CN107460175A

CN107460175A - Optimize method, recombinant bacterium and its application for carrying out Glucose oxidase secretion expression based on metabolic engineering

Info

Publication number: CN107460175A
Application number: CN201710795690.8A
Authority: CN
Inventors: 钱江潮; 魏东升; 王泽建; 段广东; 吴凡; 储炬; 庄英萍; 张嗣良; 肖慈英; 黎亮
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2017-09-06
Filing date: 2017-09-06
Publication date: 2017-12-12
Anticipated expiration: 2037-09-06
Also published as: CN107460175B

Abstract

The present invention relates to method, recombinant bacterium and its application based on metabolic engineering optimization secreting, expressing glucose oxidase (GOD).The invention provides it is a kind of being capable of efficiently secreting, expressing GOD, the method for improving its enzyme activity, methods described realizes GOD high efficient expression by co-expressing GOD and the encoding gene of malic dehydrogenase 1 (mdh1) or 6 phosphogluconolactonase encoding genes (sol3) in yeast strain.Meanwhile present invention also offers the recombinant bacterium with Pichia pastoris (Pichia pastoris) for the efficient secretion GOD of host's structure.

Description

Optimize the method for carrying out Glucose oxidase secretion expression, restructuring based on metabolic engineering Bacterium and its application

Technical field

The invention belongs to technical field of bioengineering, more particularly it relates to carry out Portugal based on metabolic engineering optimization Method, recombinant bacterium and its application of grape carbohydrate oxidase secreting, expressing.

Background technology

Glucose oxidase (Glucose oxidase, GOD) is widely used in food, chemical industry, medicine, biotechnology etc. Field.

Based on the specificity of glucose oxidase catalytic reaction, available for biology sensor.Glucose oxidase be for The nucleus of the concentration of glucose in blood sensor is detected, being fixed on GOD on sensor electrode can will be micro in blood Glucose changes into the hydrogen peroxide easily determined, and the hydrogen peroxide that platinum electrode detects is more, and electric signal is stronger.Utilize Portugal The blood glucose meter that grape carbohydrate oxidase is made can easily detect the fluctuating level of blood sugar in diabetic patients.

Glucose oxidase is widely used in food service industry.It is fresh-keeping for food deoxygenation, added in commodity vacuum bag A little glucose oxidase and glucose, so as to suppress the growth of microorganism and breeding, can prolong oxygen depletion in vacuum bag The shelf-life of long food.Remove the glucose in protein product.Containing 0.5%~0.6% glucose in the albumen of egg, During storage and processing, the carbonyl of glucose and the amino of protein can occur there occurs mailland reaction in albumen Pore.Glucose can be consumed by adding GOD, so as to ensure the quality of protein.Face can be added to as food additives In powder.Bonet etc. has found that GOD can produce hydrogen peroxide, in the help of hydrogen peroxide with the glucose response in dough Under, mucedin can form disulfide bond, and then form complicated network structure, and this structure can strengthen the chewy of dough, Lift the mouthfeel of Flour product.In addition, hydrogen peroxide can also change the color and luster of flour, make the appearance of Flour product become attractive in appearance.

In textile industry, GOD is often applied to bleaching technique.During β-D-Glucose being catalyzed due to GOD, production The hydrogen peroxide of strong oxidizing property is given birth to, it is difficult to reuse GOD.GOD is covalently fixed on alumina and glass branch by Tzanov et al. Support on thing, improve enzyme recycling rate of waterused.After about 450 minutes, the maximum concentration of hydrogen peroxide for being fixed on glass support reaches 0.35g/L, and be 0.24g/L on alumina, compared with standard bleaching process, the concentration of hydrogen peroxide on textile substantially exists Same level, add GOD repeat usage.

GOD has very big potentiality in low technique for brewing alcohol.After adding glucose oxidase in liquor production, find Alcohol content in white wine reduces 2%, white wine is become pure taste, do not produce precipitation, it is muddy phenomena such as.Fermentation process In, GOD can kill acetic acid bacteria and lactic acid bacteria, and GOD bactericidal action means that GOD can not be widely used in wine brewing production, Pickering etc. reduces ethanol content, 87% glucose changes into Portugal using the double enzyme system processing grape juices of GOD/CAT Grape saccharic acid.

At this stage, glucose oxidase is also used in biological fuel cell industry, and catalase and GOD are fixed On same electrode, because GOD catalysis glucose responses are redox reactions, so electronics can be transferred to the carbon of the other end On electrode, so as to form green biological fuel cell, it can be provided for biology sensor and man-made organ lasting The energy.

In the oral cavity, streptococcic presence would generally lift the probability of decayed tooth, and GOD, Neng Gouhe can be added in toothpaste The glucose response of oral cavity residual, caused hydrogen peroxide can play the growth and breeding for suppressing harmful bacteria, so as to reduce trouble mouth The probability of chamber disease.

Gluconic acid and its derivative are widely used in health products trade, and GOD is as the pass in gluconic acid production Key enzyme, carry out Production by Enzymes was both can be used alone, can also be during being produced using aspergillus niger by adding GOD come fast Speed synthesis grape acid, improve the yield of gluconic acid.

At present, this area produces GOD by the method for microbial fermentation, still, utilizes aspergillus niger or mould fermenting and producing GOD there is production of enzyme it is low, isolate and purify the problems such as endocellular enzyme difficulty.And Escherichia coli production GOD is utilized, GOD can not be entered Row post translational processing, the GOD of synthesis are inactive.Those skilled in the art also utilize Pichia pastoris production GOD, but yield and enzyme Activity is still not ideal enough, it is also necessary to further optimizes GOD production technology.

The content of the invention

It is an object of the invention to provide a kind of method of the secreting, expressing glucose oxidase based on metabolic engineering, restructuring Bacterium and its application.

In the first aspect of the present invention, there is provided a kind of method using yeast cell to express glucose oxidase, the side Method includes：

(1) the glucose oxidase encoding gene of external source is introduced in yeast cells；

(2) at least one gene being selected from the group of external source is introduced in the yeast cells of step (1)：Malic dehydrogenase 1 encoding gene, 6-phosphogluconic acid lactone enzyme coding gene；

(3) yeast cells of incubation step (2), glucose oxidase is expressed, described glucose oxidase is high enzyme activity Glucose oxidase.

In a preference, glucose oxidase encoding gene, the encoding gene of malic dehydrogenase 1 or 6- phosphoric acid Portugal Grape saccharic acid lactone enzyme coding gene is incorporated into the genome of yeast cells by way of homologous recombination.

In another preference, methods described includes：

(1) expression vector is provided, the sequence of glucose oxidase encoding gene is included in the expression vector, this is expressed Carrier is transferred in yeast cells；

(2) expression vector is provided, the encoding gene of malic dehydrogenase 1 or G6P are included in the expression vector The sequence of acid lactone enzyme coding gene, the expression vector is transferred in the yeast cells of step (1)；

(3) yeast cells of incubation step (2), glucose oxidase is expressed.

In another preference, described yeast cells is Pichia pastoris.

In another preference, the expression of the glucose oxidase encoding gene of external source is driven with AOX1 promoters, with The expression of the 6-phosphogluconic acid lactone enzyme coding gene of AOX1 promoters driving external source.

In another aspect of this invention, there is provided a kind of method of the expression cell for the glucose oxidase for preparing high enzyme activity, Methods described includes：

(3) the glucose oxidase encoding gene for carrying external source, at least one base being selected from the group of external source are isolated Cause：The encoding gene of malic dehydrogenase 1, the recombinant yeast cell of 6-phosphogluconic acid lactone enzyme coding gene.

In another aspect of this invention, there is provided a kind of expression cell of the glucose oxidase of high enzyme activity, it is that yeast is thin Born of the same parents, it is integrated with its genome：The glucose oxidase encoding gene of external source is (it is preferred that it drives table with AOX1 promoters Up to)；And at least one gene being selected from the group of external source：The encoding gene of malic dehydrogenase 1, in 6-phosphogluconic acid Esterase encoding gene (is expressed) it is preferred that it is driven with AOX1 promoters.

In a preference, the glucose oxidase encoding gene of described external source, the malic dehydrogenase 1 of external source is compiled Code gene or 6-phosphogluconic acid lactone enzyme coding gene are incorporated into the genome of yeast cells by way of homologous recombination In；It is preferred that described yeast cells is prepared by foregoing method.

In another aspect of this invention, there is provided a kind of kit for being used to produce the glucose oxidase of high enzyme activity, it is described Kit includes：Above any described yeast cells.

The other side of the present invention is apparent to those skilled in the art due to this disclosure 's.

Brief description of the drawings

Fig. 1, plasmid pPIC9K-GOD are built.

Fig. 2, PCR expand GOD genes.

Lane M：Marker 5,000；

Lane1,2：Using plasmid pUC57-GOD as template, GODF/GODR is primer.

Fig. 3, digestion verification plasmid pPIC9K-GOD.

Lane M：Marker 15,000；

Lane1：PPIC9K-GOD is through BamHI and SalI digestion (3982bp and 7037bp).

Fig. 4, PCR checking recombinant bacterium G/GODM.

Lane M：Marker 5,000；

Lane 1：Using P.pastoris GS115 genomic DNAs template；

Lane 2、3：Using recombinant bacterium G/GODM genomic DNAs template.

Lane1、2、3：Using GODF/GODR as primer.

Fig. 5, expression vector pAOX-sol3, pAOX-mdh1, pAOX-zwf1, pAOX-gdh3 structure schematic diagram.

Fig. 6, expression vector pAOX-zwf1, pAOX-sol3, pAOX-gdh3, pAOX-mdh1 double digestion checking.

Lane M：Marker 15,000；

Lane 1：PAOX-zwf1 digests through Xho Ι and Not Ι；

Lane 2：PAOX-sol3 digests through Xho Ι and Not Ι；

Lane 3：PAOX-gdh3 digests through Xho Ι and Not Ι；

Lane 4：PAOX-mdh1 digests through Xho Ι and Not Ι.

Fig. 7, recombinant bacterium G/GMS3, G/GMM1, G/GMZ1, G/GMG3 building process (X represent sol3, mdh1, zwf1, gdh3)。

The GOD recombinant bacteriums of Fig. 8, PCR checking coexpression molecular chaperones.

A, recombinant bacterium RH integrations are verified.Lane M：2,000bp；Lane 1、2、3、4：Respectively with G/GMZ1, G/ GMS3, G/GMG3, G/GMM1 genomic DNA are template, using RHF/AOXR as primer.

B, recombinant bacterium DDKC integrations are verified.Lane M：2,000bp；Lane 1、2、3、4：Respectively with G/GMZ1, G/ GMS3, G/GMG3, G/GMM1 genomic DNA are template, with CYCTTF2/DDKCR2 primers.

The growth curve of Fig. 9, recombinant bacterium G/GMZ1, G/GMS3, G/GMG3, G/GMM1 in culture medium BMMY.

Figure 10, recombinant bacterium G/GMZ1, G/GMS3, G/GMG3, G/GMM1 extracellular (A), intracellular (B) GOD are than living.

Figure 11,120h recombinant bacterium G/GMZ1, G/GMS3, G/GMG3, G/GMM1 enzyme activity are horizontal.

Embodiment

In order to improve yeast production GOD yield, the present inventor develops one by in-depth study and substantial amounts of screening The method for kind can effectively facilitate GOD expression, improving its enzyme activity, methods described by co-expressed in yeast strain GOD and The encoding gene of malic dehydrogenase 1 (mdh1) or 6-phosphogluconic acid lactone enzyme coding gene (sol3), by strengthening carbon generation Thank to approach, GOD efficient secretory expression is realized with this.Meanwhile present invention also offers with Pichia pastoris (Pichia Pastoris) the efficient secretion GOD built for host recombinant bacterium.

As used herein, " external source " or " heterologous " refers to two or more pieces nucleic acid or protein from separate sources Relation between sequence.For example, if the combination of promoter and objective gene sequence were not usually naturally occurring, promoter It is external source for the target gene.Particular sequence is " external source " for cell that it is inserted or organism.

Described " promoter " refers to a kind of nucleotide sequence, and it is typically found in the upstream (5 ' of target gene coded sequence End), nucleotide sequence can be guided to be transcribed into mRNA.Usually, promoter or promoter region provide RNA polymerase and correct starting The recognition site of other factors necessary to transcription.

As used herein, " sol3 ", " mdh1 " gene are for recombinantly expressing in yeast cells with the gene of bar, also wrapping Include the molecule with gene order hybridization or the family gene molecule homologous with above-mentioned numberator height under strict conditions.Should In definition also comprising under strict conditions with " sol3 ", " mdh1 " hybridize molecule or the family homologous with above-mentioned numberator height Gene molecule.

As used herein, term " stringent condition " refers to：(1) hybridization under compared with low ionic strength and higher temperature and wash It is de-, such as 0.2 × SSC, 0.1%SDS, 60 DEG C；Or added with denaturant, such as 50% (v/v) formamide during (2) hybridization, 0.1% is small Cow's serum/0.1%Ficoll, 42 DEG C etc.；Or the phase same sex of (3) only between two sequences at least 50%, preferably 55% with Above, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85% or more than 90%, more preferably Just hybridize when more than 95%.

The malic dehydrogenase of mdh1 (Mitochondrial malate dehydrogenase) gene code mitochondrial, Malic acid generation oxaloacetic acid can be catalyzed.The nucleotide sequence (PAS_chr2-1_0238) such as SEQ ID NO of mdh1 genes: Shown in 1, or or its degeneracy sequence.The invention further relates to the polynucleotides variant of " mdh1 ", and it is encoded and open country The amino acid sequence identical polypeptide (enzyme) of raw type gene code.The variant of this polynucleotides can be the equipotential naturally occurred The variant that variant or non-natural occur.These nucleotide variants include substitution variants, Deletion variants and insertion and become Allosome.As known in the art, allelic variant is the alternative forms of a polynucleotides, and it is probably one or more nucleosides Acid substitution, missing or insertion, but not from substantially change its coding polypeptide function.

Sol3 gene code 6-phosphogluconic acid lactonases, it is an enzyme in non-oxidation phosphopentose (PP) approach. The nucleotide sequence (PAS_chr3_1126) such as SEQ ID NO of sol3 genes:2 shown or its degeneracy sequences.This Invention further relates to the polynucleotides variant of " sol3 ", and it encodes the amino acid sequence identical polypeptide with wild type gene coding (enzyme).The variant of this polynucleotides can be the variant of the allelic variant or non-natural generation naturally occurred.These cores Thuja acid variant includes substitution variants, Deletion variants and insert variation.As known in the art, allelic variant is one The alternative forms of individual polynucleotides, it is probably substitution, missing or the insertion of one or more nucleotides, but not from substantial Change the function of the polypeptide of its coding.

Amino acid sequence (the Uniprot of heretofore described " GOD " gene code：P13006) such as SEQ ID NO:3 Shown in (coming from aspergillus niger), its first 22 are signal peptide sequence.The invention further relates to the polynucleotides variant of " GOD ", and it is compiled The amino acid sequence identical polypeptide (enzyme) of code and wild type gene coding.The variant of this polynucleotides can naturally be occurred Allelic variant or non-natural occur variant.These nucleotide variants include substitution variants, Deletion variants and Insert variation.As known in the art, allelic variant is the alternative forms of a polynucleotides, and it is probably one or more Substitution, missing or the insertion of individual nucleotides, but not from substantially change its coding polypeptide function.

" sol3 " of the present invention, the nucleotides full length sequence of " mdh1 " or its fragment can generally use PCR TRAPs, again Group method or artificial synthesized method obtain., can be according to relevant nucleotide sequence disclosed in this invention, especially for PCR TRAPs It is open reading frame sequence to design primer, and with commercially available cDNA storehouses or by routine side well known by persons skilled in the art CDNA storehouses prepared by method expand as template and obtain relevant sequence.

In order to improve enzymatic activity of the glucose oxidase after recombination expression, the present inventor is extensively studied, found It is suitable for the gene improved, and constructs corresponding construction.

Therefore, the invention provides a kind of construction, " GOD gene " and external source of the construction including external source The expression cassette of " sol3 ".Described expression cassette possess needed for gene expression all elements (including promoter, coding DNA and Terminator etc.), so as to intactly give expression to corresponding albumen.

Present invention also offers a kind of construction, the construction includes " the GOD genes " of external source and " mdh1 " of external source The expression cassette of gene.Described expression cassette possess needed for gene expression all necessary elements (including promoter, coding DNA with And terminator etc.), so as to intactly give expression to corresponding albumen.

Generally, described construction is located on expression vector.Therefore, present invention additionally comprises a kind of carrier, it contains described Construction.Described expression vector is generally also containing replication orgin and/or marker gene etc..Those skilled in the art is known Method can be used to building the required expression vector of the present invention.These methods include recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination technology etc..Described DNA sequences can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA Synthesis.Expression vector also includes the ribosome bind site and transcription terminator of translation initiation.

As long as it should be understood that can realize in yeast cells increase " GOD genes ", " sol3 " or " mdh1 " expression, it is more The mode of kind recombination expression " GOD genes ", " sol3 " or " mdh1 " is available.As the preferred embodiment of the present invention, GOD bases Cause, " sol3 " or " mdh1 " are integrated into the genome of yeast cells by way of homologous recombination.

In addition, expression vector preferably includes one or more selected markers, it is used to select conversion to provide The phenotypic character of host cell, dihyrofolate reductase, neomycin resistance such as eukaryotic culture.

Appropriate conventional meanses, including reagent, temperature, pressure condition etc. can be used to implement described method.The present invention Expression vector when being expressed in higher eucaryotic cells, if will be increased transcription when inserting enhancer sequence in the carrier By force.Enhancer is DNA cis-acting factors, generally about there is 10 to 300 base-pairs, acts on promoter to strengthen base The transcription of cause.Persons skilled in the art are aware that how to select appropriate carrier, promoter, enhancer and host cell.

Comprising above-mentioned appropriate polynucleotide sequence and the carrier of appropriate promoter or control sequence, can be used for turning Change appropriate host.In the method for the invention, described host is yeast cells.

It can be carried out with recombinant DNA conversion host cell with routine techniques well known to those skilled in the art, such as calcium phosphate Coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging etc..As a preferred mode, can electricity consumption The method of conversion is carried out.

The method of the present invention is simple and easy, highly efficient in productivity.Also, the result of the present invention shows, restructuring of the invention The GOD of yeast cells production enzymatic activity significantly improves.Compared with only expressing GOD, the method for the invention can improve GOD list Position thalline enzyme activity about more than 6%；More preferably about more than 11%.

Present invention also offers the kit for including recombinant expression carrier and yeast cells constructed by the present invention；Or Person includes the kit of the recombinant yeast cell constructed by the present invention.

Other reagents for being usually used in carrying out transgeneic procedure can be also comprised in described kit, to facilitate this area Technical staff uses.

In addition, it can also include the operation instructions for instructing those skilled in the art to operate in the kit.

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip Part such as J. Pehanorm Brookers etc. are write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, the condition described in 2002, or According to the condition proposed by manufacturer.

1st, material and method

1.1 plasmid

Plasmid is shown in Table 1 used in the present invention.

Table 1, plasmid

1.2 bacterial strain

Bacterial strain uses therefor is shown in Table 2 in the present invention.

Table 2, bacterial strain

1.3 primer

In the present invention 3 are shown in Table with primer.

Table 3, primer

1.4 culture medium

1.LB fluid nutrient mediums：

20g/L tryptones, 10g/L yeast extracts, 10g/L NaCl.

The preparation of solid LB media needs to add 2% agar Icing Sugar；LB resistance cultures base can add debita spissitudo Antibiotic.

2.YPD fluid nutrient mediums：

20g/L tryptones, 20g/L glucose, 10g/L yeast extracts.

The preparation of solid YPD culture mediums needs to add 2% agar Icing Sugar；YPD resistance cultures base can add appropriate dense The antibiotic of degree.

3.YPG fluid nutrient mediums：

20g/L tryptones, 10g/L glycerine, 10g/L yeast extracts.

4.MD solid mediums：

20g/L glucose, 0.4mg/L biotins, 13.4g/L YNB, 20g/L agar powders.

5.BMGY fluid nutrient mediums

10g/L glycerine, 10g/L yeast extracts, 20g/L Peptone, 13.4g/L YNB, 0.1M pH=6K₂HPO₄/ KH₂PO₄Buffer solution.

6.BMMY fluid nutrient mediums：

1% methanol, 10g/L yeast extracts, 20g/L Peptone, 13.4g/L YNB, 0.1M pH=6K₂HPO₄/ KH₂PO₄Buffer solution.

1.5 cultural method

Flat board culture：Pichia pastoris glycerol tube is taken out from -20 DEG C of refrigerators, the oese with sterilizing is containing YPD solids Rule in culture medium, be placed in 30 DEG C of incubators and cultivate.

Tube propagation：From culture dish, picking single bacterium colony Pichia pastoris, the YPD for filling 3mL test tube is inoculated into In, cultivate about 18~24h in 30 DEG C, 220rpm constant-temperature tables.

Shaking culture：Recombinant bacterium is inoculated into the BMGY culture mediums for filling 25mL, trained under the conditions of 30 DEG C, 220rpm About 18h is supported, between OD600=4~6；Shift bacterium solution into sterile 50mL centrifuge tubes, under the conditions of 4 DEG C, 4000rpm from Heart 5min, thalline is resuspended with BMMY culture mediums, and adjusts OD₆₀₀1 or so, then, it is transferred to and fills 50mL BMMY culture mediums 500mL shaking flasks in carry out Fiber differentiation, every 24h sample, and supplement 1% methanol.

1.6 determination method

The measure of dry cell weight：Zymotic fluid is taken, is diluted to certain multiple, then when wavelength is at 600, determines OD₆₀₀ =OD_Reading× extension rate n；According to OD₆₀₀With the relation of dry cell weight：DCW (g/L)=0.24 × OD₆₀₀+1.23(R²= 0.994) DCW, is calculated.

1.7 glucose oxidase GOD enzyme activity determinations

Fermentation liquor treatment：Zymotic fluid is centrifuged into 5min, the EP of transfer fermented supernatant fluid to 1.5mL under the conditions of 12000rpm In, determine enzyme activity；Then thalline is resuspended with YeastBuster Protein Extraction Reagent, be placed on rocker 30min is incubated under room temperature condition, is then centrifuged for, takes supernatant in 1.5mL EP pipes, determines intracellular enzyme activity.

End-point method determines GOD enzyme activity：2.5mL 0.21mM dianisidine is sequentially added in 10mL centrifuge tubes, 0.3mL 18% glucose, 0.1mL 90U/mL horseradish peroxidase, after 37 DEG C are incubated 5min, add into centrifuge tube Enter V₀Enzyme liquid, after reacting 3min, 2mol/L sulfuric acid terminating reactions are added, take out centrifuge tube, determine OD₅₀₀Light absorption value.

GOD standard items are diluted to 0.5,1.0,1.5,2.0,2.5,3.0U/mL, according to GOD enzyme activity determination methods, survey Surely the OD after 3min is reacted₅₀₀Value, with OD₅₀₀It is worth for abscissa, using GOD enzyme activity as ordinate, draws standard curve and be used to calculate Enzyme activity：GOD enzyme activity (U/mL)=(0.1578 × OD₅₀₀-0.0033)×V/V₀, wherein V is to react cumulative volume, V₀To add enzyme Volume.

GOD enzyme activity defines：At 37 DEG C, the enzyme amount of 1 μm of ol of catalysis per minute β-D-Glucose generation gluconic acid is 1 Individual unit enzyme activity U.

The structure of embodiment 1, recombinant bacterial strain

1st, recombinant bacterium G/GODM structure

In order to realize glucose oxidase secreting, expressing in Pichia pastoris, the present inventor have selected pPIC9K carriers.With PUC57-GOD is masterplate, by PCR, is held in GOD sequences 5 ' and introduces EcoR Ι, 3 ' ends introduce Not Ι, digestion, with same digestion PPIC9K connections, build GOD secretion expression carrier pPIC9K-GOD, structure flow chart such as Fig. 1.More specifically, with plasmid PUC57-GOD (GOD nucleotide sequences are connected on pUC57) is template, and using primer GODF and GODR, purpose is prepared by PCR Fragment GOD (1749bp, is shown in Fig. 2), while introduces two restriction enzyme sites of EcoR Ι and Not Ι, distinguishes line using restriction enzyme Property amplified production and pPIC9K carriers, be then attached reaction, finally, bacillus coli DH 5 alpha be transformed into by thermal shock and experienced In state cell, and it is coated on the LB culture dishes containing Ampicillin resistances.

From the positive colony of picking recombination bacillus coli in Ampicillin resistant panels, after carrying out amplification cultivation, extracting Plasmid is simultaneously using the checking of BamHI and SalI double digestions, as can be seen from Figure 3, the stripe size that pPIC9K-GOD occurs after digestion It is in the same size with expection, sequencing company sequencing is sent to, through comparing, sequencing result is consistent with the expected results of target gene.Plasmid PPIC9K-GOD is successfully constructed.

By the correct plasmid pPIC9K-GOD of digestion verification, linearized, and purified with restriction enzyme SalI, pass through electricity Hit conversion to enter in Pichia pastoris GS115 competent cell, be incorporated into by the method for homologous recombination on GS115 genome, The principle that can be grown using GS115 histidine deficient feature and recombinant bacterium on the culture dish without histidine, in MD Screen recombinant bacterium on solid medium culture dish, the single bacterium colony on picking MD culture dishes, use primer GODF/GODR, carry out bacterium Fall PCR checking recombinant bacteriums (Fig. 4), and successfully constructed through sequence verification recombinant bacterium G/GOD.

The recombinant bacterium in MD culture dishes is collected using sterile water washing, and is coated on containing different antibiotic concentration G418 (0,0.25,0.5,0.75,1.0,1.5,1.75,2.0,3.0,4.0mg/mL) YPD culture dishes on, screening obtains high copy Recombinant bacterium G/GODM.

2nd, recombinant bacterium G/GMS3, G/GMM1 structure

Based on recombinant bacterium G/GODM, the present inventor choose on P.pastoris GS115 RH (PAS_chr-4_0191) and DDKC (PAS_chr-4_0192) is homologous recombination site, by sol3, mdh1, zwf1, gdh3 by way of homologous recombination it is whole Close on P.pastoris genomes, it recombinates principle and sees that Fig. 6, X therein represent sol3, mdh1, zwf1 or gdh3.

Structure coexpression bacterium, using pAOX-SSN as initial vector, inserts sol3 or mdh1 in SacI and NotI, obtains pAOX-zwf1、pAOX-sol3、pAOX-gdh3、pAOX-mdh1.Directly utilize plasmid pAOX-zwf1, pAOX-sol3, pAOX- Gdh3, pAOX-mdh1 (Fig. 5), they are transformed into after being cultivated in Escherichia coli and extract plasmid, use XhoI and NotI digestion matter Grain, it is used to convert recombinant bacterium G/GODM (Fig. 7) after electrophoresis is verified.

Expression vector pAOX-sol3, pAOX-mdh1, pAOX-zwf1, pAOX-gdh3 are through Xho Ι/EcoR Ι double digestions Afterwards, isolate and purify to obtain the fragment for homologous recombination, electroporated recombinant bacterium G/GODM competent cells, from containing Picking transformant on the YPD flat boards of Zeocin resistances, using RHF/pAOXR and CYCTTF2/DDKCR2 as primer, passes through bacterium colony PCR checking positive restructuring bacterium (such as Fig. 8), and be sequenced, known by nucleic acid electrophoresis and sequencing result, recombinant bacterium G/GMS3, G/GMM1, G/GMZ1, G/GMG3 are successfully constructed.

Embodiment 3, recombinant bacterium Performance

Picking verifies correct transformant G/GMS3 and G/GMM1, is inoculated into the test tube for filling 3mL YPG culture mediums, trains After supporting about 18h, it is transferred in the 250mL shaking flasks containing 25mL BMGY culture mediums, enrichment culture to OD₆₀₀For 4~6, bacterium is collected Body, it is inoculated into the 500mL shaking flasks containing 50mL BMMY culture mediums and cultivates, originates OD₆₀₀=1, every the first of 24h supplements 1% Alcoholic solution, while OD is measured by sampling₆₀₀, investigate growth effect to recombinant bacterium of coexpression zwf1, sol3, gdh3, mdh1, enzyme activity Change etc..

1st, thalli growth

As shown in Figure 9, recombinant bacterium G/GMZ1, G/GMS3, G/GMG3, G/GMM1 growth tendencies and go out bacterium germination G/GODM ten and divide Close, average specific growth rate μ is in 0.039h^-1Left and right, illustrate that co-expression gene sol3, mdh1 expression have no effect on thalline life It is long.

2nd, enzyme activity

Known by Figure 10 A, when inducing 120h, G/GMS3 and G/GMM1 unit thalline enzyme activity are respectively 6209.25U/g DCW and 6521.2U/gDCW, improves 6.3% and 11.6% than G/GODM enzyme activity respectively.But G/GMZ1, G/GMG3 do not have There is display enzyme activity to be higher than G/GODM.

(Figure 10 B) is understood from GOD intracellular expressions amount, when inducing 96h, intracellular unit thalline enzyme activity reaches maximum, but The horizontal no significant difference of recombinant bacterium G/GMS3, G/GMM1 and G/GODM intracellular expression, highest intracellular expression level 1850~ Between 2300U/gDCW.

Calculate volume expression and understand that recombinant bacterium G/GMS3 and G/GMM1 extracellular unit volume enzyme activity are 55.21U/ ML and 60.50U/mL (Figure 11), improves 7.3% and 17.5% than G/GODM respectively.In this two plants of bacterium, GOD secretion rates are 75.4% and 75.8%, improved compared with G/GODM (74.6%).But born of the same parents are not presented in G/GMZ1, G/GMG3 The increase of other unit's volume enzyme activity, has declined on the contrary.

All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited Enclose.

Sequence table

<110>East China University of Science

<120>Optimize method, recombinant bacterium and its application for carrying out Glucose oxidase secretion expression based on metabolic engineering

<130> 176037

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

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<213>Yeast (Yeast)

<400> 1

atgttgtcca caattgccaa gcgtcaattc tcctcctctg cctctactgc ctacaaggtt 60

gccgttcttg gtgccgctgg tggaattggt cagcctttgt cgttgctgat gaagttgaac 120

cacaaggtta ctgacttagc cctgtatgac atccgtttgg ctccaggtgt agccgctgat 180

gtatcccaca tcccaaccaa ctccaccgtc accggttaca ctcctgaaga taatggtttg 240

gaaaagacac tgacgggagc tgatctggtc atcattccag ctggtgtccc aagaaagcca 300

ggtatgacca gagacgatct gttcaacacc aatgcttcta ttgtcagaga tttggccaaa 360

gctgttggtg actacagtcc tagtgctgcg gttgctatta tttctaaccc agttaactcc 420

actgttccaa ttgttgctga ggtcttgaag tccaagggtg tctacaaccc aaagaagcta 480

ttcggtgtca ccactttgga tgttctgaga gcctctcgtt tcttgtctca agtgcaaggt 540

accaacccag ccagtgagcc agtcactgtt gttggtggtc actcaggtgt cactattgtt 600

cctctgctgt ctcaatctaa gcacaaggac ttgccaaagg acacttacga cgctctggtc 660

caccgtatcc aatttggtgg tgatgaggtt gtcaaggcca aagatggtgc tggatctgct 720

acgctgtcta tggctcaggc cggtgctaga tttgccagct ccgtgttgaa cggtttggcc 780

ggtgagaatg atgtcgttga gccatctttc gtcgactctc cattgttcaa ggatgagggt 840

attgaattct tctcctccaa ggttactttg ggtccagagg gtgtcaagac catccatggt 900

ttgggagaat tgtctgctgc tgaagaggag atgatcacaa ctgccaagga gactttggcc 960

aagaacatcg ctaagggtca agagtttgtt aagcaaaacc cataa 1005

<210> 2

<211> 750

<212> DNA

<213>Yeast (Yeast)

<400> 2

atggtacaaa tctattccta tgaacgatct gatgaaattg ctaatgcagt agccaattac 60

atattagaca ttcaggatca cgtactaaaa actaatactg tttttaggat cgctgtcagt 120

ggaggctccc ttggcaaggt attaaagaag ggattgatag acaatcaaga gaacagatca 180

aaaattgcct gggataaatg gcatgtgtat ttcagtgacg aaaggttagt aaaactcaat 240

cacgaggact ccaattatgc cctattcaat gaaatggttt tgaagcctct acaaaaattc 300

aaaatgccac taccaagagt tgtcaccatc aaggaggatc tattagatga aagccgaata 360

aacgatgcaa tgatagcaag tgagtatgaa catcaccttc ccagtgtttt ggatcttgtc 420

cttttgggat gtggacctga tggtcatact tgttcccttt ttccgaacca caaactatta 480

agggaaacct caaaacgcat tgctgctata tcagattctc ccaagccacc ttcgaggaga 540

ataactttca catttccagt ccttgagaac tcctctaata tagcttttgt cgccgaagga 600

gaaggaaaat ctcctgtctt aaggcaaatt tttggagaag aaaagaccaa tttaccatgc 660

gaaatcgtaa acaaattatc tactcgagtg agttggtttg tcgataacca tgctcttagt 720

ggagtctccg tttctacttc gaaatactga 750

<210> 3

<211> 605

<212> PRT

<213>Aspergillus niger (Aspergillus niger)

<400> 3

Met Gln Thr Leu Leu Val Ser Ser Leu Val Val Ser Leu Ala Ala Ala

1 5 10 15

Leu Pro His Tyr Ile Arg Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr

20 25 30

Asp Pro Lys Asp Val Ser Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly

35 40 45

Gly Gly Leu Thr Gly Leu Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro

50 55 60

Asn Ile Ser Val Leu Val Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg

65 70 75 80

Gly Pro Ile Ile Glu Asp Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser

85 90 95

Ser Val Asp His Ala Tyr Glu Thr Val Glu Leu Ala Thr Asn Asn Gln

100 105 110

Thr Ala Leu Ile Arg Ser Gly Asn Gly Leu Gly Gly Ser Thr Leu Val

115 120 125

Asn Gly Gly Thr Trp Thr Arg Pro His Lys Ala Gln Val Asp Ser Trp

130 135 140

Glu Thr Val Phe Gly Asn Glu Gly Trp Asn Trp Asp Asn Val Ala Ala

145 150 155 160

Tyr Ser Leu Gln Ala Glu Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile

165 170 175

Ala Ala Gly His Tyr Phe Asn Ala Ser Cys His Gly Val Asn Gly Thr

180 185 190

Val His Ala Gly Pro Arg Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val

195 200 205

Lys Ala Leu Met Ser Ala Val Glu Asp Arg Gly Val Pro Thr Lys Lys

210 215 220

Asp Phe Gly Cys Gly Asp Pro His Gly Val Ser Met Phe Pro Asn Thr

225 230 235 240

Leu His Glu Asp Gln Val Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu

245 250 255

Pro Asn Tyr Gln Arg Pro Asn Leu Gln Val Leu Thr Gly Gln Tyr Val

260 265 270

Gly Lys Val Leu Leu Ser Gln Asn Gly Thr Thr Pro Arg Ala Val Gly

275 280 285

Val Glu Phe Gly Thr His Lys Gly Asn Thr His Asn Val Tyr Ala Lys

290 295 300

His Glu Val Leu Leu Ala Ala Gly Ser Ala Val Ser Pro Thr Ile Leu

305 310 315 320

Glu Tyr Ser Gly Ile Gly Met Lys Ser Ile Leu Glu Pro Leu Gly Ile

325 330 335

Asp Thr Val Val Asp Leu Pro Val Gly Leu Asn Leu Gln Asp Gln Thr

340 345 350

Thr Ala Thr Val Arg Ser Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly

355 360 365

Gln Ala Ala Trp Phe Ala Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser

370 375 380

Glu Lys Ala His Glu Leu Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu

385 390 395 400

Glu Ala Val Ala Arg Gly Gly Phe His Asn Thr Thr Ala Leu Leu Ile

405 410 415

Gln Tyr Glu Asn Tyr Arg Asp Trp Ile Val Asn His Asn Val Ala Tyr

420 425 430

Ser Glu Leu Phe Leu Asp Thr Ala Gly Val Ala Ser Phe Asp Val Trp

435 440 445

Asp Leu Leu Pro Phe Thr Arg Gly Tyr Val His Ile Leu Asp Lys Asp

450 455 460

Pro Tyr Leu His His Phe Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu

465 470 475 480

Leu Asp Leu Leu Gly Gln Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile

485 490 495

Ser Asn Ser Gly Ala Met Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro

500 505 510

Gly Asp Asn Leu Ala Tyr Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr

515 520 525

Ile Pro Tyr His Phe Arg Pro Asn Tyr His Gly Val Gly Thr Cys Ser

530 535 540

Met Met Pro Lys Glu Met Gly Gly Val Val Asp Asn Ala Ala Arg Val

545 550 555 560

Tyr Gly Val Gln Gly Leu Arg Val Ile Asp Gly Ser Ile Pro Pro Thr

565 570 575

Gln Met Ser Ser His Val Met Thr Val Phe Tyr Ala Met Ala Leu Lys

580 585 590

Ile Ser Asp Ala Ile Leu Glu Asp Tyr Ala Ser Met Gln

595 600 605

<210> 4

<211> 49

<212> DNA

<213>Primer (Primer)

<400> 4

agagaggctg aagcttacgt agaattctct aatggtattg aagcatcat 49

<210> 5

<211> 44

<212> DNA

<213>Primer (Primer)

<400> 5

taaggcgaat taattcgcgg ccgcttgcat tgaagcgtag tctt 44

<210> 6

<211> 25

<212> DNA

<213>Primer (Primer)

<400> 6

gctggaaagg tttgaggagg atctg 25

<210> 7

<211> 24

<212> DNA

<213>Primer (Primer)

<400> 7

gtaatgcgga gcttgttgca ttcg 24

<210> 8

<211> 24

<212> DNA

<213>Primer (Primer)

<400> 8

ccgctctaac cgaaaaggaa ggag 24

<210> 9

<211> 26

<212> DNA

<213>Primer (Primer)

<400> 9

gaacccaaca aattcagaga tgcctc 26

Claims

A kind of 1. method using yeast cell to express glucose oxidase, it is characterised in that methods described includes：

(1) the glucose oxidase encoding gene of external source is introduced in yeast cells；

(2) at least one gene being selected from the group of external source is introduced in the yeast cells of step (1)：Malic dehydrogenase 1 is compiled Code gene, 6-phosphogluconic acid lactone enzyme coding gene；

(3) yeast cells of incubation step (2), expresses glucose oxidase, and described glucose oxidase is the Portugal of high enzyme activity Grape carbohydrate oxidase.
2. the method as described in claim 1, it is characterised in that glucose oxidase encoding gene, malic dehydrogenase 1 encode Gene or 6-phosphogluconic acid lactone enzyme coding gene are incorporated into the genome of yeast cells by way of homologous recombination.
3. the method as described in claim 1, it is characterised in that methods described includes：

(1) expression vector is provided, the sequence of glucose oxidase encoding gene is included in the expression vector, by the expression vector It is transferred in yeast cells；

(2) expression vector is provided, included in the expression vector in the encoding gene of malic dehydrogenase 1 or 6-phosphogluconic acid The sequence of esterase encoding gene, the expression vector is transferred in the yeast cells of step (1)；

(3) yeast cells of incubation step (2), glucose oxidase is expressed.
4. the method as described in claim 1, it is characterised in that described yeast cells is Pichia pastoris.
5. the method as described in Claims 1 to 4 is any, it is characterised in that the grape glycosyloxy of external source is driven with AOX1 promoters Change the expression of enzyme coding gene, the expression of the 6-phosphogluconic acid lactone enzyme coding gene of external source is driven with AOX1 promoters.
A kind of 6. method of the expression cell for the glucose oxidase for preparing high enzyme activity, it is characterised in that methods described includes：

(1) the glucose oxidase encoding gene of external source is introduced in yeast cells；

(2) at least one gene being selected from the group of external source is introduced in the yeast cells of step (1)：Malic dehydrogenase 1 is compiled Code gene, 6-phosphogluconic acid lactone enzyme coding gene；

(3) the glucose oxidase encoding gene for carrying external source, at least one gene being selected from the group of external source are isolated：Apple Tartaric acid dehydrogenase 1 encoding gene, the recombinant yeast cell of 6-phosphogluconic acid lactone enzyme coding gene.
7. a kind of expression cell of the glucose oxidase of high enzyme activity, it is characterised in that it is yeast cells, whole in its genome Conjunction has：

The glucose oxidase encoding gene of external source；And

At least one gene being selected from the group of external source：The encoding gene of malic dehydrogenase 1,6-phosphogluconic acid lactonase are compiled Code gene.
8. cell as claimed in claim 6, it is characterised in that the glucose oxidase encoding gene of described external source, external source The encoding gene of malic dehydrogenase 1 or 6-phosphogluconic acid lactone enzyme coding gene be incorporated into by way of homologous recombination In the genome of yeast cells.
9. cell as claimed in claim 7, it is characterised in that it is prepared by the method described in claim 6.
10. a kind of kit for being used to produce the glucose oxidase of high enzyme activity, it is characterised in that the kit includes： Any described yeast cells of claim 7~9.