CN101892091A - Method for expressing FAEES by recombinant algae and in-vivo bio diesel production - Google Patents
Method for expressing FAEES by recombinant algae and in-vivo bio diesel production Download PDFInfo
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- CN101892091A CN101892091A CN2009101428928A CN200910142892A CN101892091A CN 101892091 A CN101892091 A CN 101892091A CN 2009101428928 A CN2009101428928 A CN 2009101428928A CN 200910142892 A CN200910142892 A CN 200910142892A CN 101892091 A CN101892091 A CN 101892091A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
The invention belongs to the technical field of biological energy and genetic engineering, in particular relates to algae grease and a method for producing biodiesel thereby. The novel method for expressing FAEES by recombinant algae and producing in-vivo bio diesel comprises a method for preparing the biodiesel by using grease in algae body and (external source) ethanol to perform ester exchange reaction, namely the algae organisms are soaked in a culture medium containing ethanol, FAEES gene is induced into a gene group in the algae body by using agrobacterium rhizogenes as medium for expressing, and the FAEES produced in the algae body catalyzes fatty acid of the algae body per se to react with externally added (external source) ethanol under certain conditions to obtain the biodiesel and the side product glycerol. The method has mild technical condition, low cost and easy control, and has excellent biological industrial application prospect by using in-vivo grease in the algae organisms and in-vitro ethanol.
Description
Technical field
The invention belongs to bioenergy and gene engineering technology field, specially refer to the algae grease and be used to prepare method of bio-diesel oil.Specifically, having the present invention relates to the use recombinant algae and come the production method of bio-diesel oil, is a kind of biofuel method of low input high yield.Use a kind of algae, can express the active gene of lipid acid second fat synthetic enzyme (below be referred to as FAEES) by genetic engineering technique with one changes over to, the FAEES of algae expression in vivo under certain condition in the catalysis alginite lipid acid of itself react with extraneous (external source) ethanol that adds, thereby generate biofuel in the body.Maybe will contain the algae fragmentation of FAEES gene and energy stably express, frustule added (external source) ethanol of different amounts then through the separated and collected culturing mixt, 37 ℃ of reactions 1-24 hour, after carrying out external biological transesterification enzyme reaction, obtaining the own ester of lipid acid through separation and purification is biofuel.
Background technology
Current; Increase of population makes the contradiction of ever-increasing grease demand and natural resources critical shortage be becoming increasingly acute; particularly along with the global energy shortage and the environmental degradation that are on the rise; make people have to from the angle of environment protection and development of resources; active development substitutes the renewable new forms of energy of fossil fuel, is exactly a kind of have develop on a large scale very much potentiality and eco-friendly renewable and clean energy resource as biofuel.The desired premium properties of liquid energy product such as bioenergy has that energy density, sulphur content are low, sufficient combustion, lubricity are good.Since 1988, many European countries have begun the substitute of biofuel as conventional diesel, but because higher raw materials cost, make the price of biofuel be higher than conventional diesel, this has seriously restricted the industrialization process of biofuel, therefore choose suitable, oil resource is the direction of biofuel industry development cheaply.
The research of at present relevant biodiesel manufacture concentrates on the vegetables oil aspect basically, all be to be raw material with vegetables oil or food and drink waste grease, utilize vegetables oil to be raw material, the cost height, and be raw material with the food and drink waste grease, also exist raw material to be difficult to the difficulty of collecting and transporting, so these researchs are difficult to realize industrial applications.Utilizing Vegetable oil lipoprotein to prepare in the process of biofuel, raw materials cost has just accounted for the 70%-85% of total cost, and this has seriously restricted the industrial applications of biofuel.
But the algae bio body contains a large amount of greases, in the algae bio body, prepare biofuel (being lipid acid second fat and Fatty acid methyl ester) by genetic engineering technique, this can provide cheap more raw material for the preparation of biofuel, because the scope that algae distributes is extremely wide, require not tight to envrionment conditions, adaptability is stronger, also can live having only under extremely low nutrient concentration, atomic weak intensity of illumination and the quite low temperature.Rivers, streams, lake and ocean can not only be grown in, and of short duration ponding or moist place can be grown in.From the torrid zone to the two poles of the earth, to warm spring, in not really dark soil, the algae that almost grows on trees distributes from damp ground from the high mountain of accumulated snow.And do not need a large amount of labor forces.At present, the common method of production biofuel is an ester-interchange method, promptly is added on a certain amount of low-carbon alcohol in grease, as methyl alcohol or ethanol etc., under alkali or acid catalyst effect, generates biofuel in certain temperature and time reaction, produces by-product glycerin simultaneously.The present invention combines the technology that genetic engineering technique and transesterification reaction prepare biofuel, has proposed a kind of method with preparing biodiesel from lipid in the algae bio body, has not yet to see relevant report.
Reaction conditions gentleness of the present invention, cost is low, productive rate is high, raw material resources are abundant, has land occupation not simultaneously, does not consume the advantage of oily resource, has the favorable industrial application prospect.
Summary of the invention
Purpose of the present invention: aim to provide a kind of with high productivity high efficiency production method of bio-diesel oil, comprise and use a kind of algae, can express the active gene of FAEES by genetic engineering technique with one changes over to, do not have in algae under the condition of substantive propagation, the FAEES that produces in the body interior own lipid acid of catalysis bead frond under certain conditions reacts with the extraneous ethanol that adds (external source), thus the interior generation of body biofuel.
The present invention will disclose a kind of genetic engineering technique that utilizes and change the FAEES gene in algae, thereby express FAEES, and produce a kind of way of novel production biofuel.
Phycophyta is not have real root, stem, leaf differentiation in the vegitabilia, and with the photoautotrophy life, reproductive organ is by a unicellular big monoid that constitutes and do not have fetal development.With the chlorella is example, chlorella is the unicellular kind of swimming, cell is small, circular or slightly oval, cell walls is thin, and 1 cup-shaped or bent band shape chromatophore is arranged in the cell, and chromatophore split into several piece when cell was aging, no pyrenoids has only Chlorella pyrenoidesa (Chlorella pyrenoidosa Chick.) that pyrenoids is arranged.During monogony, the protoplasma division forms 2,4,8,16 autospores, and when the parent cell wall broke, spore is emitted became new plant materials.Chlorella is widely distributed in China, moves in the water such as containing organic river, irrigation canals and ditches, pond, and distribution is also arranged on wetland.
Embodiment of the present invention is as follows:
Pass through genetic engineering technique, under the acquired prerequisite of gene of expressing FAEES, carry out gene amplification (round pcr), vitro recombination, recombinant chou and import, pass through again the colony screening and the evaluation of recombinant chou to host cell, obtain can expression alien gene FAEES algae transition.
Host microorganism according to the present invention transition is a kind of algae, and this algae is different from the prior art mushroom as the host.
One of principal character of the present invention is, can express the active gene of FAEES by genetic engineering technique with one changes over to, (external source) ethanol and the intravital lipid acid of the FAEES external adding of catalysis chlorella under certain conditions of producing in this alginite react, thereby generate biofuel.This invention is leading, is in the leading level in the world, the invention that is significant.
Embodiment
Following embodiment describes the present invention in detail, but should not be considered as the restriction of the scope of the invention.
Embodiment one: algal species cultivation
The algae kind of buying back, the LB substratum of prepared and diluted, in algae liquid and 1: 2 ratio expanding species of nutrient solution in the Erlenmeyer flask of 1000mL.Triangular flask is placed in the illumination box cultivates, control its culture condition: intensity of illumination 75.3～80.1mmol/ms, temperature is 26 ℃, ph is 6～8.Stir every day three times, each 1 minute, treat that frond reaches after certain density in algae liquid and 1: 4 ratio expanding species of nutrient solution in the Erlenmeyer flask of 3000ml, is placed on the place that temperature is lower, light is more weak, the artificial feeding through filtered air cultivated, and expanding species is 1 time about per 3 week.The program of enlarged culturing is cultivation → production pond in the thin mouthful vial → bucket of 3000mL Erlenmeyer flask → 20L.
Embodiment two: the FAEES gene clone is to shuttle vectors
Goal gene FAEES is connected with the PET28 carrier, exists, be called for short PET-FAEES with the plasmid form.As template, carry out PCR with PET-FAEES.For clone FAEES gene in PCR, synthetic following primer also uses FAEES gene amplification primer:
(a-1)5’-ccgCTCGAG?ATA?GCC?ACC?ATGCCGCCCTACACC-3’
(b-1)5’-tgcTCTAGATCACTGTTTCCCGTTGCCATT-3’
Primer (a-1) contains the Xho restriction site, and primer (b-1) contains the Xba restriction site.
PCR (Polymerase Chain Reaction) carries out as reaction reagent with " DNA thermal cycling person " and Tag archaeal dna polymerase (Takara company) in following condition.Reactant:
PCR buffer reagent, 1.25mM dNTP mixture, template, primer (upstream and downstream), Tag enzyme, sterilized water.Above composition is mixed, and make this mixture carry out the PCR reaction.
The PCR circulation:
Denaturation process: 94 ℃ 60 ℃
Annealing process: 52 ℃ 60 ℃
Extension process: 72 ℃ 120 ℃
A circulation of being made up of above process repeats 30 circulations.After reaction is finished, above reaction mixture 10ul is carried out 1% agarose gel electrophoresis, detect PCR product clip size and whether meet FAEES clip size 1.2kb.
The FAEES gene is connected with shuttle vector:
Reclaim the FAEES purpose fragment of test kit (Invitrogen company) recovery as the PCR product, the specification sheets in the detailed step reference reagent box with glue.Fragment after the recovery is connected with the pGEM-T carrier, and its linked system is: reaction buffer, pGEM-T carrier, PCR product FAEES fragment, T4 ligase enzyme.React and change 4 ligations 16 hours after 1 hour over to.Blue hickie screening connects product, and enzyme is cut and obtained purpose fragment FAEES, makes up shuttle vectors.Above ligation product is transformed into the Escherichia coli JM109 cell of competence state with the method for calcium chloride, is applied to the solid medium (10g peptone, 5g yeast powder, 5gNaCl and 16g agar are dissolved in 1 liter of distilled water) that contains 0.2M IPTG.In the positive bacterium colony amplification cultivation of this substratum picking white, collecting cell and utilize alkaline lysis to prepare plasmid in a small amount after 16 hours.Utilize Xho and Xba double digestion plasmid then and reclaim FAEES purpose fragment.This fragment is connected with the expression vector that the Xba double digestion is handled through Xho with same, and its linked system is: pKYL71, FAEES, T4 ligase enzyme, sterilized water.Connecting product is the plasmid that goal gene FAEES is connected with the pKYL71 carrier, is called for short the FAEES-pKYL plasmid.
Embodiment three: cultivation of agrobacterium tumefaciens and conversion
Utilize the FAEES-vector plasmid that builds more than the freeze-thaw method handle to change in the agrobacterium tumefaciens competent cell, coat (LB+40mg/L Rifampin+25mg/L Streptomycin sulphate+75mg/L gentamicin) on the solid medium, picking list bacterium colony, in 2mL Agrobacterium liquid substratum, 28 ℃ of overnight incubation, get the above culture of 1mL, add in the 50mL Agrobacterium liquid substratum, 28 ℃ are cultured to OD
600=1.0, the 50mL agrobacterium liquid is collected thalline through the centrifugal 8min of 3500～4000r/min, resuspended with the UM1 liquid nutrient medium, 28 ℃ are continued to cultivate 3～4h, to OD
600=0.5.
Embodiment four: the genetic transformation of algae and the screening of transformant
Above cultured algae kind is put into above-mentioned Agrobacterium nutrient solution, at 190r/min, after cultivating 10min altogether on 28 ℃ of shaking tables, with the unnecessary bacterium liquid of centrifugal removal, place on the UM1 substratum that is covered with one deck aseptic filter paper, seal culture dish with sealing film, 25 ℃, cultivate about 60h altogether in the dark place, use sterilized water afterwards successively, contain the antibiotic sterilized water of 50mg/L and Agrobacterium is washed off, be placed on the UM2 substratum, seal culture dish with sealing film with containing the antibiotic UM1 of 50mg/L, cultivate the dark place, when treating that the algae kind grows into certain density, go to and continue in the UM3 substratum to cultivate, go to afterwards in the LB substratum of prepared and diluted and cultivate, obtain algae kind transformant, but not transformant is then dead gradually in containing the antibiotic substratum of 50mg/L.
Utilize preliminary pyrolytic technique to screen total fat output height, algae kind that unsaturated fatty acid content is high is cultivated in a large number.
Embodiment five: produce in the biofuel body of algae transformant
In the substratum of the algae that contains FAEES gene and energy stably express, add (external source) ethanol of different amounts then, algae absorbs ethanol, 37 ℃ of reactions 1-24 hour, after carrying out the interior biological transesterification enzyme reaction of body, obtaining the own ester of lipid acid through separation and purification is biofuel, and by product is a glycerine.
Embodiment six: the biofuel produced in vitro of algae transformant
To contain the algae fragmentation of FAEES gene and energy stably express, frustule is through the separated and collected culturing mixt, (external source) ethanol that adds different amounts then, 37 ℃ of reactions 1-24 hour, after carrying out external biological transesterification enzyme reaction, obtaining the own ester of lipid acid through separation and purification is biofuel, and by product is a glycerine.
Claims (5)
1. biotechnological means that utilizes the algae grease and be used to prepare biofuel.It is characterized in that, comprise and utilize interior grease of alginite and the extraneous ethanol generation transesterification reaction that adds to prepare method of bio-diesel oil, promptly can express the active gene of lipid acid second fat synthetic enzyme (below be referred to as FAEES) by genetic engineering technique with one changes over to, at the FAEES of algae the expression in vivo just intravital lipid acid of catalysis algae and extraneous (external source) ethanol synthesis that adds under certain conditions, obtain biofuel, by product is a glycerine.
2. by the described biotechnological means that utilizes the algae grease and be used to prepare biofuel of claim one.It is characterized in that, described utilize the algae grease and be used to prepare the step of biofuel as follows:
1) gene clone: clone's lipid acid second fat synthase gene, as people's liver cell lipid acid ethyl acetate synthetic enzyme FAEES gene.
2) plasmid construction.The FAEES gene is connected among the plasmid pUC19, obtains FAEES+pUC19.The preparation competent escherichia coli cell, transformed into escherichia coli.
3) plasmid amplification.Plasmid increases in intestinal bacteria.Extract plasmid.
4) construction of expression vector.With Xho I and Xba I digested plasmid pUC19, electrophoresis, glue reclaims goal gene.Use Xho I and Xba I digested plasmid pKy71 again, get sticky end.J is connected to the FAEES gene among the plasmid pKy71, obtains the FAEES+ expression vector.
5) transform Agrobacterium.Preparation Agrobacterium competent cell obtains plasmid with previous step and transforms Agrobacterium.
6) transfection algae: Agrobacterium and algae after the conversion are cultivated altogether, and the FAEES gene is inserted in the algae genome.
7) genetic expression: behind the flush away Agrobacterium, cultivate the transgenosis algae in a large number.
8) algae bio is cultivated in containing the alcoholic acid substratum in a large number.
9) detection of fatty-acid ethyl ester
Detect the content of lipid acid.When content no longer changed, broken frustule was through the separated and collected culturing mixt, and with Catalytic processes production biofuel, by product is a glycerine.
3. press the biofuel of the described production of claim two.
4. claim comprises lipid acid second fat synthetic enzyme and Fatty acid methyl ester synthetic enzyme, uses with external biological is synthetic in the body of biofuel (lipid acid second fat and Fatty acid methyl ester).
5. comprise chlorella by the described algae of claim, little algae, common algae and other uncommon algae, fresh water and salt water algae.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2009101428928A CN101892091A (en) | 2009-05-20 | 2009-05-20 | Method for expressing FAEES by recombinant algae and in-vivo bio diesel production |
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| CN2009101428928A CN101892091A (en) | 2009-05-20 | 2009-05-20 | Method for expressing FAEES by recombinant algae and in-vivo bio diesel production |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120214212A1 (en) * | 2011-02-17 | 2012-08-23 | Dr. Chifu Huang | Expression and application of recombinant FAEES in algae as a novel method to produce biodiesel in vivo |
| CN104293824A (en) * | 2013-07-15 | 2015-01-21 | 丰益(上海)生物技术研发中心有限公司 | Transformation method of Crypthecodinium cohnii |
| CN105087629A (en) * | 2015-09-18 | 2015-11-25 | 宜春学院 | Method for expressing HuIFN-alpha 2b genes by chlorella |
-
2009
- 2009-05-20 CN CN2009101428928A patent/CN101892091A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120214212A1 (en) * | 2011-02-17 | 2012-08-23 | Dr. Chifu Huang | Expression and application of recombinant FAEES in algae as a novel method to produce biodiesel in vivo |
| CN104293824A (en) * | 2013-07-15 | 2015-01-21 | 丰益(上海)生物技术研发中心有限公司 | Transformation method of Crypthecodinium cohnii |
| CN105087629A (en) * | 2015-09-18 | 2015-11-25 | 宜春学院 | Method for expressing HuIFN-alpha 2b genes by chlorella |
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Application publication date: 20101124 |