CN106086055B - The strain of DNC wireless algae and its construction method that a kind of pair of alcohol resistance significantly improves - Google Patents

The strain of DNC wireless algae and its construction method that a kind of pair of alcohol resistance significantly improves Download PDF

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CN106086055B
CN106086055B CN201610478393.6A CN201610478393A CN106086055B CN 106086055 B CN106086055 B CN 106086055B CN 201610478393 A CN201610478393 A CN 201610478393A CN 106086055 B CN106086055 B CN 106086055B
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陈谷
丁清龙
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South China University of Technology SCUT
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Abstract

The present invention discloses the strain of DNC wireless algae and its construction method that a kind of pair of alcohol resistance significantly improves, and belongs to industrial microorganism field.The present invention knocks out the sll0687 gene in DNC wireless by the method for homologous recombination, obtains the DNC wireless algae strain ISm5 that a kind of pair of alcohol resistance significantly improves.Under the ethyl alcohol stress of 1.5% (v/v), the growth conditions of algae strain are substantially better than wild type algae strain.The alcohol resistance algae strain that the present invention obtains has important theoretical and practical significance to the genetic engineering bacterium of building production alcohol fuel, is with a wide range of applications.

Description

The strain of DNC wireless algae and its building that a kind of pair of alcohol resistance significantly improves Method
Technical field
The invention belongs to industrial microorganism fields, and in particular to the cytoalgae that a kind of pair of alcohol resistance significantly improves The strain of PCC6803 algae and its construction method.
Background technique
In order to cope with the energy crisis got worse, the ethyl alcohol as bio-fuel, which becomes, substitutes oil-fired selection One.Need food crops as fuel since heterotrophic fermentation generates ethyl alcohol, the increase of ethyl alcohol demand can aggravate food shortage and ask Topic.Using photosynthetic cyanobacteria can be carried out as bioreactor by CO2It is converted to ethyl alcohol, is solve the problems, such as this important Scheme.DNC wireless is easy to carry out transgeneic procedure, is to carry out the good genetic engineering bacterium of the research.Such as it will Pyruvate decarboxylase (pdc) gene and alcohol dehydrogenase II (adh) channel genes cytoalgae in Zymomonas mobilis In PCC6803, and the two gene expressions are driven with psbAII promoter, DNC wireless can be made to generate low concentration Ethyl alcohol.
But the ethanol content that transgenic Synechocystis PCC6803 is generated at present is not met by wanting for ethanol industry metaplasia production It asks, one of key factor is exactly that DNC wireless is poor to the tolerance of ethyl alcohol.Containing 1.5% (v/v) ethyl alcohol Culture medium in, DNC wireless cell can be assembled, growth rate reduce by 50% or more.Therefore, it develops resistance to ethyl alcohol It is the key that utilize photosynthesis industrial production of ethyl alcohol by the better DNC wireless genetic engineering bacterium of property.
Summary of the invention
In order to overcome the disadvantages and deficiencies of the prior art, the primary purpose of the present invention is that providing a kind of pair of alcohol resistance The DNC wireless algae strain significantly improved.Algae strain can be used for constructing the genetic engineering bacterium of production alcohol fuel.
It is a further object of the present invention to provide the structures for the DNC wireless algae strain that a kind of pair of alcohol resistance significantly improves Construction method.
The purpose of the invention is achieved by the following technical solution:
The construction method for the DNC wireless algae strain that a kind of pair of alcohol resistance significantly improves, includes the following steps:
(1) using SEQ ID NO:1 in sequence table and SEQ ID NO:2 as upstream and downstream primer, with wild type cytoalgae PCC6803 genome is template, sll0687 gene upstream sequence sigI-up is obtained by PCR amplification, such as SEQ in sequence table Shown in ID NO:7；Using SEQ ID NO:3 and SEQ ID NO:4 as upstream and downstream primer, with wild type DNC wireless gene Group is template, sll0687 downstream of gene sequence sigI-down is obtained by PCR amplification, as shown in SEQ ID NO:8；With SEQ ID NO:5 and SEQ ID NO:6 is that upstream and downstream primer is included by PCR amplification using pCDFDuet-1 plasmid as template Streptomycin gene and its upstream and downstream partial sequence Smr, as shown in SEQ ID NO:9；
(2) the sigI-up segment obtained with restriction enzyme EcoRI and KpnI processing pUC118 carrier and step (1), SigI-up is inserted on pUC118 and obtains pUC118-up plasmid；It is handled with restriction enzyme BamHI and HindIII The sigI-down segment that pUC118-up and step (1) obtain, sigI-down is inserted on pUC118-up and obtains pUC118- Up-down plasmid；The Sm obtained with restriction enzyme BamHI and KpnI processing pUC118-up-down and step (1)rPiece Section, by SmrIt is inserted on pUC118-up-down and obtains pUC118-up-down-SmrHomologous double-crossover plasmid；
(3) by pUC118-up-down-SmrPlasmid is transferred in DNC wireless in a manner of Natural Transformation, is obtained Transformant is screened with the streptomysin of various concentration, and is identified on DNA level, and it is complete to finally obtain sll0687 gene The monoclonal algae strain knocked out entirely, i.e., the DNC wireless algae strain significantly improved to alcohol resistance, is named as ISm5.
The DNC wireless algae strain that a kind of pair of alcohol resistance significantly improves, constructs to obtain by above-mentioned construction method.
The DNC wireless algae strain ISm5 significantly improved to alcohol resistance, is containing 1.5% (v/v) ethyl alcohol BG11 culture medium in growth conditions be substantially better than the strain of wild type algae.
The DNC wireless algae strain significantly improved to alcohol resistance can be used for constructing production alcohol fuel Genetic engineering bacterium.
The present invention compared with the existing technology, have following advantages and effects
The present invention knocks out the sll0687 gene in DNC wireless by the method for homologous recombination, obtains one The DNC wireless algae strain ISm5 that kind significantly improves alcohol resistance.Under the ethyl alcohol stress of 1.5% (v/v), algae strain Growth conditions be substantially better than the strain of wild type algae.Base of the alcohol resistance algae strain that the present invention obtains to building production alcohol fuel Because engineering bacteria has important theoretical and practical significance, it is with a wide range of applications.
Detailed description of the invention
Fig. 1 is pUC118-up-down-SmrThe structural schematic diagram of plasmid.
Fig. 2 is the agarose electricity that PCR identification is carried out using SEQ ID NO:1 and SEQ ID NO:3 as primer pair ISm5 algae strain Swimming figure；Swimming lane 1 is using wild type gene group as template in figure, and swimming lane 2 is the M marker using ISm5 genome as template, arrow The position of direction is 2042bp.
Fig. 3 is growth curve of DNC wireless wild type WT and ISm5 the algae strain under 1.5% (v/v) ethyl alcohol stress Scheme, E represents ethyl alcohol in figure.
Fig. 4 is the face of algae solution of DNC wireless wild type WT and ISm5 the algae strain under 1.5% (v/v) ethyl alcohol stress Color, the state that algae is the 4th day in growth curve in figure.
Fig. 5 is that full cell of DNC wireless wild type WT and ISm5 the algae strain under 1.5% (v/v) ethyl alcohol stress is inhaled Receive figure；A figure is wild type WT, and the full cell under B figure is ISm5 and wild type WT ethyl alcohol stress absorbs figure.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited In this.
The strain of DNC wireless wild type algae used in the embodiment of the present invention isolates and purifies to obtain from ATCC27184, ATCC is the abbreviation of American Type Culture Collecti, and 27184 be strain number.Plasmid pUC118 is purchased from Takara company, PCDFDuet-1 is purchased from Novagen company.
Embodiment 1
Homologous recombination double crossing over plasmid pUC118-up-down-SmrBuilding:
(1) amplification of Insert Fragment:
Using SEQ ID NO:1 in sequence table and SEQ ID NO:2 as upstream and downstream primer, with wild type DNC wireless Genome is template, sll0687 gene upstream sequence sigI-up is obtained by PCR amplification, such as SEQ ID NO:7 in sequence table It is shown；Using SEQ ID NO:3 and SEQ ID NO:4 as upstream and downstream primer, using wild type DNC wireless genome as mould Plate obtains sll0687 downstream of gene sequence sigI-down by PCR amplification, as shown in SEQ ID NO:8；With SEQ ID NO: 5 and SEQ ID NO:6 is upstream and downstream primer, using pCDFDuet-1 plasmid as template, is obtained by PCR amplification comprising streptomysin Gene and its upstream and downstream partial sequence Smr, as shown in SEQ ID NO:9.The genome of DNC wireless extracts in the present invention Using plant genome DNA rapidly extracting kit (new east station of Guangzhou wins Biotechnology Co., Ltd, article No. N1191), plasmid is extracted Using high purity plasmid Mini Kit (new east station of Guangzhou wins Biotechnology Co., Ltd, article No. N1011).
PCR reaction uses 20 μ L systems: 1 μ L, 10 × PCR Buffer (Mg of template2+Plus) 2 μ L, dNTP Mixture (each 2.5mM) 1.6 μ L, 1 μ L of upstream primer (10 μM), 1 μ L of downstream primer (10 μM), rTaq enzyme 0.2 μ L, ddH2O 13.2μL。 Each reactive component is added into PCR pipe, after of short duration centrifugation, is placed in PCR instrument and carries out amplification reaction.Amplification program are as follows: initial denaturation, 94 DEG C, 3min；Denaturation, 98 DEG C, 10s；Annealing, temperature is 5~10 DEG C generally lower than primer Tm, time 15s；Extend, 72 DEG C, Every amplification 1kb DNA needs 1min；Circulation, denaturation-annealing-extension circulation 38；72 DEG C, 5min；16 DEG C, 10min.
PCR product size is verified through agarose electrophoresis, consistent with theoretical length.Before carrying out subsequent experimental, each PCR product Also need glue recovery purifying.
(2) the digestion connection of Insert Fragment and plasmid
The sigI-up segment and the progress pair of pUC118 carrier that step (1) is obtained with restriction enzyme EcoRI and KpnI Digestion.The double digestion of Insert Fragment uses 30 μ L systems: DNA 10 μ L, 10 × Buffer 3 μ L, two kinds of quick each 1 μ of digestion enzyme L, ddH2O 15μL.The double digestion of plasmid uses 20 μ L systems: 2 μ L of DNA10 μ L, 10 × Buffer, two kinds of quick digestion enzymes are each 1 μ L, ddH2O 6μL.Endonuclease reaction temperature is 37 DEG C, time 1h.After reaction, 80 DEG C of warm bath 5min, inactivator.Digestion Product is attached reaction after purification by gel, with T4DNA ligase.Connection reaction uses 20 μ L systems: Insert Fragment DNA 12 μ L, Plasmid DNA 5 μ L, 10 × Buffer 2 μ L, 1 μ L of enzyme.Connecting reaction temperature is 16 DEG C, after reacting 8h, by connection product Conversion is into bacillus coli DH 5 alpha.To the transformant grown identified whether successful connection, successful connection plasmid warp Sanger sequencing confirmation, to obtain plasmid pUC118-up.With identical endonuclease reaction and connection reaction condition, by sigI- Down segment is connected with pUC118-up plasmid, and restriction enzyme site is BamHI and HindIII.The plasmid of successful connection is tested Card, to obtain pUC118-up-down plasmid.Finally, by SmrSegment is connected with pUC118-up-down plasmid, restriction enzyme site For BamHI and KpnI, homologous recombination double crossing over plasmid pUC118-up-down-Sm is finally obtainedr。
Obtain homologous recombination double crossing over plasmid pUC118-up-down-SmrMiddle sigI-up, sigI-down and SmrConnection Sequence is as shown in Figure 1.
Embodiment 2
Acquisition to the DNC wireless algae strain that alcohol resistance significantly improves:
(1) plasmid converts
By pUC118-up-down-SmrAfter 0.22 μm of filtering with microporous membrane degerming of plasmid, it is packed into 2mL sterile centrifugation Guan Zhong.A certain amount of BG11 culture medium (HEPES buffer solution has been added) is added thereto, making plasmid final concentration is about 10ng/ μ L.It takes 30mL is in wild type PCC6803,6000rpm the centrifugation 7min of logarithmic phase, removes supernatant.It is resuspended with the fresh BG11 culture medium of 20mL Algal gel, 6000rpm are centrifuged 7min, remove supernatant.Algal gel is resuspended with the culture medium containing plasmid.By the algae solution of resuspension at 29 DEG C, 150rpm, 1400Lux continuous illumination culture 6h.Algae solution is applied to illumination cultivation in the solid medium for being covered with composite fibre filter membrane After 1 day (just setting culture), by film transfer in the solid medium containing 10 μ g/mL streptomysins, illumination cultivation a couple of days to film table Face grows single algae and falls.It is transferred in the 20mL BG11 bottle culture medium containing same concentrations streptomysin finally, the algae grown is fallen Culture is transferred after length to logarithmic phase.
(2) Strain selection
Algae solution obtained in (1) is subjected to switching culture, condition of culture is 29 DEG C, 150rpm, 1400Lux continuous illumination. When switching, antibiotic concentration in BG11 culture medium is increased to 20 μ g/mL.It transfers again to long to logarithmic phase, later every time The concentration of antibiotic improves 10 μ g/mL in switching culture medium.When the antibiotic concentration in culture medium reaches 50 μ g/mL, by algae Liquid plate streaking.Fall behind to grow single algae on plate, picking list algae drops down onto the BG11 culture medium containing corresponding antibiotic concentration Culture.After algae length to logarithmic phase, the genome of the algae is extracted.Using this genome as template, with SEQ ID NO:1 and SEQ ID NO:3 is primer progress PCR, while using wild type gene group as control.PCR reaction system and program and implementation in the present invention It is identical in example 1.PCR product is subjected to agarose electrophoresis, identifies the sll0687 gene in the algae genome whether completely by Smr It replaces.Using SEQ ID NO:1 and SEQ ID NO:3 as primer, the segment expanded in wild type is sll0687 gene And its upstream and downstream sequence, length 2042bp；In target algae strain, the segment expanded is SmrGene and sll0687 gene Upstream and downstream sequence, length 2787bp.If replacement completely, Strain selection is completed.Otherwise, continue to improve antibiotic concentration progress Screening and identification.Obtained sll0687 gene is screened completely by SmrThe algae strain replaced is to significantly improve to alcohol resistance DNC wireless algae strain, is named as ISm5.
Fig. 2 is the agarose electricity that PCR identification is carried out using SEQ ID NO:1 and SEQ ID NO:3 as primer pair ISm5 algae strain Swimming is schemed, and swimming lane 1 is using wild type gene group as template in figure, and swimming lane 2 is using ISm5 genome as template.It can from figure Out, the segment of wild type is approached in 2000bp or so with the 2042bp length of theoretical calculation；The expanding fragment length of ISm5 is than wild Raw type it is big, the 2787bp's of position and theoretical calculation is closely located to, while no band at 2042bp.In addition to this, wild The sequence of raw type and the PCR product of ISm5 Sanger sequence verification, it is consistent with theory.This explanation, the sll0687 in ISm5 Gene is by SmrIt replaces.
BG11 culture medium prescription used in the present invention: contain NaNO in 1L culture medium31.5g, K2HPO40.04g, MgSO4·7H2O 0.075g, EDTA 0.001g, CaCl2·2H2O 0.036g, citric acid 0.006g, ferric citrate 0.006g, Na2CO30.02g, H3BO30.00286g, MnCl2·4H2O 0.00181g, ZnSO4·7H2O 0.000222g, Na2MoO4·2H2O 0.00039g, CuSO4·5H2O 0.000079g, Co (NO3)2·6H2O 0.0000494g.In use, every The HEPES buffer solution (pH=7.5) of 1mL 1mol/L is added in 50mL culture medium.When preparing solid medium, 2% is added Agar.
Embodiment 3
Growth conditions analysis of the ISm5 algae strain under ethyl alcohol stress:
ISm5 algae strain obtained in DNC wireless wild type and embodiment 2 is measured to coerce in 1.5% (v/v) ethyl alcohol Under growth curve: using the algae for growing to logarithmic phase as seed liquor, be seeded to containing in 50mL BG11 culture medium, every bottle of algae Starting OD730=0.1.Continuous culture 4 days, OD is surveyed in sampling daily730Value draws growth curve.Condition of culture is 29 DEG C, 150rpm, 1400Lux continuous illumination.When starting culture, ethyl alcohol is added in the culture medium of wild type and ISm5 experimental group to end Concentration is 1.5% (v/v).Experimental group is parallel with each three of control group.
The full cell absorption spectrum of DNC wireless wild type and the strain of ISm5 algae under 1.5% (v/v) ethyl alcohol stress is surveyed It is fixed: it takes in the measurement of 2mL growth curve and cultivates to the 4th day algae solution, carry out length scanning with UV-2300 ultraviolet specrophotometer, Scanning range is 400~800nm, scanning speed 400nm/min.Before measurement, baseline correction first is carried out with BG11 culture medium.With Wavelength is abscissa, draws full cell abosrption spectrogram by ordinate mapping of corresponding OD value.The full cell of each sample absorbs Spectrogram is normalized with the OD value at 730nm.
Fig. 3 is the growth curve chart of DNC wireless wild type and the strain of ISm5 algae under 1.5% (v/v) ethyl alcohol stress, WT represents wild type in figure, and E represents ethyl alcohol.It can be seen from the figure that the growth curve of ISm5 is apparently higher than under ethyl alcohol stress Wild type.
Fig. 4 is the color of the algae solution of DNC wireless wild type and the strain of ISm5 algae under 1.5% (v/v) ethyl alcohol stress, The state that algae is the 4th day in growth curve in figure.It can be seen from the figure that the wild type color of ethyl alcohol stress with normally cultivate Wild type color distinction is larger, the yellowish of the algae of the wild type of ethyl alcohol stress, and color is light.And the algae of the ISm5 of ethyl alcohol stress Color and the color of the not ISm5 algae of alcohol treatment are close, no significant difference.
Fig. 5 is that the full cell of DNC wireless wild type and the strain of ISm5 algae under 1.5% (v/v) ethyl alcohol stress absorbs Figure, A figure is wild type, and B figure is ISm5, and the dotted line in B figure is the wild type under ethyl alcohol stress.It can be seen that from A figure Under ethyl alcohol stress, the chlorophyll peak and phycocyanin peak of wild type are significantly reduced.In B figure, the leaf of the ISm5 of ethyl alcohol stress Green element peak and phycocyanin peak only have subtle reduction compared with the algae under normal condition, and than the wild type under ethyl alcohol stress Peak is significantly high, illustrates that ethyl alcohol influence photosynthetic on ISm5 is smaller than wild type.
The data of complex chart 3, Fig. 4 and Fig. 5 can illustrate that the tolerance that ISm5 coerces ethyl alcohol is substantially better than wild type.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

Claims (4)

1. the construction method for the DNC wireless algae strain that a kind of pair of alcohol resistance significantly improves, it is characterised in that including such as Lower step:
(1) using SEQ ID NO:1 in sequence table and SEQ ID NO:2 as upstream and downstream primer, with wild type DNC wireless Genome is template, sll0687 gene upstream sequence sigI-up is obtained by PCR amplification, such as SEQ ID NO:7 in sequence table It is shown；Using SEQ ID NO:3 and SEQ ID NO:4 as upstream and downstream primer, using wild type DNC wireless genome as mould Plate obtains sll0687 downstream of gene sequence sigI-down by PCR amplification, as shown in SEQ ID NO:8；With SEQ ID NO: 5 and SEQ ID NO:6 is upstream and downstream primer, using pCDFDuet-1 plasmid as template, is obtained by PCR amplification comprising streptomysin Gene and its upstream and downstream partial sequence Smr, as shown in SEQ ID NO:9；
(2) the sigI-up segment obtained with restriction enzyme EcoRI and KpnI processing pUC118 carrier and step (1), will SigI-up, which is inserted on pUC118, obtains pUC118-up plasmid；It is handled with restriction enzyme BamHI and HindIII The sigI-down segment that pUC118-up and step (1) obtain, sigI-down is inserted on pUC118-up and obtains pUC118- Up-down plasmid；The Sm obtained with restriction enzyme BamHI and KpnI processing pUC118-up-down and step (1)rPiece Section, by SmrIt is inserted on pUC118-up-down and obtains pUC118-up-down-SmrHomologous double-crossover plasmid；
(3) by pUC118-up-down-SmrPlasmid is transferred in DNC wireless in a manner of Natural Transformation, obtained conversion Son is screened with the streptomysin of various concentration, and is identified on DNA level, is finally obtained sll0687 gene and is struck completely The monoclonal algae strain removed, i.e., the DNC wireless algae strain significantly improved to alcohol resistance, is named as ISm5.
2. the DNC wireless algae strain that a kind of pair of alcohol resistance significantly improves, it is characterised in that by described in claim 1 Construction method construct to obtain.
3. the DNC wireless algae strain according to claim 2 significantly improved to alcohol resistance, it is characterised in that: The DNC wireless algae strain that alcohol resistance is significantly improved, in the BG11 culture medium of the ethyl alcohol containing 1.5%v/v Growth conditions be substantially better than the strain of wild type algae.
4. the application of the DNC wireless algae strain described in claim 2 or 3 significantly improved to alcohol resistance, feature Be: the DNC wireless algae strain significantly improved to alcohol resistance is for constructing the gene of production alcohol fuel Engineering bacteria.
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