CN106399114B - A kind of method and application improving DNC wireless ethanol tolerance ability - Google Patents
A kind of method and application improving DNC wireless ethanol tolerance ability Download PDFInfo
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Abstract
A kind of method and application for improving DNC wireless ethanol tolerance ability of the present invention, belongs to industrial microorganism field.The DNC wireless algae strain significantly improved to alcohol resistance is obtained using this method, algae strain can be used for constructing the genetic engineering bacterium of production alcohol fuel.Method of the invention is to be knocked out the sll0687 gene in DNC wireless by homologous recombination, and the DNC wireless algae strain IK2 that adopting said method obtains significantly improves the tolerance of ethyl alcohol.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
Technical field
The invention belongs to industrial microorganism fields, and in particular to a kind of to improve DNC wireless ethanol tolerance ability
Method and application.
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 converts generation ethyl alcohol, it is short that the increase of ethyl alcohol demand can aggravate grain
The problem of lacking.And using photosynthetic cyanobacteria can be carried out as bioreactor by CO2It is converted to ethyl alcohol, is to solve the problems, such as this
One of important aspect.DNC wireless is easy to carry out transgeneic procedure, is to carry out the good genetic engineering bacterium of the research.
Such as by pyruvate decarboxylase (pdc) gene and alcohol dehydrogenase II (adh) channel genes collection in Zymomonas mobilis
In born of the same parents algae PCC6803, and the two gene expressions are driven with psbAII promoter, DNC wireless can be made to generate low dense
The ethyl alcohol of degree.
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, research improves collection born of the same parents
The method of algae PCC6803 ethanol tolerant ability using photosynthesis industrial production of ethyl alcohol for being of great significance.
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 raising cytoalgae
The method of PCC6803 ethanol tolerance ability.The DNC wireless significantly improved to alcohol resistance has been obtained using this method
Algae strain, algae strain can be used for constructing the genetic engineering bacterium of production alcohol fuel.
The purpose of the invention is achieved by the following technical solution:
A method of DNC wireless ethanol tolerance ability is improved, is included 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 upstream and downstream primer, using pET-30b plasmid as template, is obtained by PCR amplification comprising blocking that
Mycin gene and its upstream and downstream partial sequence Kmr, 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 Km obtained with restriction enzyme BamHI and KpnI processing pUC118-up-down and step (1)rPiece
Section, by KmrIt is inserted on pUC118-up-down and obtains pUC118-up-down-KmrHomologous double-crossover plasmid；
(3) by pUC118-up-down-KmrPlasmid is transferred in DNC wireless in a manner of Natural Transformation, is obtained
Transformant is screened with the kanamycins of various concentration, and is identified on DNA level, and sll0687 gene is finally obtained
The monoclonal algae strain knocked out completely, i.e., the DNC wireless algae strain significantly improved to alcohol resistance, is named as IK2.
The DNC wireless algae strain that a kind of pair of alcohol resistance significantly improves, is obtained by the above method.
The DNC wireless algae strain IK2 constructed by the above method significantly improves the tolerance of ethyl alcohol, is containing
Growth conditions in the BG11 culture medium of 1.5% (v/v) ethyl alcohol are substantially better than wild type algae strain.
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
Method of the invention is to be knocked out the sll0687 gene in DNC wireless by homologous recombination, application
The DNC wireless algae strain IK2 that the method obtains significantly improves the tolerance of ethyl alcohol.It is coerced in the ethyl alcohol of 1.5% (v/v)
Under compeling, the growth conditions of algae strain are substantially better than wild type algae strain.The alcohol resistance algae strain that the present invention obtains produces building
The genetic engineering bacterium of alcohol fuel has important theoretical and practical significance, is with a wide range of applications.
Detailed description of the invention
Fig. 1 is pUC118-up-down-KmrThe structural schematic diagram of plasmid.
Fig. 2 is the agarose electrophoresis that PCR identification is carried out using SEQ ID NO:1 and SEQ ID NO:3 as primer pair IK2 algae strain
Figure；Swimming lane 1 is using wild type gene group as template in figure, and swimming lane 2 is using IK2 genome as template, and M marker, arrow refers to
To position be 2042bp.
Fig. 3 is growth curve of DNC wireless wild type WT and IK2 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 IK2 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 IK2 the algae strain under 1.5% (v/v) ethyl alcohol stress absorbs
Figure；A figure is wild type, and full cell absorbs figure under B figure is IK2 and wild type WT ethyl alcohol stress.
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, pET-30b
Purchased from Novagen company.
Embodiment 1
Homologous recombination double crossing over plasmid pUC118-up-down-KmrBuilding:
(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 pET-30b plasmid as template, is obtained by PCR amplification comprising kanamycins base
Cause and its upstream and downstream partial sequence Kmr, as shown in SEQ ID NO:9.The genome extraction of DNC wireless is adopted in the present invention
With plant genome DNA rapidly extracting kit (new east station of Guangzhou wins Biotechnology Co., Ltd, article No. N1191), plasmid extraction is adopted
With 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 KmrSegment is connected with pUC118-up-down plasmid, restriction enzyme site
For BamHI and KpnI, homologous recombination double crossing over plasmid pUC118-up-down-Km is finally obtainedr。
Obtain homologous recombination double crossing over plasmid pUC118-up-down-KmrMiddle sigI-up, sigI-down and KmrConnection
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-KmrAfter 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 kanamycins, illumination cultivation a couple of days to film
Surface grows single algae and falls.Finally, the algae grown to be fallen to the 20mL BG11 bottle culture being transferred to containing same concentrations kanamycins
It cultivates in base, transfers 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 Kmr
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 KmrGene and sll0687 gene
Upstream and downstream sequence, length 3059bp.If replacement completely, Strain selection is completed.Otherwise, continue to improve antibiotic concentration progress
Screening and identification.Obtained sll0687 gene is screened completely by KmrThe algae strain replaced is to significantly improve to alcohol resistance
DNC wireless algae strain, is named as IK2.
Fig. 2 is the agarose electrophoresis that PCR identification is carried out using SEQ ID NO:1 and SEQ ID NO:3 as primer pair IK2 algae strain
Scheme, swimming lane 1 is using wild type gene group as template in figure, and swimming lane 2 is using IK2 genome as template.It can be seen from the figure that wild
The segment of raw type is approached in 2000bp or so with the 2042bp length of theoretical calculation；The expanding fragment length of IK2 is than wild type
Greatly, the 3059bp of position and theoretical calculation is closely located to, while at 2042bp no band.In addition to this, wild type and
The sequence of the PCR product of IK2 Sanger sequence verification, it is consistent with theory.This explanation, sll0687 gene in IK2 by
KmrIt 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 IK2 algae strain under ethyl alcohol stress:
IK2 algae strain obtained in DNC wireless wild type and embodiment 2 is measured under 1.5% (v/v) ethyl alcohol stress
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
Originate 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, it is dense to end that ethyl alcohol is added in the culture medium of wild type and IK2 experimental group
Degree 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 IK2 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 IK2 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 IK2 is apparently higher than open country under ethyl alcohol stress
Raw type.
Fig. 4 is the color of the algae solution of DNC wireless wild type and the strain of IK2 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 IK2 of ethyl alcohol stress
Color and the color of the not IK2 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 IK2 algae under 1.5% (v/v) ethyl alcohol stress absorbs
Figure, A figure is wild type, and B figure is IK2, 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 IK2 of ethyl alcohol stress is green
Plain peak and phycocyanin peak also decrease compared with normal algae, but the peak height of the wild type than ethyl alcohol stress, illustrate ethyl alcohol pair
The photosynthetic influence of IK2 is smaller than wild type.
The data of complex chart 3, Fig. 4 and Fig. 5 can illustrate that the tolerance that IK2 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. a kind of method for improving DNC wireless ethanol tolerance ability, it is characterised in that include 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 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 pET-30b plasmid as template, is obtained by PCR amplification comprising kanamycins base
Cause and its upstream and downstream partial sequence Kmr, 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 Km obtained with restriction enzyme BamHI and KpnI processing pUC118-up-down and step (1)rPiece
Section, by KmrIt is inserted on pUC118-up-down and obtains pUC118-up-down-KmrHomologous double-crossover plasmid；
(3) by pUC118-up-down-KmrPlasmid is transferred in DNC wireless in a manner of Natural Transformation, obtained conversion
Son is screened with the kanamycins of various concentration, and is identified on DNA level, and it is complete to finally obtain sll0687 gene
The monoclonal algae strain of knockout, i.e., the DNC wireless algae strain significantly improved to alcohol resistance, is named as IK2.
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
Method 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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| CN109022285B (en) * | 2018-07-25 | 2021-05-14 | 华南理工大学 | Method for improving tolerance capacity of Synechocystis PCC6803 ammonium salt and application thereof |
| CN109706104B (en) * | 2018-12-25 | 2020-12-22 | 华南理工大学 | Application of sll0528 gene in improvement of ethanol tolerance of synechocystis PCC6803 |
| CN113025506B (en) * | 2019-12-24 | 2022-12-09 | 中国科学院天津工业生物技术研究所 | Recombinant filamentous fungus for producing ethanol and construction and application thereof |
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| CN103014053A (en) * | 2012-12-10 | 2013-04-03 | 山东省农业科学院高新技术研究中心 | Synechocystis efficient double homologous recombinant vector as well as construction method and application thereof |
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| CN103014053A (en) * | 2012-12-10 | 2013-04-03 | 山东省农业科学院高新技术研究中心 | Synechocystis efficient double homologous recombinant vector as well as construction method and application thereof |
| CN104293803A (en) * | 2014-09-29 | 2015-01-21 | 天津大学 | Ethanol-tolerant related gene slr0982 of synechocystis 6803 and applications of gene |
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