CN101372669B - Cyanobacteria modified by gene engineering and use thereof for producing ethanol - Google Patents
Cyanobacteria modified by gene engineering and use thereof for producing ethanol Download PDFInfo
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Abstract
The invention relates to the technical field of biological energy source, in particular to a cyanobacteria synechocystis mutant, a method for screening the synechocystis mutant, a plasmid expression vector introduced with a promoter sequence which is encoded with pyruvate decarboxylase polynucleotide and/or is inducted by nitrate, the synechocystis mutant introduced with the promoter sequence which is encoded with the pyruvate decarboxylase polynucleotide and/or is inducted by the nitrate, and the application of the mutant to prepare ethanol.
Description
Technical field
The present invention relates to the bioenergy technical field, be specifically related to a kind of cyanobacteria cytoalgae mutant strain, the method for screening cyanobacteria cytoalgae mutant strain, the plasmid expression vector that has imported coding pyruvic carboxylase polynucleotide and/or nitrate inductive promoter sequence, the above-mentioned cytoalgae mutant strain that has imported coding pyruvic carboxylase polynucleotide and/or nitrate inductive promoter sequence and the application of said mutation strain in preparation ethanol.
Background technology
Since the Industrial Revolution of Britain, development of human society is just closely bound up with the use of the energy before more than 250 years.Along with the continuous development of global economy, with oil, coal and Sweet natural gas are that the exploitation of main fossil energy and use problem seem and become increasingly conspicuous: one, and oil, coal and Sweet natural gas etc. all is non-renewable resource, they can be consumed exhaustion in the near future; Its two, developed countries such as the U.S. make the geographic upheaval of world local can produce the great variety of oil price to the dependence of Imported oil, directly have influence on Economic development, social stability or even national security; Its three, use traditional fossil energy can discharge great amount of carbon dioxide isothermal chamber gas, cause serious environmental to pollute and global climate change.So energy problem and environmental problem have become the most important subject under discussion of international summit now.In order to ensure energy security, protect environment, walk the road of Sustainable development, substitute energy and renewable energy source have obtained various countries and have more and more paid attention to.
In the renewable energy source, biomass energy with the fastest developing speed.Biomass (Biomass) mainly refer to all kinds of plants of occurring in nature, comprise farm crop, become chemical energy to contain in plant organism solar energy converting by photosynthesis.Being based upon the fuel ethanol production of biomass ferment method, is to be raw material with cereal, corn, wheat, potato class, molasses etc., makes through fermentation, distillation, and the fermentation method ethanol back of further dewatering is formed denatured fuel ethanol.There are many shortcoming parts in the biomass alcohol production.At first, it will be fought for and consume numerous plant resourceses, make the supply of grain, feed occur in short supply.Secondly, biomass production ethanol need be considered raw material and ethanol transportation problem, problems such as biomass pre-treatment and solid waste processing.Moreover the greenhouse gases carbonic acid gas of biomass production ethanol production process discharging will aggravate the rising of global temperatures.
For overcoming the problem that above-mentioned new forms of energy production exists, be necessary to research and develop the new bio technology in order to produce ethanol.To this, patent (U.S. Patent number 6,306,639, the calendar year 2001 of Canadian Ennol Energy Inc. application; With 6,699,696,2004) and the United States Patent (USP) (" Methods and Composition for Ethanol Producing Cyanobacteria " of Hawaii, America university application, pending, 2007) all relate to by genetic engineering modified cyanobacteria and produce ethanol.But there is following defective in Canadian Ennol Energy Inc. patented technology:
A, cyanobacteria unit cell algae Synechococcus PCC 7942 use the salt water nutrient solution, and production line is easy cleaning not, and equipment and pipeline are subject to the salinity corrosion;
B, promotor are temperature-induced, and necessary elevated temperature is to express the alcohol production gene.This can influence cyanobacteria unit cell algae activity;
C, no heat-resisting anti-alcohol performance;
D, alcohol production concentration are low;
E, alcohol production gene pdc and adh are present in the unit cell algae 7942 with the plasmid form, and stability is bad.Need to use a large amount of microbiotic to keep the stability of plasmid in the strain of unit cell phycomycete, thereby improved production cost.
In addition, for Hawaii, America university patented technology, the problem of its existence is:
The alcohol production genetic expression promotor of a, cyanobacteria cytoalgae 6803 is photoinduction, alcohol production decrease in efficiency when night or cloudy day;
B, no resistance toheat insert that (LmrA LmrCD) improves anti-alcohol performance and do not obtain experimental data and confirm from the omrA gene of Oenococcus oeni or homologous gene;
C, alcohol production concentration are not high enough.
Summary of the invention
The present inventor has carried out unremitting research in order to address the above problem, and the result passes through orthogenesis cyanobacteria cytoalgae 6803 (available from U.S. American Type Culture Collection (ATCC), ATCC
Number:27184
TM) and obtain its mutant strain Synechocystis strictus (abbreviating S.strictus as), this mutant strain has stronger heat-resisting anti-alcohol performance.Further, the inventor operates this bacterial strain by engineered means, makes that finally the cytoalgae mutant strain can light harvesting cooperation usefulness, and carbon dioxide collection and alcohol production three functions are in one, thereby has finished the present invention.
The present invention relates to a kind of mutant strain of cyanobacteria cytoalgae, the method for screening the mutant strain of this cytoalgae, the plasmid expression vector that has imported coding pyruvic carboxylase polynucleotide and/or nitrate inductive promoter sequence, the above-mentioned cytoalgae mutant strain that has imported coding pyruvic carboxylase polynucleotide and/or nitrate inductive promoter sequence and the application of said mutation strain in preparation ethanol.
Particularly, the invention provides following technical scheme:
1, cyanobacteria cytoalgae, its preserving number are CGMCC NO.2137.
2, the method for screening above-mentioned 1 described cyanobacteria cytoalgae, it has used the method for heat-shocked.
3, cyanobacteria cytoalgae, it has the polynucleotide of the active polypeptide of pyruvic carboxylase for having imported to encode in above-mentioned 1 described cyanobacteria.
4, above-mentioned 3 described cyanobacteria cytoalgaes, wherein said polynucleotide are:
(a) polynucleotide, it contains by sequence number: the polynucleotide that 1 base sequence is formed;
(b) polynucleotide, it contains and sequence number: polynucleotide or sequence number that 1 base sequence is formed: polynucleotide hybridize under stringent condition that the complementary base sequence of 1 base sequence is formed and coding have the active proteinic polynucleotide of pyruvic carboxylase; Or
(c) polynucleotide, it is a sequence number: the polynucleotide that 1 base sequence or its complementary base sequence are formed.
5, above-mentioned 4 described cyanobacteria cytoalgaes, it further contains nitrate inductive promotor.
6, above-mentioned 5 described cyanobacteria cytoalgaes, wherein said promotor is:
(a) polynucleotide, it contains by sequence number: the polynucleotide that 2 base sequences are formed;
(b) polynucleotide, it contains and sequence number: polynucleotide or sequence number that 2 base sequence is formed: polynucleotide hybridize under stringent condition that the complementary base sequence of 2 base sequence is formed and coding have the polynucleotide of nitrate inductive promoter activity; Or
(c) polynucleotide, it is a sequence number: the polynucleotide that 2 base sequence and/or its complementary base sequence are formed.
7, polynucleotide and/or promotor that any described cyanobacteria cytoalgae of above-mentioned 3-6, wherein said coding have an active polypeptide of pyruvic carboxylase are carried by plasmid expression vector.
8, above-mentioned 7 described cyanobacteria cytoalgaes, wherein said plasmid expression vector sequence is a sequence number: the sequence shown in 3.
9, the application of any described cyanobacteria of above-mentioned 1-8 in producing ethanol.
Description of drawings
The structural representation of Fig. 1 recombinant plasmid pSKBPDC.
The structural representation of Fig. 2 recombinant plasmid pPDCNIRA.
Fig. 3 utilizes bioreactor to observe the test of ethanol production rate out of doors, and wherein Fig. 3 a is a temperature variation curve, and Fig. 3 b is cell concn and alcohol production ability.
Embodiment
Can produce alcoholic acid microorganism cells (for example intestinal bacteria, yeast, motion fermentation sporangium etc.) for all, known alcohol production path be pyruvic acid from glycolytic pathway through acetaldehyde again to terminal meta-bolites ethanol.Pyruvic acid is controlled by Pyruvate Decarboxylase Gene pdc to the reaction process of acetaldehyde, and acetaldehyde is controlled by alcohol dehydrogenase gene adh to the alcoholic acid reaction process.Cyanobacteria exists the earth existing more than 3,500,000,000 years, is one of biology the most ancient on the earth.Its adaptive faculty is very strong, can stand high temperature, and is freezing, anoxic, dry and high salinity, many abominable life conditions such as severe radiation.Owing to do not have Pyruvate Decarboxylase Gene pdc in its genome, cause the alcohol production path incompleteness of wild strain cytoalgae 6803, thereby do not have the alcohol production ability.The present invention adopts engineered method, and a kind of aquatic green plants cyanobacteria such as low (Cyanobacteria is called for short cyanobacteria, is commonly called as blue-green algae or blue-green algae) is transformed.Thereby make cyanobacteria absorb the energy in the sunlight, directly carbonic acid gas is converted to ethanol.The cyanobacteria cytoalgae Synechocystis sp.PCC 6803 (being designated hereinafter simply as " cytoalgae 6803 ") that the present invention uses is a kind of non-thread and non-fixed nitrogen fresh water bacterial strain, has autotrophy and heterotrophic growth ability concurrently.Cytoalgae 6803 is that (Ikeuchi et al., Tanpakushitsu Kakusan Koso 1996,41 (16): 2579-2583), this has established important foundation for the engineered development of algae to first genome light autotrophic microorganism of being checked order fully.Cytoalgae 6803 can merge foreign DNA, by homologous recombination foreign DNA is incorporated on the chromosomal DNA.Because can transform naturally, its genome can accurately regulate by recombinant DNA technology (Aoki et al., J MicrobiolMethods 2002,49 (3): 265-74., Vermaas et al., Proc Natl Acad Sci U S A, 1986,83 (24): 9474-9477, Williams, Methods Enzymol., 1988,167:766-778).So cytoalgae 6803 is idealized systems that a kind of research and utilization plant is put photosynthetic biology of oxygen and metabolic process.It also is an expression system commonly used in the research of algae genetically engineered.People detoxifying gene, people source superoxide dismutase gene and mouse metallothionein gene successfully have been transferred in the cytoalgae 6803 (Sun Jun. biotechnology progress, 1994,14 (6): 39～42).
The present invention utilizes genetic engineering technique synthetic pyruvic carboxylase pdc and nirA promotor, it is connected with high-expression plasmid carrier PSKAIIKS, constructed recombinant plasmid pPDCNIRA, be inserted into the cyanobacteria cytoalgae mutant strain S.strictus that screens with " heat-shocked " orthogenesis then, thereby be communicated with the ethanol synthesis path, construct recombinant bacterial strain, wherein said cyanobacteria cytoalgae mutant strain S.strictus has very strong heat-resisting anti-ethanol ability.This recombinant bacterial strain that constructs can be induced with nitrate, utilizes solar energy and carbon dioxide production ethanol efficiently, thereby makes that this cytoalgae recombinant bacterial strain can light harvesting cooperation usefulness, and carbon dioxide collection and alcohol production three functions are in one.
The present inventor improves cytoalgae 6803 heat-resisting anti-ethanol abilities by the method for " orthogenesis ".Use carbonic acid gas (CO
2) need adopt the industrial photosynthetic organism reactor assembly of outdoor closed conduit cyanobacteria cytoalgae and gather the demand that sunlight satisfies the cyanobacteria photosynthetic response in the daytime as the extensive fuel ethanol production of carbon source.The growth temperature of general cytoalgae is about 30 ℃, and noon in summer, external temperature may be up to more than 40 ℃, and this normal growth and fuel ethanol production ability to cytoalgae has been brought adverse influence.For example, wild strain cytoalgae 6803 is not cultivated in adding temperature controlled outdoor photosynthetic organism reactor, uses the BG-11 nutrient solution.Since the very fast whole inactivations of cyanobacteria chlorophyll that just make of high temperature in the daytime, thus " albefaction " phenomenon produced, lose energy for growth; Moreover wild strain cytoalgae 6803 adds in the photosynthetic organism reactor of temperature controlled laboratory to be cultivated, and gathers 50 milliliters and places bottle, puts into 45 ℃ of water baths " heat-shocked " and takes out after two hours.Put back to 30 ℃ of shaking tables and cultivate, cyanobacteria has produced completely " albefaction " phenomenon equally after three days.
The alcohol fuel industrial production increases temperature control if desired, will additionally increase the investment of equipment and energy aspect.The present inventor has adopted the method for " orthogenesis " to improve the cyanobacteria temperature capacity, makes it and produce alcohol fuel at normal growth under the hot conditions, thereby has avoided temperature controlled needs.When the cyanobacteria mutant strain improves temperature capacity, also can improve anti-ethanol ability.
The cyanobacteria cytoalgae mutant strain that is obtained by " orthogenesis " also has anti-ethanol ability except temperature capacity.A big problem of using cyanobacteria to produce alcohol fuel is to run up to certain concentration (for example 5% concentration) when ethanol in nutrient solution, will hinder the growth of cyanobacteria.When alcohol concn continuation increase, cyanobacteria may begin death.Improving the anti-ethanol ability of cyanobacteria cytoalgae becomes a key factor of fuel ethanol production.Current research is the result show, temperature raises, extraneous unfavorable factors such as alcohol concn increase, pair cell can produce similar stress, all can make the interior heat shock protein(HSP) height of cell express (Roy et al., JOURNAL OF BACTERIOLOGY, 180 (15): 3997-4001,1998; Glatz et al, ActaBiologica Szegediensis.46 (3-4): 53,2002).Simultaneously, result of study shows also and shows that heat-resisting anti-ethanol has interactivity (Michel et al., 1986).Work as intestinal bacteria, when the heat resistance of yeast and cyanobacteria increased, their anti-ethanol ability also increased (Horvath et al., Biochemistry, 95:3513-3518,1998) simultaneously.In wild strain cyanobacteria and " orthogenesis " cyanobacteria bacterial strain, add 5% ethanol and cultivate, find after three days that " albefaction " appears in wild strain cyanobacteria cytoalgae 6803, and the cytoalgae mutant strain of " orthogenesis " is uninfluenced.
The cyanobacteria cytoalgae mutant strain called after cyanobacteria cytoalgae Cyanobacteria Synechocystis (abbreviating S.strictus as) that the present invention will screen by above-mentioned orthogenesis, this microorganism on August 22nd, 2007 at the common micro-organisms center (CGMCC of China Committee for Culture Collection of Microorganisms, the address: carried out preservation Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica), deposit number is: CGMCCNO.2137).
The present invention adopts the Pyruvate Decarboxylase Gene pdc (sequence number: 1) with available nitrate inductive nirA promotor (sequence number: 2) with the homology high-expression plasmid carrier PSKAIIKS of cyanobacteria cytoalgae 6803 be connected of molecule clone technology with synthetic, constructed recombinant plasmid pPDCNIRA, change above-mentioned cyanobacteria cytoalgae mutant strain again over to, thereby construct recombinant bacterial strain, this project bacterium can be induced with nitrate, utilizes solar energy and carbon dioxide production ethanol efficiently.The present invention adopts the nirA promotor (not being subjected to temperature, illumination effect) of chemical induction to reach the alcohol production genetic expression of stability and high efficiency, improves alcohol production efficient; And the present invention adopts " heat-shocked " orthogenesis to obtain the cyanobacteria cytoalgae mutant strain with heat-resisting anti-alcohol performance.In addition, Comparatively speaking, the experimental data of Ennol Energy Inc. and University of Hawaii all produces photoproduction reactor in the laboratory, and the inventor also uses outdoor experimental installation to obtain than they higher alcohol production results except with the photoproduction reactor in the laboratory.
For obtaining to produce alcoholic acid cyanobacteria cytoalgae of the present invention, the polynucleotide that coding can be had the active polypeptide of pyruvic carboxylase import the above-mentioned cyanobacteria cytoalgae mutant strain that obtains, as long as the polynucleotide that import can be encoded and be had the active polypeptide of pyruvic carboxylase and just can realize purpose of the present invention, such polynucleotide are preferably sequence number: the sequence shown in 1.
On the other hand, those of ordinary skill in the art should be understood that, keto acid decarboxylase gene used in the present invention and available nitrate inductive promoter sequence are not limited to sequence number: 1 and sequence number: what 2 described sequences, coding had active proteinic other polynucleotide of keto acid decarboxylase or nucleic acid and other can all be can be used for the present invention by nitrate inductive promoter sequence.
For example, Pyruvate Decarboxylase Gene of the present invention and available nitrate inductive promoter sequence also comprise following polynucleotide, it contains and sequence number: 1 or sequence number: the polynucleotide hybridize under stringent condition that the complementary base sequence of 2 base sequences is formed, and have the polynucleotide of pyruvic carboxylase activity or nitrate inductive promoter activity, perhaps it contains and sequence number: 1 or sequence number: the polynucleotide hybridize under stringent condition that 2 base sequences are formed, and polynucleotide with pyruvic carboxylase activity or nitrate inductive chemistry promoter activity.
" polynucleotide of hybridize under stringent condition " herein, be meant by sequence number: 1 or sequence number: all or part of of the polynucleotide that the complementary base sequence of 2 base sequence is formed is probe, by the polynucleotide (for example DNA) that use colony hybridization method, plaque hybridization method or Southern hybrid method etc. to obtain.Hybridizing method for example can utilize Molecular Cloning 3rd Ed., Current Protocols inMolecular Biology, John Wiley ﹠amp; Method described in the Sons 1987-1997 etc.
For " stringent condition " described in this specification sheets, for example be 5 * SSC, 5 * Denhardt solution, 0.5%SDS, 50% methane amide, 32 ℃ condition; Perhaps, for example be 5 * SSC, 5 * Denhardt solution, 0.5%SDS, 50% methane amide, 42 ℃ condition; Perhaps, for example, be 5 * SSC, 5 * Denhardt solution, 0.5%SDS, 50% methane amide, 50 ℃ condition.In above-mentioned condition, improve temperature more, can expect the polynucleotide (for example DNA) that acquisition efficiently has high homology more.The factor of influence hybridization severity can be multiple factors such as temperature, concentration and probe concentration, probe length, ionic strength, time, salt concn, and those skilled in the art can realize same stringent condition by suitable these factors of selecting.
It is also to be noted that ethanol as herein described not only refers to alcohol fuel, the ethanol of other purposes is also contained within the present invention.
Below, be described in detail the present invention by embodiment, but the invention is not restricted to following examples.
Embodiment 1
Concrete operations: wild strain cytoalgae 6803 is seeded in that (shaking bottle by built-in 6 150ML of repacking shaking table forms in the photosynthetic organism set of reactors, working fluid is 50ML), and cultivate by the BG-11 nutrient solution, the shaking table built-in light source, the reactor surface light intensity is about 200Einstein/m
2/ s.The shaking table temperature adjusting is being lower than 30 ℃, and speed is 200RPM.Put into the hot water bath cabinet heat-shocked of heating after 5 days.Put back to then in 30 ℃ of photosynthetic organism reactors, wait to have produced completely (about 3 days) after " albefaction " phenomenon, centrifugation cytoalgae 6803 cells are outwelled supernatant liquor, use fresh BG-11 nutrient solution to cultivate cytoalgae 6803 cells of " albefaction ".The cytoalgae cell that has adapted to heat-shocked will regenerate chlorophyll, and then recovers photosynthesis and energy for growth.Simultaneously, the metabolic mechanism of cell can take place gradual, and the cytoalgae mutant strain is with the rising of energy adaptive temperature.The inventor has spent " orthogenesis " process that year carries out above-mentioned cyanobacteria cytoalgae cell heat-shocked-albefaction-recovery repeatedly, increases temperature (being raised to 47 ℃ from 35 ℃) and heat-shocked time (being raised to 4 hours from 30 minutes) gradually.Obtained the cytoalgae mutant strain of preservation of the present invention at last, it can be at normal growth under the outdoor high temperature condition in summer (＞45 ℃).
Embodiment 2: Pyruvate Decarboxylase Gene pdc and the design of nirA promotor
By the method for information biology, be easy to obtain the sequence (the search engine http://www.kegg.com/ that the present inventor uses Japanese Kyoto Encyclopedia of Genes and Genomes to provide obtains the sequence of Pyruvate Decarboxylase Gene pdc and nirA promotor) of Pyruvate Decarboxylase Gene pdc and nirA promotor.1) and nirA (sequence number: 2) can be successively obtain (Roy, et al.European Journal of Biochemistry.191 (3): 647-652,1990 Pdc (sequence number: with artificial synthetic method; Shabalina, et al.Nucleic Acids Res.34:2428-2437,2006; Satya et al.Proc IEEE Comput SocBioinform Conf.2:294-305,2003; Kim, et al.Gene.199:293-301,1997).The sequence of above-mentioned acquisition is analyzed with Sanger method (F.Sanger, Science, 214,1215,1981), to confirm base sequence.
Embodiment 3:pdc gene transformation cytoalgae mutant strain S.strictus
According to position and the sequence of ampicillin resistant gene, aphX and type froctosan saccharase gene sacB among the cyanobacteria cytoalgae 6803 endogenous plasmid PSBAIIKS, for transform Pyruvate Decarboxylase Gene pdc designed one couple of PCR primers P1 (sequence number: 4) and P2 (sequence number: 5):
P1 (5 end primer): 5 '-GGAGTAAG
CATATGAGTTATACTG-3 '
NdeI
P2 (3 ' the end primer): 5 '-GGATCTCGACTCTAGA
GGATCC-3 '
BamHI
Wherein primer P1 has designed a NdeI restriction enzyme site, and its sequence is CA ↓ TATG, and primer P2 has designed a BamHI restriction enzyme site, and its sequence is GGATC ↓ C.To get the pdc dna fragmentation P of 1410bp after the conventional pcr amplification method amplification.PCR reactant: in the aseptic centrifuge tube of 0.5mL, add successively: deionized water 36 μ l; 10XTaq enzyme buffer liquid 5 μ l; 4XdNTP (2.5mmol/L) 5 μ l; P1 primer 1 μ l; P2 primer 1 μ l; Template (being the pdc of synthetic) 1 μ l; Taq enzyme (3U/ μ l) 1 μ l.Totally 50 μ l.
PCR reaction heat loop parameter: at first 94 ℃ of pre-sex change are 5 minutes.; Follow 94 ℃ of sex change 30 seconds.; 50 ℃ of renaturation 45 seconds.; 72 ℃ were extended 50 seconds; 35 circulations; 72 ℃ were extended 5 minutes again.; 4 ℃ of terminations.
After reaction finishes, get 2.5 μ l pcr amplification products and carry out 1% agarose gel electrophoresis (2XTAE, 100V voltage, 40 minutes), a treaty 1410bp bright band on electrophoretogram, occurs, show that what will increase is the pdc gene fragment that expection will transform.Electrophoresis reclaims the 1410bp fragment.According to the restriction enzyme site in the primer, with restriction enzyme NdeI and BamHI while double digestion fragment P and carrier PSBAIIKS, connect with the T4DNA ligase enzyme then, connector transformation receptor bacterium E.coli C600, obtain recombinant plasmid pSKBPDC by the Amp screening, this plasmid size is 5.58kb (Fig. 1).As shown in Figure 1, the Pyruvate Decarboxylase Gene pdc of synthetic is inserted into the downstream of promotor psbAII, the position of promptly original aphX and sacB gene.Utilize nature to transform integrating expression vector is imported cytoalgae mutant strain S.strictus, and change the Pyruvate Decarboxylase Gene pdc that homologous recombination makes synthetic by single cross and be incorporated on the cytoalgae mutant strain S.strictus karyomit(e).After the conversion, the BG-11 nutrient solution that will contain cyanobacteria cytoalgae mutant strain S.strictus cell earlier is coated on the topped Millipore filter membrane on the BG-11 solid medium that does not contain penbritin (Amp) to be cultivated 20 hours, shifted filter membrane then to containing the enterprising row filter cultivation of BG-11 solid medium that penbritin concentration is 15 μ g/ml.After one week, the green single algae that occurs anti-Amp on the filter membrane falls, and is and transforms the cytoalgae bacterial strain.Repeat the cultivation of BG-11 solid medium and progressively improve penbritin concentration, can screen the transgenosis cytoalgae mutant strain S.strictus that obtains stabilization characteristics of genetics.
In order to prove that foreign gene has been incorporated on the cytoalgae mutant strain S.strictus karyomit(e), thereby whole ethanol synthesis path (pyruvic acid → acetaldehyde → ethanol) is connected, uses the generation of Schiff's reagent (Schiff ' s reagent) test acetaldehyde.Wild strain cytoalgae 6803 does not have the pdc gene, can not the catalysis pyruvic acid → acetaldehyde reaction, when Schiff's reagent is added in wild strain cytoalgae 6803 bacterium colonies on the BG-11 solid medium, there is not colour-change.Yet when Schiff's reagent was added in transgenosis cytoalgae mutant strain S.strictus bacterium colony on the BG-11 solid medium, bacterium colony and periphery became garnet.The acetaldehyde and the Schiff's reagent that show the cytoalgae output react.
Embodiment 4:nirA promotor transforms cytoalgae
The inventor further induces psbAII promotor among the nirA promoter replacement pSKBPDC with nitrate, thereby the change photoinduction is chemical induction.The design of nirA promoter primer is as follows:
T1 (sequence number: 6) (5 ' the end primer):
5’-GAGAGAGAG
CTGCAGAGCGTTCCAGTGGATATT-3’
PstI
T2 (sequence number: 7) (3 ' the end primer):
5’-ATTATATATAT
CATATGTTCATCTGCCTACAAAGCAGC-3’
NdeI
Wherein primer T1 has designed a PstI restriction enzyme site, and its sequence is CTGCA ↓ G, and primer T2 has designed a NdeI restriction enzyme site, and its sequence is CA ↓ TATG.To get the nirA fragment T of 528bp after the conventional pcr amplification method amplification.
PCR reactant: in the aseptic centrifuge tube of 0.5mL, add successively: deionized water 36 μ l; 10XTaq enzyme buffer liquid 5 μ l; 4XdNTP (2.5mmol/L) 5 μ l; P1 primer 1 μ l; P2 primer 1 μ l; Template (being the nirA promotor of synthetic) 1 μ l; Taq enzyme (3U/ μ l) 1 μ l.50 μ l altogether.
The PCR reaction conditions is identical with the pdc dna fragmentation P of amplification 1410bp.
After reaction finishes, get 2.5 μ l pcr amplification products and carry out 1% agarose gel electrophoresis (2XTAE, 100V voltage, 40 minutes), a treaty 528bp bright band on electrophoretogram, occurs, show that what will increase is the nirA promoter fragment that expection will transform.Electrophoresis reclaims the 528bp fragment.According to the restriction enzyme site in the primer, with restriction enzyme PstI and NdeI while double digestion fragment T and vector plasmid pSKBPDC, connect with the T4DNA ligase enzyme then, connector transformation receptor bacterium E.coli C600, screen with Amp, obtain recombinant plasmid pPDCNIRA (Fig. 2), this plasmid size is 5.96kb.Utilize nature to transform integrating expression vector is imported cytoalgae mutant strain S.strictus.After the conversion, the BG-11 nutrient solution that will contain cyanobacteria cytoalgae S.strictus cell earlier is coated on the Millipore filter membrane on the BG-11 solid medium that does not contain penbritin (Amp), and cultivated 20 hours, shift filter membrane then to containing the enterprising row filter cultivation of BG-11 solid medium that penbritin concentration is 5 μ g/ml.One after thoughtful 10 days, and the green single algae that occurs anti-Amp on the filter membrane falls, and are and transform the cytoalgae bacterial strain.Repeat the BG-11 solid medium cultivate and progressively improve penbritin concentration (to concentration be 15 μ g/ml), can screen the cytoalgae mutant strain S.strictus engineering bacteria that obtains stabilization characteristics of genetics.The different algaes that it is good that picking is grown fall to being inoculated into 6 150ML and shake the bottle composition, and BG-11 nutrient solution work liquid level is that the photosynthetic organism set of reactors of 50ML is cultivated, the shaking table built-in light source, and the reactor surface light intensity is about 200Einstein/m2/s.The shaking table temperature adjusting is being lower than 30 ℃, and speed is 200RPM.After 5 days, respectively get 1ml suspension, the centrifugal supernatant liquor that gets from 6 reactors.With enzyme reaction reagent (R-Biopharm, Inc., Marshall, MI, USA) survey alcoholic acid generation concentration.Through relatively getting ethanol high yield cytoalgae mutant strain S.strictus bacterial strain.
Embodiment 5: bioreactor is cultivated and is observed the ethanol production rate in the laboratory
The cytoalgae mutant strain S.strictus engineering bacteria that obtains among the liquid culture embodiment 4 can determine that not only foreign gene is efficiently expressed, but also the possibility of optimizing bioprocesses is provided.Bioreactor is 1 liter of (CelliGen in this laboratory
Plus, New Brunswick Scientific Inc., Edison, NJ, USA).This system carries temperature, pH, rotating speed, measurement and controllers such as dissolved oxygen.On this basis, install adjustable light sources additional, make reactor outer wall light intensity reach 1000Einstein/m
2/ s.And design a calculating machine sampling and Controlling System, software is then used LabVIEW6.0 (National Instruments Corp., Austin, TX, USA) programming.In this system, it is to reach OD in cytoalgae engineering bacteria cell optical density (OD) that nitrate induces ethanol synthetic
730Began in=0.5 o'clock.As a result, the alcohol concn of cyanobacteria cytoalgae engineering bacteria production can reach 14mM.
Embodiment 6: utilize outdoor bioreactor to observe the ethanol production rate
Use the cyanobacteria cytoalgae mutant strain S.strictus engineering bacteria that obtains among 10 liters of bioreactor suspension culture embodiment 4 out of doors.Control the add-on of carbonic acid gas with the mode of control pH.Temperature does not add control.This self-control reactor is made by glass, adds in the upcast to put internals and can promote bubbles dispersion effectively, improves the mixing of gas-liquid two-phase, strengthens the carbonic acid gas transmittance process.Fig. 5 a is a temperature variation curve, and Fig. 5 b is cell concn and alcohol production ability.It is to reach OD in cytoalgae engineering bacteria cell optical density (OD) that nitrate induces ethanol synthetic
730Began in=0.5 o'clock.Alcohol concn can reach 50mM as a result, has improved about 5 times than room light bio-reactor result.
In order more to be expressly understood the present invention, the present invention and two other intraclass correlation invention are contrasted, the result is as follows:
| Ennol | The University of Hawaii | This patent |
| The cyanobacteria original strain: | ||
| Synechococcus PCC 7942 (salt water bacterial strain, 2004 finish gene order-checking) | Synechocystis PCC 6803 (fresh water bacterial strain, 1996 finish gene order-checking) | Synechocystis PCC 6803 (fresh water bacterial strain, 1996 finish gene order-checking) |
| Foreign gene: | ||
| Pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) | Pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) | Pyruvate decarboxylase (pdc) |
| The structure of gene transformation carrier: | ||
| PCB4-CPpa, pCB4-LRpa, pCB4-LR (TF) pa | The two homologous recombination methods of pMota | The pPDCNIRA homologous recombination method |
| The promotor that is used for genetic expression: | ||
| From the thermoinducible CI-857rbcLS promotor of Synechococcus with from the lacZ promotor of E.coli | PsbAII photoinduction promoter from Synechocystis | The nirA nitrate evoked promoter of synthetic |
| Alcohol concn: | ||
| Reached 5mM in the laboratory in about 30 days | Reached 12mM in the laboratory in about 5 days | Reached 50mM in outdoor about 8 days |
| Gene integration: | ||
| Exist with the Synechococcus plasmid | Be integrated into Synechocystis karyomit(e) | Be integrated into Synechocystis karyomit(e) |
| The heat-resisting anti-alcohol performance of cyanobacteria bacterial strain: | ||
| Do not have | No resistance toheat.(LmrA LmrCD) improves anti-alcohol performance from the omrA gene of Oenococcus oeni or homologous gene in insertion. | " heat-shocked " orthogenesis |
Sequence table
Claims (8)
1. cyanobacteria cytoalgae (Cyanobacteria Synechocystis) PCC 6803, its preserving number is CGMCC NO.2137.
2. cyanobacteria cytoalgae, it has the polynucleotide of the active polypeptide of pyruvic carboxylase for imported coding in the described cyanobacteria cytoalgae of claim 1.
3. cyanobacteria cytoalgae according to claim 2, wherein said polynucleotide are:
(a) polynucleotide, it contains by sequence number: the polynucleotide that 1 base sequence is formed; Or
(b) polynucleotide, it is a sequence number: the polynucleotide that 1 base sequence or its complementary base sequence are formed.
4. cyanobacteria cytoalgae according to claim 3, it further contains nitrate inductive promotor.
5. cyanobacteria cytoalgae according to claim 4, wherein said promotor is:
(a) polynucleotide, it contains by sequence number: the polynucleotide that 2 base sequences are formed; Or
(b) polynucleotide, it is a sequence number: the polynucleotide that 2 base sequence and/or its complementary base sequence are formed.
6. according to any described cyanobacteria cytoalgae of claim 2-5, polynucleotide and/or promotor that wherein said coding has an active polypeptide of pyruvic carboxylase are carried by plasmid expression vector.
7. cyanobacteria cytoalgae according to claim 6, wherein said plasmid expression vector sequence is a sequence number: the sequence shown in 3.
8. the application of any described cyanobacteria cytoalgae of claim 2-7 in producing ethanol.
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Non-Patent Citations (2)
| Title |
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| 王永红 等.集胞藻6803 在封闭式光生物反应器中的混合营养培养.《高技术通讯》.2001,全文. * |
| 王永红.集胞藻6803封闭式光生物反应器混合营养培养及其生理学研究.《中国优秀博士学位论文数据库基础科学辑》.2006,(第11期),全文. * |
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