·L-1, the overexpression of destination protein is induced, is then transferred to 28-30 DEG C, 225rpm continues culture 18-24 hours；Training
Bacterium solution centrifuging and taking supernatant after supporting is evaporated under reduced pressure after removing n-hexane and biogasoline is made with isometric n-hexane extraction.
Compared with prior art, the invention has the following advantages that the production efficiency of alkane is greatly enhanced, and
Preparation is simple, and alkane secretion, convenient for separation and purifying, can be suitable for industrialized production outside protecting.
Specific embodiment
The clone of 1 bacillus cereus dehydrogenase of embodiment and the building of carrier
The clone of bacillus cereus bacterium dehydrogenase gene
Total mRNA of bacillus cereus CMCC 63303, then reverse transcription is cDNA, according to GenBank:KZD31264
Restriction enzyme site is added at primer both ends in design primer, and PCR amplification clones its dehydrogenase gene BcDHD, recycles glue reclaim reagent
Box recycles target gene.
Glue dehydrogenase gene BcDHD after the recovery and pACYCDuet-1 carrier (Novagen) are used into BamH I and Sal 1
Carry out double digestion, carrier and exogenous sequences in molar ratio 1: 5 ratio, 4 DEG C of connections are overnight, connection product Transformed E .coli DH5
α, PCR screening positive clone reflect after extracting recombinant plasmid pA-BcDHD in positive colony, then through restricted digestion and sequencing
It is fixed.By identification, construction of recombinant plasmid is correct.
In addition, preferably replacing the site for improving enzyme activity according to having for having verified that, while will be corresponding by multiplex PCR
Mutational site is introduced into gene order, to obtain different mutants gene, utilizes above-mentioned identical method, building expression
Carrier is respectively designated as: pA-BcDHD-G63A, pA-BcDHD-N70S, pA-BcDHD-K84T, pA-BcDHD-I93D, pA-
BcDHD-S110G、pA-BcDHD-R119F、pA-BcDHD-D128S、pA-BcDHD-L137D、pA-BcDHD-I 145S、pA-
BcDHD-K146K、pA-BcDHD-L181G、pA-BcDHD-S182H、pA-BcDHD-T196Q、pA-BcDHD-V243I、pA-
BcDHD-A252S、pA-BcDHD-S263P、pA-BcDHD-F303G、pA-BcDHD-Y362T、pA-BcDHD-E379M、pA-
BcDHD-L404W、pA-BcDHD-K422E。
The building of 2 recombination bacillus coli of embodiment
It obtains according to the method for CN 101899412A embodiment 1 containing pA-accABCD and pET-acr1/BTE/CER1
Two expression vectors.
The recombinant plasmid pA-BcDHD and pA-accABCD and pET-acr1/BTE/CER1 tri- that embodiment 1 is prepared
Thermal shock Transformed E .coli BL21 (DE3) competent cell is extracted a expression vector by PCR screening acquisition positive colony together
Pass through digestion and sequencing identification after the plasmid of positive colony again.It thereby is achieved containing pA-BcDHD, pA-accABCD and pET-
The engineering colon bacillus of tri- expression vectors of acr1/BTE/CER1.Engineering colon bacillus after conversion, using TargeTronTMBase
The fadE gene of Escherichia coli is knocked out because knocking out system (Sigma-Aldrich).The bacterial strain is DHD/accABCD/
Acr1/BTE/CER1/-fadE Escherichia coli, it is simple to name are as follows: BcDHD bacterial strain.
The substituted expression vector being prepared in embodiment 1, also uses above-mentioned similar method, and replacement pA-BcDHD is right
It is big to convert the corresponding engineering of building acquisition respectively together for this two expression vectors of pA-accABCD and pET-acr1/BTE/CER1 afterwards
Enterobacteria.Using TargeTronTMGene knockout system (Sigma-Aldrich) knocks out the fadE gene of Escherichia coli.
The title in the site after BcDHD replaces is respectively adopted to name the bacterial strain.
3 fermenting and producing of embodiment
The culture of engineering colon bacillus
(50 μ gmL are included for being inoculated into M9 liquid medium after the engineering bacteria built activation in 1: 100 ratio-1
Kanamycins and 34 μ gmL-1Chloramphenicol), 37 DEG C, shaken cultivation under the conditions of 225rpm works as OD600nmWhen for 0.6-0.8, In
Inducer IPTG to final concentration 0.1mmolL is added in bacterium solution-1, then it is transferred at 30 DEG C, under the conditions of 225rpm, continues to cultivate
24h.Bacterium solution after Fiber differentiation is centrifuged 10min under the conditions of 12000g, collects supernatant, and extracted with isometric n-hexane
It takes 2-3 times, up to middle short chain alkanes after rotary evaporation removing n-hexane.N-hexane recycles.Obtained alkane is passed through into GC-
MS measures its composition and content.It is found by detection, a variety of Escherichia coli of Examples 1 and 2 building can effectively produce alkane
Hydrocarbon, specific content are as follows:
As can be seen from the above results, by having imported the Escherichia coli of bacillus cereus dehydrogenase in paraffin production
Improving aspect has obvious action, and output increased has the effect of significant more than 100%, and effect is can not be pre-
Material.
In addition, finding by mass spectrum, the structure of the alkane of the Escherichia coli production of bacillus cereus dehydrogenase has been imported
It is more nearly with the component of gasoline, is more suitable for the burning of gasoline, heating ratio can achieve the hot ratio of gasoline
95.3%, it is substituted for gasoline substantially.The hot ratio of alkane of Escherichia coli production without importing the dehydrogenase is
The 77.8% of gasoline, efficiency is slightly weaker.
Specific embodiment listed above is the explanation carried out to the present invention.It should be pointed out that above embodiments are only used
In the invention will be further described, protection scope of the present invention is not represented, other people prompts according to the present invention are made non-
The modification and adjustment of essence, still fall within protection scope of the present invention.
Sequence table
110 > Men Dongdong of <
A kind of method for producing bio-fuel of 120 > of <
〈160〉1
<210>1
<211>436
<212>PRT
<400> dehydrogenase
1 MKHFDVAIVG GGLAGLTASI YLAKAGRKVI VLEKSSHFGG RGMTINKNGI CMNLGAHALY
61 RGGEAFITFN ELGVNLPGGI PSTKAHGIWK GDIYTIPTDF RSILSTPLLS WSAKVQFARL
121 MIHLGNLDVE KVPKISLTTW AENEIKDPMV RNIFYALCRT TTYTYAPTIQ LASSVLKQIQ
181 LSMKEGVLYV DGGWETIITN LRGIANTSGV QFLAKKHVLK IEHCEGKQRI HCFDDEVFEA
241 GAVIVTTPPK EACKIIKGTE ETSLQKWSDQ SIQVTVAALD IGLKQLPNPL HHFALGLDQP
301 IFFTNQSRAA KLSEDGSIVV SLIKYHNPVL ELNHIQEEKE QLENMMELLH PNWKREVVAQ
361 QYLPKITVVH DFPHIDRVEK PGPNIPEMPG VYVAGDWAGY DEILADAAVA SGKRAALHIL
421 KKFESEAVHH GNGAVI
Claims (3)
1. a kind of engineering colon bacillus for being used to prepare biogasoline, is obtained by following methods: passing through polymerase chain reaction
Expand acetyl-C0A carboxylase gene, thioester enzyme gene, acyl-CoA reductase gene, fatty aldehyde decarboxylase gene and amino
Acid sequence is Bacillus cereus dehydrogenase gene shown in SEQ ID NO:1, recycles target fragment with plastic recovery kit, so
Double digestion target fragment and prokaryotic expression carrier pET-30a (+) or pACYCDuet-1 carrier, the recycling of carrier segment glue are distinguished afterwards
Afterwards, by carrier: genetic fragment is the ratio mixing of 1-2:4-5 in molar ratio, and 4-7 DEG C of connection 15-30 after T4DNA ligase is added
Hour, 38-42 DEG C of thermal shock Transformed E .coli DH5a competent cell of connection product, spread plate overnight incubation, PCR screening sun
Property clone；Positive colony extracts Plasmid DNA, and after digestion and sequencing identification, thermal shock Transformed E .coli BL21 (DE3) is finally knocked out
Up to purpose project Escherichia coli after the fadE gene of the engineering colon bacillus.
2. Escherichia coli as described in claim 1, it is characterised in that: there is a mutation on the site of SEQ ID NO:1, it is described prominent
Become G63A, N70S, K84T, I93D, S110G, R119F, D128S, L137D, I145S, K146K, L181G, S182H,
Any one of T196Q, V243I, A252S, S263P, F303G, Y362T, E379M, L404W or K422E.
3. a kind of enzyme, amino acid sequence is on the basis of SEQ ID NO:1 there are a mutation, it is described sport G63A,
N70S、K84T、I93D、S110G、R119F、D128S、L137D、I145S、K146K、L181G、S182H、T196Q、V243I、
Any one of A252S, S263P, F303G, Y362T, E379M, L404W or K422E.
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