CN105087602A - Application of heat shock protein gene in construction of high-heat-tolerance escherichia coli - Google Patents

Abstract

The invention discloses a pyrococcus horikoshii heat shock protein gene (PhHSPS) with high heat tolerance. The nucleotide sequence of the mutant PhHSPS with high heat tolerance is shown as SEQ ID NO 1, and the coding amino acid sequence of the mutant PhHSPS with high heat tolerance is shown as SEQ ID NO2. In process of transforming mutant PhHSPS obtained by means of molecular evolution into escherichia coli, heat tolerance capacity of escherichia coli can be improved greatly by means of the mutant PhHSPS, and accordingly, the mutant PhHSPS can be applied to construction of bacterial strains of high heat tolerance.

Description

The application of a kind of heat shock protein gene in high temperature resistant intestinal bacteria build

Technical field

The invention belongs to genetically engineered field, be specifically related to ancient bacterium (Pyrococcushorikoshii) the heat shock protein gene PhHSPS of a kind of extreme thermophilic through in-vitro directed molecular evolution and application thereof, particularly relate to the application of described gene in high-temperature resistant strain builds.

Background technology

Heat shock protein(HSP) (HeatShockProtein, HSP) be that biology is subject to occurring to reflect when physics in environment, chemistry, biology, spirit etc. stimulate and the protein that synthesizes, have another name called stress protein (StressProtein, SP), be the protein of a class high conservative, be prevalent in protokaryon and eukaryote.Report the earliest about HSP sees 1962, and Ritossa observes and to be brought up to DEG C from 25 DEG C by the raising temperature of drosophila larvae, after 30min, its salivary gland chromosome has occurred special " puff ".Studied discovery afterwards, the transcribing of the generation of this puff and this zone gene is strengthened relevant, the increase that certain albumen may be had to synthesize during prompting heat-shocked.This phenomenon is called heat shock response or Heat shock response (heatshockresponse, HSR) by people.

The effect of heat shock protein(HSP) in heat defence mainly refers to that the hot tolerance of biology or cell strengthens, no matter be vegetable cell or some unicellular lower eukaryotes, after living through once moderate beat exposure process in advance, just can tolerate the thermal treatment that thereafter one is strong.In other words, sub-lethality heat-shocked can make cell obtain and produce heat tolerance to lethality thermal treatment subsequently, thus greatly strengthen the viability of cell.Many experiments directly prove, the generation of heat tolerance is relevant with HSP, the disappearance of heat tolerance is consistent with the degraded of HSP, and in the heat shock protein(HSP) not derivative etap, organism is to its sensitivity thermoae, heat tolerance can not be set up, when a certain amount of HSP exists, do not need to carry out heat-shocked process in advance, cell also can produce heat tolerance, can not obtain the induction of mutant without HSP of heat tolerance, the generation blocking HSP can stop the acquisition of heat tolerance.

At present because traditional energy is to the pollution of environment, many places, the world have started with clean reproducible ethanol as important energy source widespread use.A lot of microorganism can be applicable to produce ethanol, they are Microbial cell factories very important during ethanol fermentation is produced, but traditional fermentable temperature is lower, (leavening temperature that such as yeast saccharomyces cerevisiae is the suitableeest is 28 ~ 33 DEG C, generally be no more than 36 DEG C), this is the very large bottleneck that restriction ethanol fermentation is produced, and its can improve alcohol production cost greatly.If can select withstand high temperatures microorganism, then have and reduce microbiological contamination probability, shortening fermentation period, raising fermentation rate and reduce the advantages such as water coolant cost of use, this will be significant with reduction production cost to raising leavening property.

Summary of the invention

In the present invention, we have transformed the heat shock protein(HSP) that one derives from the ancient bacterium (Pyrococcushorikoshii) of extreme thermophilic phHSP, through in-vitro directed molecular evolution, obtain the heat shock protein(HSP) of sudden change phHSPSand utilize genetic engineering technique to be successfully transferred in intestinal bacteria by this gene, and through a series of test, find that transformed mutator gene can improve the colibacillary high temperature resistant ability of coercing significantly, thus provide a kind of scheme of solution for improving the high temperature resistance of microorganism.

Therefore, one of technical problem to be solved by this invention, is to provide a kind of bacterium (Pyrococcushorikoshii) heat shock protein gene more ancient than wild-type extreme thermophilic phHSPthere is the heat shock protein gene through sudden change of higher temperature capacity phHSPS.

Technical problem two to be solved by this invention, is to provide the described heat shock protein gene deriving from the ancient bacterium (Pyrococcushorikoshii) of extreme thermophilic phHSPSmicroorganism is high temperature resistant coerce in application, be about to sudden change after phHSPSgene proceeds in intestinal bacteria, and carries out functional verification to this transgenic escherichia coli, with prove it have improve intestinal bacteria thermotolerances function.

In order to achieve the above object, the present invention realizes by the following technical solutions.

The heat shock protein gene of the ancient bacterium (Pyrococcushorikoshii) of the described extreme thermophilic through DNA-shuffling phHSPS, its nucleotide sequence is as shown in SEQIDNO1, and the aminoacid sequence of its coding is as shown in SEQIDNO2.The long 519bp of this heat shock protein gene, containing 519 bases, the amino acid/11 of coding 72.

The heat shock protein gene of the ancient bacterium (Pyrococcushorikoshii) of described extreme thermophilic phHSPI, adopt manual method synthesis.Namely according to PTDS(PCR-basedtwo-stepDNAsynthesis, PTDS) method clone is derived from the heat shock protein gene of the ancient bacterium (Pyrococcushorikoshii) of extreme thermophilic phHSP, carry out chemosynthesis, in maintenance phHSPon the basis that the aminoacid sequence of gene is constant, design primer synthesizes the heat shock protein gene of the ancient bacterium (Pyrococcushorikoshii) of extreme thermophilic of the present invention phHSPI.

Synthetic phHSPIcarry out in-vitro directed molecular evolution, by obtained sudden change phHSPIgene proceeds to intestinal bacteria, proceeds to overnight growth at 55 DEG C, within second day, choose well-grown bacterium colony extracting DNA, and check order, finally obtain through 37 growths after 1 hour phHSPSsequence.By what obtain phHSPSgene transformation intestinal bacteria, through high temperature resistant evaluation, acquisition has phHSPSgene is high temperature resistant coli strain.

Illustrate thus: the heat shock protein gene coming from the ancient bacterium (Pyrococcushorikoshii) of extreme thermophilic of the present invention phHSPSobtain the gene that can improve intestinal bacteria high-temperature tolerance through in-vitro directed molecular evolution, this makes this gene become possibility for improving microorganism high temperature resistance.

Accompanying drawing explanation

Fig. 1 mutator gene phHSPSwith phHSPIamino acid alignment result.

Fig. 2 screens through high temperature, the positive single bacterium of intestinal bacteria of acquisition.

Sudden change high-temperature resistant strain and wild type gene growing state is obtained after Fig. 3 high temperature (53 DEG C) process.

Embodiment

Technical scheme of the present invention is described in detail below in conjunction with accompanying drawing.Embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to the technical scheme of invention or equivalent replacement, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in right of the present invention.

If the reagent unexplained reference that the present invention is used, all purchased from Sigma-Aldrich (Sigma-Aldrich) company.

The present invention relates to molecular biology experiment, as not dated especially, all with reference to from " molecular cloning " book (J. Pehanorm Brooker, E.F. be Ritchie, T. Manny A Disi work not, 1994, Science Press).

Embodiment 1 is extremely high temperature resistant gene phHSPIsynthetic

According to PTDS(PCR-basedtwo-stepDNAsynthesis, PTDS) method clone is derived from the heat shock protein gene of the ancient bacterium (Pyrococcushorikoshii) of extreme thermophilic phHSP, carry out chemosynthesis, at maintenance heat shock protein gene phHSPthe constant basis of aminoacid sequence on, design primer synthesizes heat shock protein gene of the present invention phHSPI, the primer of design is as follows:

1.PHHSPI-1:Tm=54,60mer

ATG,CGT,GGT,GGT,GAA,CGT,ATG,GTT,CGT,CGT,CGT,CGT,TGG,GAC,ATC,TGG,GAC,CCA,TTC,GAC

2.PHHSPI-2:Tm=54,60mer

CCC,ATT,CGA,CTT,GAT,TCG,TGA,AAT,CCA,AGA,AGA,GAT,CGA,TGC,CAT,GTT,CGA,CGA,GTT,CTT,C

3.PHHSPI-3:Tm=54,60mer

GAC,GAG,TTC,TTC,AGT,CGT,CCA,CGT,CTC,TGG,ACC,TAT,CGT,CGT,TGG,AAG,GAA,CCA,GAA,CTC,TAC,GAA,G

4.PHHSPI-4:Tm=54,60mer

GAA,CTC,TAC,GAA,GAG,GGA,ACA,GGT,GAA,GTC,TGG,CGT,GAA,CCA,TTC,GTT,GAC,ATC,TTC,GAT,CGT,G

5.PHHSPI-5:Tm=54,60mer

CAT,CTT,CGA,TCG,TGG,TGA,TGA,GCT,AGT,TGT,CAT,AGC,TGA,GTT,ACC,AGG,AGT,CCG,TAA,GGA,AGA,CAT,CAA,G

6.PHHSPI-6:Tm=54,60mer

GAA,GAC,ATC,AAG,GTC,CGT,GTC,ACT,GAG,GAC,AGC,GTC,TAC,ATC,GAA,GCC,ATA,GTA,CGT,CGT,GAG,AAG

7.PHHSPI-7:Tm=54,60mer

GTA,CGT,CGT,GAG,AAG,GAA,CTC,GAA,GAA,GAA,GGA,GCA,GTC,CGT,GTT,GAG,CGT,TAC,TAC,AGC,GGT,TAT,CGT,C

8.PHHSPI-8:Tm=54,60mer

CAG,CGG,TTA,TCG,TCG,TGT,CAT,CCG,TCT,TCC,AGA,AGA,AGT,CAT,CCC,AGA,GAA,GGC,TAA,GGC,TAA,GTA,CAA,C

9.PHHSPI-9:Tm=54,60mer

GGC,TAA,GTA,CAA,CAA,CGG,TGT,CCT,TGA,GAT,CCG,TAT,TCC,AAA,GAA,GAA,CCC,AAC,AAA,GAA,G

10.PHHSPI-10:Tm=54,60mer

TTA,TTC,GAT,CTT,GAC,CTC,GAA,TCC,CTC,TCC,TTC,CTT,CTT,TGT,TG

PCR is utilized to carry out phHSPgene amplification, in 100 μ l reaction systems, phHSPI-2 to phHSPIthe addition of-9 totally 8 primers is 2ng, Outside primer phHSPI-1 and phHSPI-10 additions are 30ng, and amplification condition is: 94 DEG C of preheating 1min; 94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 2min, the Taq DNA polymerase of use is KODFXtaq enzyme (Toyobo company, Japan), totally 25 circulations.

After PCR terminates, 1% agarose gel reclaims, and gets 10 μ l and is directly connected (the precious biotech firm in Dalian) with T/A cloning vector.4 DEG C of connections are spent the night, in Efficient Conversion DH5 α competence, obtain positive colony through Shanghai Sani company sequencing analysis, gained sequence be heat shock protein gene of the present invention ( phHSPI), the long 519bp of this heat shock protein gene, containing 519 bases, the amino acid/11 of coding 72.

The 2-in-1 one-tenth of embodiment phHSPIgene DNA-shuffling suddenlys change

1) by the method for multidigit point rite-directed mutagenesis, eliminate phHSPIin gene bamHi and sacthe restriction endonuclease sites such as I.

2) according to phHSPImeasure sequences Design two ends primer, at gene two ends bamhI and saci increases restriction enzyme site, with phHSPIgene is that template carries out pcr amplification, and test kit reclaims am-plified fragments.

3) be separated through 12% polyacrylamide gel electrophoresis with the product of DNaseI degraded, Bag filter method reclaims below 100bp small segment.

4) with the small segment reclaimed for template is carried out without primer PCR (PrimerlessPCR), 50 circulation after, amplified production is through 1.0%Agrose electrophoresis detection.

5) according to the result of PrimelessPCR electrophoresis, get the product after PrimelessPCR amplification, after adding primer, carry out custom primer PCR reaction, pcr amplification product is through 1.0%Agrose electrophoresis, and saturating suction bag method recovery suddenlys change through DNAShuffling phHSPIgene.

6) with the DSN enzyme pair from Crab phHSPImutator gene carries out homogenization process, will increase considerably after homogenization phHSPIthe kind of mutator gene and ratio.

7) by sudden change phHSPI and G251carrier connects, and builds the prokaryotic expression library of mutator gene.

The in-vitro directed molecular evolution of embodiment 3 mutator gene

1) prepare esherichia coli EG50 Competent, method is with reference to Molecular Cloning: A Laboratory guide (Rainerietal., 1990).

2) get 50 μ L intestinal bacteria EG50 competent cells, add 1 μ L mutant DNA, fully mix, the 2YT be coated in by mixture after 20min containing penbritin is dull and stereotyped, and first renewal cultivation 1 hour (37 DEG C), is then placed in 55 DEG C of incubators and grows 12h. by flat board

3) the high temperature resistant clone of picking in the bacterium that 2YT plate grows from step 2, after enzyme cuts qualification correctly, serves the order-checking of extra large sunny company.

4) by order-checking, high temperature resistant mutator gene and original synthetic gene are carried out sequence alignment, obtains the gene of high temperature resistant relevant mutational site phHSPS.

Embodiment 4 is high temperature resistant, and intestinal bacteria build and high thermal resistance checking

1) prepare esherichia coli EG50 Competent, method is with reference to Molecular Cloning: A Laboratory guide (work J. Sha nurse Brooker Huang training hall is translated)

2) wild type gene is carried out high temperature resistant comparison with mutator gene in contrast, concrete grammar is: get 50 μ LGV3101 competent cells, add the wild-type DNA of 1 μ L mutant DNA or synthesis, abundant mixing, the 2YT be coated in by mixture after 20min containing penbritin is dull and stereotyped, cultivates 12 hours (37 DEG C), and then picking list bacterium colony is placed in 2ml LB liquid medium cultivation 12 hours, measure OD value, and adjust mutator gene OD value to close with wild-type.

3) 1 μ L bacterium liquid point is drawn on the 2YT flat board containing penbritin, in 37 DEG C of incubators, cultivation proceeds in 53 DEG C of incubators after 1 hour and cultivates 12h, evaluate resistance to elevated temperatures by the upgrowth situation of bacterial plaque, thus obtain high temperature resistant intestinal bacteria and corresponding mutator gene phHSPS, its nucleotide sequence is as shown in SEQIDNO1, and the aminoacid sequence of its coding is as shown in SEQIDNO2.

Claims (3)

1., through the heat shock protein(HSP) mutator gene PhHSPS deriving from the ancient bacterium (Pyrococcushorikoshii) of extreme thermophilic of in-vitro directed molecular evolution, it is characterized in that nucleotide sequence is as shown in SEQIDNO1.

2. sudden change heat shock protein gene PhHSPS according to claim 1, it is characterized in that, its aminoacid sequence is as shown in SEQIDNO2.

3. the sudden change heat shock protein gene PhHSPS described in claim 1 or 2, through transformation of E. coli, can be used for building high temperature resistant coli strain.