CN1212393C - Gene engineering strain MI033WZ expression M1033 GI mutation enzyme GIG 38P and construction method thereof - Google Patents

Abstract

The present invention relates to a gene engineering strain M1033WZ for expressing the mutant enzyme GIG138P of M1033GI and a construction scheme thereof, namely a gene engineering strain for expressing the glucose isomerase and a construction method. With a homologous recombination method of gene knockout and gene substitution, tricyclic chromosome frame modification is made to a gene frame for expressing GI in the chromosome of No. 7 diastasic streptomycete M1033 to implement chromosome molecule mutation, namely that a structural gene 138 site is introduced into a GIG 138P mutant site to construct the gene engineering strain. Experiments on gene levels, protein levels and strain passages show that M1033WZ enzyme activity expression capacity constructed by the method is extremely stable; compared with the thermal stability of wild enzymes, the thermal stability of a mutant enzyme GIG138P expressed by the expression capacity is greatly improved to bring mature industrial production conditions for recombinant glucose isomerase with solid reaction columns.

Description

Express engineering strain M1033WZ and the construction process thereof of the mutant enzyme GIG138P of M1033GI

Technical field:

The present invention relates to express the engineering strain of glucose isomerase, and the gene engineering method that makes up this bacterial strain.

Background technology:

Glucose isomerase (Glucose Isomerse, be called for short GI) prepares the key enzyme of high fructose syrup as industrial large-scale amylase, has extremely important economic worth.The wild-type GI that obtains from natural strains separation purifying has been used for industrial production and has obtained huge economic and social benefit.But the thermostability of wild-type GI under higher reaction temperatures is desirable not enough, and temperature is high more in the isomerization reaction, and thermodynamic(al)equilibrium trends towards the fructose aspect more.So the thermostability that improves wild-type GI can increase the output of fructose in the molecular balance process, thereby reduce the thalline input amount, simplify technical process, increase economic efficiency.

Chinese patent " the mutant enzyme GIG138P of SM33GI and a kind of sudden change scheme of the GI of raising thermostability " (95112782.9) has provided mutant enzyme GIG138P and the sudden change scheme thereof that glucose isomerase SM33GI (also the claiming M1033GI) house of correction that suddenlys change to No. 7 streptomyces diastaticus SM33 (also claiming M1033) gets, and not can be applicable to the industrial stable and engineering strain that efficiently expresses but provide.

Summary of the invention:

The purpose of this invention is to provide a kind of stability and high efficiency express GIG138P, can be used for industrial engineering strain M1033WZ, and the construction process of this engineering strain.

Technical solution of the present invention is as follows:

A kind of engineering strain M1033WZ that expresses the mutant enzyme GIG138P of M1033GI, the 138th glycine of the glucose isomerase GI nucleotide sequence in its karyomit(e) changes proline(Pro) into.

The construction process of said gene engineering strain is: at first, gene order structure according to M1033GI, design also makes up the homologous recombination vector pTR that is used for the GI gene knockout, utilize the double-stranded segment of its sex change realize its with M1033 karyomit(e) on the homologous recombination of GI gene, acquisition displaced type glucose isomerase deficient strain M1033LJ; Secondly, make up displaced type homologous recombination plasmid pXL, its alkaline denaturation plasmid changes the M1033LJ protoplastis over to, obtains the GI enzyme bacterial strain M1033XL that recovers alive of insert type homologous recombination; At last, utilize the negative screen method of dull and stereotyped switching resistance to obtain to express the engineering strain M1033WZ of GIG138P mutant enzyme.

Innovation part of the present invention is to utilize the dna homolog recombinant technology in the genetically engineered to make M1033GI obtain the karyomit(e) molecular modification.

Streptomycete is accumulating suitable rich experience as the existing production history more than 30 years of the important suitability for industrialized production bacterial strain of a class aspect plant-scale cultivation and the fermentation technique.When determining to make up the scheme of engineering strain M1033WZ, once attempt having carried out the structure of streptomycete expression of recombinant plasmid bacterial strain and strepto-site-specific type integration bacterial strain respectively, but exist the unstable of recombinant plasmid copy number and integrative vector structure integration trip to become the variation problem respectively.The stability of considering recombinant bacterial strain is vital for industrial production, so the method for selective staining body molecular modification makes up engineering strain M1033WZ.

The homologous recombination technique of gene (homologous recombination), claim gene targeting (gene targeting) again, thereby refer to reorganization take place between the DNA on foreign gene DNA and the recipient cell karyomit(e) and be incorporated into the method that changes the cytogenetics characteristic on the predetermined site.Feature according to reorganization back target gene can be divided into two types: one, because the introducing of exogenous gene sequence or part replacement, target gene (being the gene of recipient cell karyomit(e)) original structure is destroyed, and this is gene disruption or gene knockout (gene disruption or knockout).Two, the full sequence of target gene is replaced by new gene, and this is gene substitution (gene replacement).The characteristics of homologous recombination technique are: 1. can introduce foreign gene on the specific fragment of the chromosomal DNA of recipient cell.Under situation reasonable in design, can carry out meticulous transformation to the recipient cell chromogene.2. after the homologous recombination the new gene of introducing with chromosomal DNA duplicate and stable duplicating.This emerging technology has been proved to be and has been the most effectual way of modifying factor group accurately at present.Theoretically, it will make the mankind can according to specific design philosophy to extremely complicated cellular genome structure fix a point, quantitative change, thereby directionally change the genetic construction and the feature of cell.Thereby this is for the main research contents in present molecular biology and the genetically engineered field---the researchs such as gene therapy of gene structure and function, genetic expression and regulation and control and human inheritance's disease all have very important meaning.The present invention is the practice of this emerging technology in the molecular modification of streptomyces gene engineering strain karyomit(e) just.

But because the frequency that homologous recombination takes place is very low, thereby the cell of screening and enrichment homologous recombination (just seeking suitable gene knockout and gene substitution recombinant vectors) is the technology of most critical in this field always.This Progress in technique makes homologous recombination research have landmark breakthrough and is divided into three developmental stage: the homologous recombination of the special gene of the sign that 1. self can elect.This is the method that commitment adopts.1981, the foundation and the vitro culture technology of embryonic stem cell line (Embronic stem cells is called for short the ES cell) were the solid basis that homologous recombination technique has been laid cell levels.2. adopt positive and negative selection (positive and negative selection, be called for short PNS or+/-select) homologous recombination of system.Because its application of method that serves as a mark of the special gene self that adopts of stage is subjected to great limitation in early days, the PNS method becomes the method that extensively adopts at present.3. target site is carried out the homologous recombination of accurate operation.Though by intracellular homologous recombination enzyme system, can produce the correction of gene original position, utilize DNA repair enzyme system defective and during induced mutation, can not be tested control owing to being attended by unmarked sudden change, so be unsuitable for meticulous gene correction.Therefore, in recent years+/-this accurate operation new technology that the system of selection base growth is got up, not only make operation become possibility, and make specific site operation also become possibility specific gene to specific gene in the complicated genome.In the accurate operation technology, its elementary tactics can be divided into two big classes: (1) hit and run strategy (hit and run strategy).This is a kind of two step homologous recombination strategies of using insertional vector.(2) tag-and-exchange strategy (tag and exchangestrategy).Use two kinds of substituted type carriers that are called as mark and sudden change respectively, in the homologous sequence of labeled vector, contain flag sequence, in the homologous sequence of mutational vector, contain mutant nucleotide sequence.

The earliest homologous recombination is to utilize to finish by the plasmid of self-replicating in streptomycete in streptomycete.Another kind of streptomycete recombinant vectors is the phage or the plasmid of responsive to temperature type.The third recombinant vectors is non-replicating plasmid vector pDH5.PDH5 is a kind of colibacillary phagemid, it can not be in streptomycete self-replicating, but contain the resistant gene tsr of thiostrepton on it, can be in order to the screening recon.Because the homologous recombination rate of single stranded DNA is than high one or two order of magnitude of double-stranded DNA, so use the success ratio that this carrier system can improve homologous recombination in the streptomycete greatly in streptomycete.Find further that on this basis the dna single chain that obtains by thermally denature or alkaline denaturation can reach the effect that improves recombination fraction equally in order to transform streptomycete.This discovery means that the homologous recombination operation in the streptomycete not necessarily will use specific recombinant vectors, and can directly utilize the escherichia coli plasmid that contains homologous fragment to carry out, thereby has expanded the range of application of streptomycete homologous recombination greatly.Simultaneously, this method also can solve when using the pDH5 carrier strand, and to prepare productive rate low and exist to methylate in some streptomycetes and modify the problem of obstacle, and the operation of homologous recombination is further simplified.

When carrying out homologous recombination in streptomycete, its main problem is: to have lost the recon of carrier sequence in order obtaining exchanging, must to have carried out a large amount of screening operations from the first step is recombinated the offspring of recon of gained.It is much to screen at random the situation that obtains a recon that double exchange taken place the clone from 1000-5000.Thereby in streptomycete, find a kind of negative selectable marker that is similar to thymidine kinase (HSV-tk) gene in the eukaryotic cell to become the developing direction of streptomycete homologous recombination technique research in the last few years.There have been at present two kinds of genes to be used in the gene substitution of streptomycete as the negative marker gene of selecting.First kind of gene is glucokinase gene (glk), and second kind of gene is ribosomal protein gene (rpsL), but its screening amount is still bigger.

The present invention has adopted brand-new mark-exchange-mark-negative sieve strategy in the karyomit(e) molecular modification of streptomyces gene engineering strain, promptly the method for taking when gene substitution is that karyomit(e) self reorganization is in conjunction with the negative sieve of resistance, the big problem of screening operation amount when having solved gene substitution in the streptomycete has been avoided genetic manipulation complicated in the said gene replacement process simultaneously.

That is to say that the present invention designs and made up rational gene knockout and gene substitution recombinant vectors according to the gene order of M1033GI, by protoplast transformation, in M1033GI karyomit(e), successively realize the GI gene mark, knock out and replace.By this three-wheel homologous recombination process, introduce mutational site G138P in the chromosomal GI gene of M1033GI 138 sites, thereby obtain engineering strain M1033WZ.

Molecule checking to engineering strain M1033WZ comprises polymerase chain reaction (PCR) amplification, nucleotide sequencing and hybridization (Southern) etc., and the albumen checking comprises protein electrophoresis (SDS-PAGE), GI separation and purification, determination of activity, heat stability test etc.The molecule experimental result shows: mutational site G138P has been introduced in the chromosomal GI gene of the engineering strain M1033WZ that is obtained 138 sites; The albumen confirmatory experiment is the result show: M1033WZ can express mutant enzyme GIG138P by stability and high efficiency, and the heat inactivation transformation period of mutant enzyme GIG138P is the twice of wild-type enzyme in the time of 80 degrees centigrade.And according to theoretical prediction, if the thermostability of enzyme improves 50%, just will make the industrial production cost reduce by 15%, so engineering strain M1033WZ possesses the huge industrial production value of potential.

At present, this engineering strain M1033WZ has submitted China Committee for Culture Collection of Microorganisms to, its preservation accession designation number is CGMCC No.0637, No. 7 streptomyces diastaticus Streptomycesdiastaticus of classification called after No.7, engineering strain called after M1033WZ, storage life are 30 years that 2001.9.27 rises.

Description of drawings:

Accompanying drawing 1: the design of graphics that is used for the homologous recombination vector pTR of gene knockout;

Accompanying drawing 2: the design of graphics that is used for the homologous recombination vector pXL of gene substitution;

Accompanying drawing 3:M1033 chromogene knocks out and obtains M1033LJ homologous recombination synoptic diagram;

The displacement of accompanying drawing 4:M1033LJ chromogene obtains M1033XL homologous recombination synoptic diagram;

The negative sieve of accompanying drawing 5:M1033XL karyomit(e) obtains M1033WZ homologous recombination synoptic diagram;

Accompanying drawing 6:M1033 and M1033WZ karyomit(e) enzyme are cut results of hybridization (Southern);

The total DNA enzyme of 1:M1033 bacterial strain is cut results of hybridization;

The total DNA enzyme of 2:M1033WZ bacterial strain is cut results of hybridization;

The thermostability curve of the pure enzyme of GI of accompanying drawing 7:M1033 and M1033WZ;

The thermostability curve of the pure enzyme of GI of 1:M1033;

The thermostability curve of the pure enzyme of GIG138P of 2:M1033WZ;

The protein electrophoresis of accompanying drawing 8:M1033 and M1033WZ (SDS-PAGE);

1: albumen lower molecular weight standard;

The pure enzyme of the GI of 2:M1033;

The pure enzyme of the GIG138P of 3:M1033WZ;

Embodiment:

Below in conjunction with accompanying drawing, the concrete building process of M1033WZ is described below:

(1) structure of glucose isomerase gene knockout deficient strain M1033LJ:

1) structure of gene knockout homologous recombination plasmid pTR

The pUB1 plasmid contains the non-regulating and controlling sequence of complete GI structure gene, promotor and 3 ' hold.At first cut pUB1 with the XhoI enzyme, it is stand-by that Klenow mends the big segment that reclaims 4.9Kb in flat back.Cut pIJ702 with the BclI enzyme simultaneously, mend the tsr gene segment that flat rear electrophoresis reclaims 1.1Kb; Two flush ends connections obtain recombinant plasmid pTR.In the recombinant plasmid the 700th of GI structure gene to 3 ' end back the 400th by tsr gene substitution.The tsr gene that inserts interrupts GI structure gene on the one hand, and one side is as the selected marker of recombinant bacterial strain after the homologous recombination.(see figure 1)

2) the double-stranded method of sex change realizes that homologous recombination obtains M1033LJ

The pulsating alkaline denaturation form of EcoRI-HindIII of taking pTR transforms the M1033 protoplastis as the homologous recombination unit.Screen transformant with the thiostrepton resistance.The result shows that the transformation frequency of taking the double-stranded method of sex change to obtain the resistance transformant is 101-102/ug DNA.Live by the enzyme that shakes bottle survey biopsy survey transformant, found the bacterial strain M1033LJ of a GI loss of activity.Protein electrophoresis and blot hybridization (Western Blotting) have further confirmed the result of active detection, promptly do not express the GI albumen of 43Kd in M1033LJ.With the sphI segment of the GI gene probe that serves as a mark, find that with M1033LJ and the hybridization of M1033 karyomit(e) that the SalI/SphI enzyme is cut former GI gene has carried out two-way replacement with recomposition unit on the M1033LJ karyomit(e).Because how metathetical tsr gene has made in the GI gene SalI site, M1033LJ is shown as the hybrid belt of a 1.1Kb, and M1033 is a 2.3Kb hybrid belt.Can prove in view of the above and successfully utilize the two-way replacement homologous recombination to realize knocking out of GI gene in the M1033 karyomit(e).(see figure 3)

(2) insert type homologous recombination GI enzyme is lived and is recovered the structure of bacterial strain M1033XL:

1) structure of displaced type homologous recombination plasmid pXL

Obtain wherein to contain the fragment of neomycin resistance gene aph I with Xhol I, Bcl I double digestion streptomycete plasmid pIJ680, in pGM-7Zf (+)/Xho I BamHI, obtain recombinant plasmid pHERO by blue hickie screening with the mode subclone of two cohesive ends.Consider that pUB-GI2 goes up the restriction of restriction enzyme site, take three step subclones that the aphI gene on the pHERO is introduced 3 ' end that pUB1-GI1 goes up the GI gene, concrete steps are: cut pUB1-GI1 with the SphI enzyme, recovery contains the 4.8kb carrier segments of GI gene 3 ' end and the 1.1kb GI gene fragment of 3 ' terminal deletion respectively.The carrier segments of 4.8kb is connected certainly, transformed into escherichia coli Dh5a competent cell, through the Amp resistance screening, plasmid extraction, enzyme is cut checking, obtains recombinant plasmid pBL.Cut pHERO with the SacII enzyme again, reclaiming the aphI gene fragment of 1.0kb also scabbles with T4 Polymerase, cut pBL and mend flat with the Klenow enzyme with the XhoI enzyme simultaneously, then they are connected, transformed into escherichia coli Dh5a competent cell is through the Amp resistance screening, the screening of plasmid Rapid identification, plasmid extraction, enzyme is cut checking, obtains recombinant plasmid pBH.Cut pBH with the SphI enzyme at last, it is connected with the GI gene fragment of 3 ' terminal deletion, transformed into escherichia coli Dh5a competent cell, through the Amp resistance screening, plasmid extraction, enzyme is cut checking, obtains displaced type homologous recombination plasmid pXL.(see figure 2)

2) the double-stranded method of plasmid pXL sex change realizes that homologous recombination obtains M1033XL

The alkaline denaturation form of taking the pXL plasmid transforms the M1033LJ protoplastis as the homologous recombination unit.Neomycin resistance screening transformant, the result obtains the resistance transformant.Live by the enzyme that shakes bottle survey biopsy survey transformant, select the bacterial strain called after M1033XL of a GI activation recovering.Protein electrophoresis has further confirmed the result of active detection, has promptly recovered to express the GI albumen of 43kD in M1033XL.Owing to find that M1033XL also maintains the thiostrepton resistance, so can infer that between plasmid pXL and the chromosomal homologous fragment of bacterial strain M1033LJ single cross having taken place changes homologous recombination.(see figure 4)

(3) structure of engineering strain M1033WZ:

Negative sieve method obtains GI engineering strain M1033WZ

Consider the unstable of insert type homologous recombination pattern, designed negative sieve method.M1033XL is passed three plates continuously in non-resistance R2YE substratum, the preparation spore suspension, dilution is coated on the neomycin resistance flat board, after waiting to grow spore, transfer simultaneously and connect on the silk rhzomorph resistance plate at neomycin resistance plate and sulphur, pick out and can not grow in an even silk rhzomorph resistance plate of sulphur, but the bacterial strain that can on the neomycin resistance plate, grow.Screened with this method that sulphur is connected the silk rhzomorph is responsive and have the active bacterial strain M1033WZ of a GI (see figure 5).Protein electrophoresis has confirmed the (see figure 8) as a result of active detection.With the SphI fragment of the GI gene probe that serves as a mark, find with M1033WZ and the hybridization of M1033 karyomit(e) that the SalI enzyme is cut, on the M1033LJ karyomit(e) disrupted-GI gene of about 2.6kb with recomposition unit pXL on corresponding homology zone carried out two-way replacement.How made in the GI gene SalI site owing to replace aphI gene on the karyomit(e), M1033WZ is shown as the hybrid belt of a 2.0kb, and M1033 is the hybrid belt (see figure 6) of a 3.2kb.With the method for PCR, be primer with P1, P2, the total DNA to M1033WZ and M1033 increases respectively, and both all amplify the 1.2kb band of complete GI structure gene.Sequencing result shows that 138 mutational sites have introduced M1033WZ karyomit(e) (seeing sequence table).Can prove that in view of the above the displacement that utilizes the interior homologous recombination of karyomit(e) to realize two copy GI genes in the M1033XL karyomit(e) obtains M1033WZ.

(4) albumen of engineering strain M1033WZ checking

Spore inoculating M1033WZ and M1033 are in 1L liquid YEME substratum, 30 ℃ of shaking culture 70h, centrifugal collection thalline ultrasonic disruption prepares crude enzyme liquid in the born of the same parents, pure enzyme purification adopts the method for " Acta Biochimica et Biophysica Sinica " (27 (5) 470 pages of nineteen ninety-fives), with the DEAE-Sepharose FF and the Sephacryl HR S-300 of Sweden Fa Maxi (Pharmacia) company, the enzyme that above-mentioned streptomycete is expressed carries out separation and purification and obtains GI and the pure enzyme of GIG138P respectively.By the method in No. 95112782.9 Chinese patents, with glucose substrate, detect its thermostability under 80 ℃ of heat treated conditions, find that the GIG138P heat inactivation transformation period of M1033WZ expression is improved about two times of (see figure 7)s.Under the condition of no Xin Meisu, M1033WZ is carried out the solid cultivation of going down to posterity.After dull and stereotyped cultivation of eight times commentaries on classics and liquid culture, the offspring still keeps the resistance of Xin Meisu and GI activity, and the GI displaced type bacterial strain M1033WZ after this proof reorganization is highly stable.

The nucleotides sequence of GI tabulation (in the square frame is mutant nucleotide sequence) among the M1033WZ:

(videing infra)

30 60

M：ATG AGC TAC CAG CCC ACC CCC GAG GAC AAG TTC ACG TTC GGC CTG TGG ACC GTC GGC TGG 60

M：CAG GGA CGG GAC CCC TTC GGC GAC GCC ACC CGC GGC GCC CTC GAC CCG GCC GAG TCC GTC 120

M：CGC CGC CTC GCC GAG CTC GGC GCC CAC GGC GTG ACG TTC CAC GAC GAC GAC CTC ATC CCC 180

M：TTC GGC GCG AGC GAC AGC GAG CGC GCC GAG CAC ATC AAG CGG TTC CGC CAG GCG CTG GAC 240

M：GAG ACC GGC ATG AAG GTC CCG ATG GCG ACC ACC AAC CTG TTC ACC CAC CCG GTG TTC AAG 300

M：GAC GGC GGC TTC ACC GCG AAC GAC CGT GAC GTG CGC CGT TAC GCC GTG CGC AAG ACC ATC 360

M：GAG GGC GCC GAG TCC GGC GCC GCC AAG GAC GTC CGG GTC GCC CTC GAC CGC ATG AAG GAG 480

M：GCT TTC GAC CTC CTC GGC GAG TAC GTC ACC TCC CAG GGC TAC GAC ACT CCG TTC GCC ATC 540

M：GAG CCC AAG CCG AAC GAG CCC CGC GGC GAC ATC CTC CTG CCG ACG ATC GGC CAC GCC CTC 600

M：GCC TTC ATC GAC GGC CTC GAG CGC CCG GAG CTG TAC GGC GTC AAC CCG GAG GTC GGC CAC 660

M：GAG CAG ATG GCC GGG CTC AAC TTC CCG CAC GGC ATC GCC CAG GCC CTG TGG GCG GGC AAG 720

M：CTG TTC CAC ATC GAC CTC AAC GGC CAG TCC GGC ATC AAG TAC GAC CAG GAC CTC CGC TTC 780

M：GGG CCG GGC GAC CTG GCC GCC GCG TTC TGG CTG GTG GAC CTG CTG GAG TCG GCC GGC TAC 840

M：GAG GGC CCG CGC CAC TTC GAC TTC AAG CCG CCG CGG ACC GAG GAC TTC GAC GGC TTC GAC 900

M：TTC AAG CCG CCG CGG ACC GAG GAC TTC GAC GGC GTC TGG GCC TCC GCG GCC GGC TGC ATG 960

M：CGC AAC TAC CTG ATC CTC AAG GAG CGC GCG GCC GCC TTC CGT GCG GAC CCG GAG GTG CAG 1020

M：GAG GCG CTG CGC GCG GCC CGG CTG GAC GAG CTC GCC CAG CCG ACG GCG GGC GAC GGC CTC 1080

M：CAG GCC CTG CTG CCC GAC CGC TCG GCG TTC GAG GAC TTC GAC CCG GAC GCG GCG GCG GCC 1140

M：CGT GGC ATG GCC TTC GAG CGG CTG GAC CAG CTG GCG ATG GAC CAC CTG CTG GGC GCG CGG 1200

M：GGC TAG

Claims (1)

1, a kind of engineering strain M1033WZ that expresses the mutant enzyme GIG138P of M1033GI is characterized in that the 138th glycine of the glucose isomerase GI nucleotide sequence in the karyomit(e) changes proline(Pro) into.

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