CN106318894B - A method of manufacture bio-fuel - Google Patents
A method of manufacture bio-fuel Download PDFInfo
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- CN106318894B CN106318894B CN201610697329.7A CN201610697329A CN106318894B CN 106318894 B CN106318894 B CN 106318894B CN 201610697329 A CN201610697329 A CN 201610697329A CN 106318894 B CN106318894 B CN 106318894B
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N9/93—Ligases (6)
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- C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
- C12Y103/01008—Acyl-CoA dehydrogenase (NADP+) (1.3.1.8)
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- C12Y301/02—Thioester hydrolases (3.1.2)
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- C12Y604/00—Ligases forming carbon-carbon bonds (6.4)
- C12Y604/01—Ligases forming carbon-carbon bonds (6.4.1)
- C12Y604/01002—Acetyl-CoA carboxylase (6.4.1.2)
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
Abstract
The invention belongs to bio-fuel fields, more particularly to a kind of method for preparing bio-fuel, vibrio parahemolyticus dehydrogenase is imported into the inverted Escherichia coli for having accABCD/acr1/BTE/CER1 gene and fadE gene knockout, expression can be obtained corresponding alkane.The production efficiency of alkane of the present invention is greatly enhanced, and prepares simply, and alkane secretion separates outside protecting and purifying, can be suitable for industrialized production.
Description
Technical field
The invention belongs to bio-fuel fields, and in particular to a method of manufacture 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 102277211 A of CN.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 101165188 A of CN 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 vibrio parahemolyticus 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 vibrio parahemolyticus 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 350 kinds of substitutions, finally screen to obtain P122, A124,
L133、A135、V183、Q184、T244、K259、G267、D302、L324、L365、A422、F481、V482、T543、I549、
This 21 kinds of sites Q588, T601, D613, K669 are enzyme activity critical sites, can enhance corresponding enzyme activity after being substituted, and remaining
Cannot cause enzyme activity improve some even result in enzyme activity reduce 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 P122S, A124P, L133H, A135Q, V183S, Q184G, T244V, K259H, G267P, D302S,
L324V,L365D,A422M,F481A,V482P,T543R,I549A,Q588G,T601S,D613Q,K669P.Wherein P122S table
Show that the P of the 122nd position in the amino acid sequence of SEQ ID NO:1 is substituted by S.
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 vibrio parahemolyticus dehydrogenase GI (NCBI): KYX36827 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 T4 DNA 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 vibrio parahemolyticus dehydrogenase of embodiment and the building of carrier
The clone of vibrio parahemolyticus dehydrogenase gene
Total mRNA of vibrio parahemolyticus WP-1 is extracted, then reverse transcription is cDNA, is set according to GenBank:KYX36827
It counts primer and restriction enzyme site is added at primer both ends, PCR amplification clones its dehydrogenase gene DHD, and plastic recovery kit is recycled to return
Receive target gene.
By glue dehydrogenase gene DHD after the recovery and pACYCDuet-1 carrier (Novagen) with BamH I and EcoR1 into
Row double digestion, carrier and exogenous sequences in molar ratio 1: 5 ratio, 4 DEG C connection overnight, connection product Transformed E .coli DH5 α,
PCR screening positive clone is identified after extracting recombinant plasmid pA-DHD in positive colony, then through restricted digestion and sequencing.
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-DHD-P122S, pA-DHD-A124P, pA-DHD-L133H, pA-DHD-A135Q, pA-DHD-
V183S、pA-DHD-Q184G、pA-DHD-T244V、pA-DHD-K259H、pA-DHD-G267P、pA-DHD-D302S、pA-
DHD-L324V、pA-DHD-L365D、pA-DHD-A422M、pA-DHD-F481A、pA-DHD-V482P、pA-DHD-T543R、
pA-DHD-I549A、pA-DHD-Q588G、pA-DHD-T601S、pA-DHD-D613Q、pA-DHD-K669P。
The building of 2 recombination bacillus coli of embodiment
It obtains according to the method for 101899412 A embodiment 1 of CN containing pA-accABCD and pET-acr1/BTE/
Two expression vectors of CER1.
The recombinant plasmid pA-DHD 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 positive expression vector by PCR screening acquisition positive colony together
Property clone plasmid after again by digestion and sequencing identification.It thereby is achieved containing pA-DHD, pA-accABCD and pET-acr1/
The engineering colon bacillus of tri- expression vectors of BTE/CER1.Engineering colon bacillus after conversion, using TargeTronTMClpp gene
Except fadE gene of the system (Sigma-Aldrich) to Escherichia coli knocks out.The bacterial strain is DHD/accABCD/acr1/
BTE/CER1/-fadE Escherichia coli, it is simple to name are as follows: DHD bacterial strain.
The substituted expression vector being prepared in embodiment 1 also uses above-mentioned similar method, replaces pA-DHD respectively
It is big that the corresponding engineering of building acquisition respectively is converted together with this two expression vectors of pA-accABCD and pET-acr1/BTE/CER1
Enterobacteria.Using TargeTronTMGene knockout system (Sigma-Aldrich) knocks out the fadE gene of Escherichia coli.
The title in the site after DHD- 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 bacterium of Examples 1 and 2 building can effectively produce
Alkane, specific content are as follows:
As can be seen from the above results, by having imported the Escherichia coli of vibrio parahemolyticus dehydrogenase in paraffin production
Improving aspect has obvious action, and output increased has the effect of significant more than 90%, and effect is to fail to predict
's.
In addition, finding by mass spectrum, the structure of the alkane of the Escherichia coli production of vibrio parahemolyticus 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
93%, it is substituted for gasoline substantially.The hot ratio of alkane without importing the Escherichia coli production of the dehydrogenase is vapour
The 78% of oil, 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 manufacturing bio-fuel of 120 > of <
〈160〉1
<210>1
<211>713
<212>PRT
<400> dehydrogenase
1 MKQILQDMAK GGSNLVQAPA PKATTGNMLI DTSISLISAG TERMLVDFGK ASLLDKARQQ
61 PDKVKMVLEK VQTDGLMTTL EAVQSKLAQP LPLGYCNVGV VNAVGKGVQG FKEGDRVVSN
121 GPHADVVRVP KNLCALIPDN VSDEEASFTV VASIGLQGIR LAEPTLGEAF VVTGVGLIGL
181 LTVQLLRAQG CRVLAIDYDD AKLELAKQFG AEVCNPGKGE DPVAAGMAFS RGNGVDGVII
241 TASTKSNDPV TQAARMSRKR GRIILVGVTG LELNRADFYE KELTFQVSCS YGPGRYDANY
301 EDNGQDYPYA FVRWTEQRNF EAILDMMASG QVDVKPLITH RFKFEDAEQA YDLLTSDKSA
361 LGILLQYQSD SSARHEKSIT LNSEATFEAS KPVVGFVGAG NYASRMLIPA FKSGGAQLHT
421 IATSGGINGV THGEKAGFAK ATTDTSAMIS DSEINTIAIV TRHNSHAHFV SEALNAGKNV
481 FVEKPLAVTL EELAEVEATY YSKASTASAP KLMVGFNRRF SPQVQKMKEL LSSVKEPKSF
541 MMTMNAGAIP ADHWTQDNEI GGGRIIGEAC HFIDLMRYLA DSDIVSVQCR RMGDVDSVDI
601 TEDKAAIILG FADGSFGTIH YLANGAANFP KERVEVFAAG RVLQLDNFRK LKGFGWPGFS
661 KMNLWKQDKG QTGCAAAFLS AIESGKESPI PPRELFEVAR VTIEIAEQLR KQQ
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 vibrio parahemolyticus 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 in the sequence of SEQ ID NO:1, it is described prominent
Become P122S, A124P, L133H, A135Q, V183S, Q184G, T244V, K259H, G267P, D302S, L324V, L365D,
Any one of A422M, F481A, V482P, T543R, I549A, Q588G, T601S, D613Q or K669P.
3. a kind of enzyme, amino acid sequence is to have a mutation on the site of SEQ ID NO:1, it is described sport P122S,
A124P、L133H、A135Q、V183S、Q184G、T244V、K259H、G267P、D302S、L324V、L365D、A422M、
Any one of F481A, V482P, T543R, I549A, Q588G, T601S, D613Q or K669P.
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| CN201610697329.7A CN106318894B (en) | 2016-08-20 | 2016-08-20 | A method of manufacture bio-fuel |
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| CN106318894B true CN106318894B (en) | 2019-11-05 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899411A (en) * | 2009-08-12 | 2010-12-01 | 青岛生物能源与过程研究所 | Engineering escherichia coli for preparing medium-carbon fatty alcohol |
| CN101899412A (en) * | 2009-08-12 | 2010-12-01 | 青岛生物能源与过程研究所 | Engineering colon bacillus for preparing biological gasoline |
-
2016
- 2016-08-20 CN CN201610697329.7A patent/CN106318894B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899411A (en) * | 2009-08-12 | 2010-12-01 | 青岛生物能源与过程研究所 | Engineering escherichia coli for preparing medium-carbon fatty alcohol |
| CN101899412A (en) * | 2009-08-12 | 2010-12-01 | 青岛生物能源与过程研究所 | Engineering colon bacillus for preparing biological gasoline |
Non-Patent Citations (1)
| Title |
|---|
| dehydrogenase [Vibrio parahaemolyticus];WP_062907030;《NCBI Reference Sequence: WP_062907030.1》;20160407;全文 * |
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| Publication number | Publication date |
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| CN106318894A (en) | 2017-01-11 |
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