CN107460175B

CN107460175B - Method for carrying out glucose oxidase secretory expression based on metabolic engineering optimization, recombinant bacterium and application thereof

Info

Publication number: CN107460175B
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: 2021-04-23
Anticipated expiration: 2037-09-06
Also published as: CN107460175A

Abstract

The invention relates to a method for optimizing secretion expression of Glucose Oxidase (GOD) based on metabolic engineering, a recombinant strain and application thereof. The invention provides a method capable of efficiently secreting and expressing GOD and improving enzyme activity of the GOD, which realizes the efficient expression of the GOD by co-expressing the GOD and a malate dehydrogenase 1 encoding gene (mdh1) or a 6-phosphogluconolactonase encoding gene (sol3) in a yeast strain. Meanwhile, the invention also provides a recombinant bacterium which is constructed by taking Pichia pastoris (Pichia pastoris) as a host and can efficiently secrete GOD.

Description

Method for carrying out glucose oxidase secretory expression based on metabolic engineering optimization, recombinant bacterium and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a method for carrying out secretion expression of glucose oxidase based on metabolic engineering optimization, a recombinant bacterium and application thereof.

Background

Glucose Oxidase (GOD) is widely applied to the fields of food, chemical industry, medicine, biotechnology and the like.

Based on the specificity of the glucose oxidase catalytic reaction, the method can be used for biosensors. Glucose oxidase is a core component of a sensor for detecting the concentration of glucose in blood, GOD immobilized on a sensor electrode can convert a trace amount of glucose in blood into hydrogen peroxide which can be easily measured, and the more hydrogen peroxide detected by a platinum electrode, the stronger the electric signal. The glucometer made of the glucose oxidase can conveniently detect the fluctuation level of the blood sugar of the diabetic.

Glucose oxidase is widely used in the food industry. The oxygen-removing and fresh-keeping method is used for oxygen-removing and fresh-keeping of food, and a small amount of glucose oxidase and glucose are added into a commodity vacuum bag, so that oxygen in the vacuum bag can be exhausted, growth and reproduction of microorganisms are inhibited, and the shelf life of the food is prolonged. Removing glucose from the protein preparation. The egg albumen contains 0.5-0.6% of glucose, and during the storage and processing, the carbonyl of the glucose and the amino of the albumen generate Maillard reaction, and small black spots appear in the albumen. The addition of GOD consumes the glucose, thus ensuring the quality of the protein. Can be added into flour as food additive. Bonet et al found that GOD can react with glucose in the dough to generate hydrogen peroxide, and with the help of the hydrogen peroxide, mucedin can form disulfide bonds, so that a complex network structure is formed, and the structure can enhance the strength of the dough and improve the mouthfeel of the flour product. In addition, the hydrogen peroxide can change the color of the flour, so that the appearance of the flour product becomes beautiful.

In the textile industry, GOD is often used in bleaching and decolorizing processes. The GOD is difficult to reuse because the strong oxidizing hydrogen peroxide is generated in the process of catalyzing beta-D-glucose by the GOD. Tzanov et al covalently immobilized GOD on alumina and glass supports to increase enzyme reuse efficiency. After about 450 minutes, the maximum hydrogen peroxide concentration fixed to the glass support reached 0.35g/L compared to 0.24g/L on alumina, and the hydrogen peroxide concentration on the textile was approximately the same level compared to the standard bleaching process, increasing GOD reuse.

GOD has great potential in low alcohol brewing processes. After the glucose oxidase is added in the production of the white spirit, the content of the alcohol in the white spirit is reduced by 2 percent, so that the white spirit has pure taste and does not generate the phenomena of precipitation, turbidity and the like. In the fermentation process, the GOD can kill acetic acid bacteria and lactic acid bacteria, the sterilization effect of the GOD means that the GOD cannot be widely applied to wine production, Pickering and the like use a GOD/CAT dual-enzyme system to treat grape juice, the ethanol content is reduced, and 87% of glucose is converted into gluconic acid.

At present, glucose oxidase is also applied to the biofuel cell industry, catalase and GOD are fixed on the same electrode, and because GOD catalyzes glucose reaction to be redox reaction, electrons can be transferred to the carbon electrode at the other end, so that a green environment-friendly biofuel cell is formed, and continuous energy can be provided for biosensors and artificial organs.

In the oral cavity, the existence of streptococcus usually can improve the probability of decaying teeth, GOD can be added into toothpaste and can react with residual glucose in the oral cavity, and the generated hydrogen peroxide can inhibit the breeding and reproduction of harmful bacteria, thereby reducing the probability of suffering from oral diseases.

Gluconic acid and derivatives thereof are widely applied to the health care product industry, and GOD, as a key enzyme in the production of the gluconic acid, can be used independently for enzymatic production, and can also be added to rapidly synthesize the gluconic acid in the production process of Aspergillus niger so as to improve the yield of the gluconic acid.

Currently, the GOD is produced by a microbial fermentation method in the field, but the GOD produced by fermentation of Aspergillus niger or Penicillium has the problems of low enzyme yield, difficulty in separation and purification of intracellular enzymes and the like. And the GOD produced by using escherichia coli cannot be subjected to post-translational processing, and the synthesized GOD is inactive. The skilled person also uses pichia pastoris to produce GOD, but the yield and enzyme activity are not ideal enough, and further optimization of GOD production process is needed.

Disclosure of Invention

The invention aims to provide a metabolic engineering-based method for secretory expression of glucose oxidase, a recombinant strain and application thereof.

In a first aspect of the invention, there is provided a method of expressing glucose oxidase using a yeast cell, the method comprising:

(1) introducing exogenous glucose oxidase coding gene into yeast cells;

(2) introducing into the yeast cells of step (1) an exogenous at least one gene selected from the group consisting of: a malate dehydrogenase 1 encoding gene, a 6-phosphogluconolactonase encoding gene;

(3) and (3) culturing the yeast cells in the step (2) to express glucose oxidase, wherein the glucose oxidase is high-enzyme-activity glucose oxidase.

In a preferred embodiment, the glucose oxidase-encoding gene, the malate dehydrogenase 1-encoding gene or the 6-phosphogluconolactonase-encoding gene is integrated into the genome of the yeast cell by means of homologous recombination.

In another preferred example, the method comprises:

(1) providing an expression vector, wherein the expression vector contains a sequence of a glucose oxidase coding gene, and transferring the expression vector into a yeast cell;

(2) providing an expression vector which comprises a sequence of a malate dehydrogenase 1 encoding gene or a 6-phosphogluconolactonase encoding gene, and transferring the expression vector into the yeast cell in the step (1);

(3) and (3) culturing the yeast cells in the step (2) to express glucose oxidase.

In another preferred embodiment, the yeast cell is a pichia pastoris cell.

In another preferred embodiment, the AOX1 promoter is used to drive the expression of the exogenous gene encoding glucose oxidase, and the AOX1 promoter is used to drive the expression of the exogenous gene encoding 6-phosphogluconolactonase.

In another aspect of the present invention, there is provided a method for preparing a cell expressing a high-enzyme-activity glucose oxidase, the method comprising:

(1) introducing exogenous glucose oxidase coding gene into yeast cells;

(3) isolating a gene encoding glucose oxidase carrying an exogenous gene, the exogenous gene being selected from at least one of the following: a gene encoding malate dehydrogenase 1 and a gene encoding 6-phosphogluconolactonase.

In another aspect of the present invention, there is provided a high enzyme activity glucose oxidase-expressing cell, which is a yeast cell having integrated in its genome: an exogenous glucose oxidase encoding gene (preferably, it is expressed from the AOX1 promoter); and an exogenous at least one gene selected from the group consisting of: malate dehydrogenase 1 encoding gene, 6-phosphogluconolactonase encoding gene (preferably, it is expressed from AOX1 promoter).

In a preferred embodiment, the exogenous glucose oxidase encoding gene, the exogenous malate dehydrogenase 1 encoding gene or the 6-phosphogluconolactonase encoding gene is integrated into the genome of the yeast cell by means of homologous recombination; preferably, the yeast cell is prepared by the method described above.

In another aspect of the present invention, there is provided a kit for producing a high-enzyme-activity glucose oxidase, comprising: a yeast cell as described in any preceding paragraph.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, plasmid pPIC9K-GOD construction.

FIG. 2, PCR amplification of GOD gene.

Lane M：Marker 5,000；

Lane1, 2: plasmid pUC57-GOD was used as a template, and GODF/GODR was used as a primer.

FIG. 3, restriction enzyme verification plasmid pPIC 9K-GOD.

Lane M：Marker 15,000；

Lane 1: pPIC9K-GOD was digested with BamHI and SalI (3982bp and 7037 bp).

FIG. 4, PCR verification of recombinant G/GODM.

Lane M：Marker 5,000；

Lane 1: using P.pastoris GS115 genome DNA as a template;

lane 2, 3: recombinant strain G/GODM genome DNA is used as a template.

Lane1, 2, 3: GODF/GODR is used as a primer.

FIG. 5 is a schematic diagram showing the construction of expression vectors pAOX-sol3, pAOX-mdh1, pAOX-zwf1 and pAOX-gdh 3.

FIG. 6, double restriction enzyme verification of expression vectors pAOX-zwf1, pAOX-sol3, pAOX-gdh3, pAOX-mdh 1.

Lane M：Marker 15,000；

Lane 1: pAOX-zwf1 was digested by Xho I and Not I;

lane 2: pAOX-sol3 was digested by Xho I and Not I;

lane 3: pAOX-gdh3 was digested by Xho I and Not I;

lane 4: pAOX-mdh1 was digested by Xho I and Not I.

FIG. 7 shows the construction process of recombinant bacteria G/GMS3, G/GMM1, G/GMZ1 and G/GMG3 (X represents sol3, mdh1, zwf1 and gdh 3).

FIG. 8 shows PCR verification of GOD recombinant bacteria co-expressing chaperones.

And A, carrying out RH site integration verification on the recombinant bacteria. Lane M: 2,000 bp; lane1, 2, 3, 4: genomic DNAs of G/GMZ1, G/GMS3, G/GMG3 and G/GMM1 were used as templates and RHF/AOXR as primers, respectively.

And B, carrying out integration verification on the DDKC locus of the recombinant strain. Lane M: 2,000 bp; lane1, 2, 3, 4: genomic DNAs of G/GMZ1, G/GMS3, G/GMG3 and G/GMM1 were used as templates, and CYCTTF2/DDKCR2 primers were used.

FIG. 9 growth curves of recombinant bacteria G/GMZ1, G/GMS3, G/GMG3 and G/GMM1 in BMMY medium.

FIG. 10 shows the specific activities of recombinant bacteria G/GMZ1, G/GMS3, G/GMG3, G/GMM1 extracellular (A) and intracellular (B) GOD.

FIG. 11 and 120h show the enzyme activity levels of recombinant bacteria G/GMZ1, G/GMS3, G/GMG3 and G/GMM 1.

Detailed Description

In order to increase the yield of GOD produced by yeast, the inventors of the present invention conducted extensive research and extensive screening to develop a method capable of effectively promoting expression of GOD and increasing the enzyme activity thereof by co-expressing GOD and a malate dehydrogenase 1-encoding gene (mdh1) or a 6-phosphogluconolactonase-encoding gene (sol3) in a yeast strain and enhancing the carbon metabolic pathway, thereby achieving efficient secretory expression of GOD. Meanwhile, the invention also provides a recombinant bacterium which is constructed by taking Pichia pastoris (Pichia pastoris) as a host and can efficiently secrete GOD.

As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, a promoter is foreign to a gene of interest if the combination of the promoter and the sequence of the gene of interest is not normally found in nature. A particular sequence is "foreign" to the cell or organism into which it is inserted.

By "promoter" is meant a nucleic acid sequence, usually present upstream (5' to) the coding sequence of a gene of interest, capable of directing transcription of the nucleic acid sequence into mRNA. Generally, a promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary to properly initiate transcription.

As used herein, the "sol 3" and "mdh 1" genes are genes for recombinant expression in yeast cells, and also include molecules that hybridize to the gene sequences under stringent conditions, or family gene molecules that are highly homologous to the above-mentioned molecules. Also included in this definition are molecules that hybridize under stringent conditions to "sol 3", "mdh 1", or family gene molecules that are highly homologous to the above-described molecules.

As used herein, the term "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 50%, preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, more preferably 95% or more.

mdh1 (mitochondral matrix dehydrogenase) gene encodes Mitochondrial malate dehydrogenase, which catalyzes the production of oxaloacetate from malate. The nucleotide sequence of mdh1 gene (PAS _ chr2-1_0238) is shown in SEQ ID NO. 1, or can be a degenerate sequence thereof. The invention also relates to polynucleotide variants of "mdh 1" which encode a polypeptide (enzyme) which is identical to the amino acid sequence encoded by the wild-type gene. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.

The sol3 gene encodes gluconolactonase 6-phosphate, an enzyme in the non-oxidative Pentose Phosphate (PP) pathway. The nucleotide sequence of sol3 gene (PAS _ chr3_1126) is shown in SEQ ID NO:2, and can also be a degenerate sequence thereof. The present invention also relates to polynucleotide variants of "sol 3" which encode a polypeptide (enzyme) having the same amino acid sequence as that encoded by the wild-type gene. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.

The amino acid sequence (Uniprot: P13006) of the GOD gene code in the invention is shown in SEQ ID NO. 3 (from Aspergillus niger), and the front 22 position is a signal peptide sequence. The invention also relates to polynucleotide variants of a "GOD" which encode a polypeptide (enzyme) having the same amino acid sequence as that encoded by the wild-type gene. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.

The full-length nucleotide sequence of the "sol 3" or the "mdh 1" or the fragment thereof of the present invention can be obtained by a PCR amplification method, a recombinant method or a synthetic method. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates.

In order to improve the enzyme activity of glucose oxidase after recombinant expression, the present inventors have conducted extensive studies, found a gene suitable for improvement, and constructed a corresponding construct.

Accordingly, the present invention provides a construct comprising an exogenous "GOD gene" and an exogenous expression cassette of "sol 3". The expression cassette has all elements required for gene expression (including promoter, coding DNA, terminator, etc.), so that the corresponding protein can be completely expressed.

The invention also provides a construct comprising expression cassettes for an exogenous "GOD gene" and an exogenous "mdh 1" gene. The expression cassette has all the necessary elements (including promoter, coding DNA and terminator, etc.) for gene expression, so that the corresponding protein can be completely expressed.

Typically, the construct is located on an expression vector. Thus, the invention also includes a vector comprising the construct. The expression vector usually further contains an origin of replication and/or a marker gene and the like. Methods well known to those skilled in the art can be used to construct the expression vectors required by the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

It is to be understood that various means for recombinantly expressing "GOD gene", "sol 3" or "mdh 1" may be used, as long as increased expression of "GOD gene", "sol 3" or "mdh 1" in the yeast cell can be achieved. As a preferred mode of the present invention, the GOD gene, "sol 3" or "mdh 1" is integrated into the genome of the yeast cell by means of homologous recombination.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance for eukaryotic cell culture.

The methods may be carried out using suitable conventional means, including reagents, temperature, pressure conditions, and the like. When the expression vector of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene. It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Vectors comprising the appropriate polynucleotide sequences described above, together with appropriate promoter or control sequences, may be used to transform an appropriate host. In the method of the present invention, the host is a yeast cell.

Transformation of a host cell with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art, such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like. In a preferred embodiment, the method may be carried out by electrotransformation.

The method is simple and easy to implement and has high production efficiency. Moreover, the results of the invention show that the enzymatic activity of GOD produced by the recombinant yeast cell is obviously improved. Compared with the method only expressing GOD, the method can improve the unit bacterial enzyme activity of the GOD by about more than 6 percent; more preferably about 11% or more.

The invention also provides a kit containing the recombinant expression vector and the yeast cell constructed by the invention; or a kit comprising the recombinant yeast cell constructed according to the present invention.

Other reagents commonly used to perform transgenic procedures may also be included in the kit for ease of use by those skilled in the art.

In addition, the kit may further comprise instructions for use to instruct a person skilled in the art to perform the method.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

1. Materials and methods

1.1 plasmids

The plasmids used in the present invention are shown in Table 1.

TABLE 1 plasmids

1.2 strains

The strains used in the present invention are shown in Table 2.

TABLE 2 strains

1.3 primers

Primers used in the present invention are shown in Table 3.

TABLE 3 primers

1.4 Medium

LB liquid medium:

20g/L tryptone, 10g/L yeast extract, 10g/L NaCl.

The preparation of the solid LB culture medium needs to be supplemented with 2 percent of agar sugar powder; LB-resistant medium may be supplemented with appropriate concentrations of antibiotics.

YPD liquid Medium:

20g/L tryptone, 20g/L glucose, 10g/L yeast extract.

The preparation of the solid YPD culture medium needs to be supplemented with 2 percent of agar sugar powder; YPD-resistant medium can be supplemented with appropriate concentrations of antibiotics.

YPG liquid Medium:

20g/L tryptone, 10g/L glycerol, 10g/L yeast extract.

MD solid Medium:

20g/L glucose, 0.4mg/L biotin, 13.4g/L YNB and 20g/L agar powder.

BMGY liquid Medium

10g/L of glycerol, 10g/L of yeast extract, 20g/L of Peptone, 13.4g/L of YNB, 0.1MpH ═ 6K₂HPO₄/KH₂PO₄And (4) a buffer solution.

BMMY liquid medium:

1% methanol, 10g/L yeast extract, 20g/L Peptone, 13.4g/L YNB, 0.1M pH 6K₂HPO₄/KH₂PO₄And (4) a buffer solution.

1.5 culture method

Plate culture: taking out the Pichia pastoris glycerol tube from a refrigerator at-20 ℃, streaking the Pichia pastoris glycerol tube in a YPD-containing solid medium by using a sterilized inoculating loop, and culturing the Pichia pastoris glycerol tube in an incubator at 30 ℃.

And (3) test tube culture: and (3) selecting a single colony of Pichia pastoris cells from the culture dish, inoculating the single colony of Pichia pastoris cells into a test tube containing 3mL of YPD, and culturing the single colony of Pichia pastoris cells in a constant temperature shaking table at 30 ℃ and 220rpm for about 18-24 hours.

And (3) shake flask culture: inoculating the recombinant bacteria into a container with a volume of 25mCulturing in BMGY medium L at 30 ℃ and 220rpm for about 18h until OD600 is between 4 and 6; transferring the bacterial liquid into a sterile 50mL centrifuge tube, centrifuging at 4 deg.C and 4000rpm for 5min, resuspending the bacterial cells with BMMY medium, and adjusting OD₆₀₀At around 1, then, transfer to a 500mL shake flask containing 50mL BMMY medium for induction culture, sample every 24h and supplement with 1% methanol.

1.6 detection and analysis method

Measurement of the Dry weight of the cells: taking the fermentation liquid, diluting to a certain multiple, and measuring OD at 600 wavelength₆₀₀＝OD_{Reading number}X is a dilution multiple n; according to OD₆₀₀Relationship with the dry weight of the cells: DCW (g/L) ═ 0.24 XOD₆₀₀+1.23(R²0.994), DCW is calculated.

1.7 determination of enzyme Activity of glucose oxidase GOD

And (3) treating fermentation liquor: centrifuging the fermentation liquor at 12000rpm for 5min, transferring the fermentation supernatant to 1.5mL of EP, and measuring enzyme activity; then the thalli is resuspended by YeastBuster Protein Extraction Reagent, put on a shaking plate and incubated for 30min under the condition of room temperature, then centrifuged, and the supernatant is taken out to be put in a 1.5mL EP tube to measure the intracellular enzyme activity.

Determining GOD enzyme activity by an end-point method: 2.5mL of 0.21mM o-dianisidine, 0.3mL of 18% glucose, 0.1mL of 90U/mL horseradish peroxidase were sequentially added to a 10mL centrifuge tube, and after incubation at 37 ℃ for 5min, V was added to the centrifuge tube₀Reacting enzyme solution for 3min, adding 2mol/L sulfuric acid to terminate the reaction, taking out the centrifuge tube, and measuring OD₅₀₀The absorbance of (a).

Diluting GOD standard into 0.5, 1.0, 1.5, 2.0, 2.5, 3.0U/mL, and determining OD after 3min reaction according to GOD enzyme activity determination method₅₀₀Value in OD₅₀₀The value is an abscissa, the GOD enzyme activity is an ordinate, and a standard curve is drawn for calculating the enzyme activity: GOD enzyme activity (U/mL) ═ 0.1578 XOD₅₀₀-0.0033)×V/V₀Wherein V is the total volume of the reaction, V₀The volume of enzyme added.

GOD enzyme activity definition: the enzyme amount for catalyzing 1 mu mol of beta-D-glucose to generate gluconic acid per minute at 37 ℃ is 1 unit of enzyme activity U.

Example 1 construction of recombinant strains

1. Construction of recombinant strain G/GODM

To achieve secreted expression of glucose oxidase in pichia, the present inventors selected the pPIC9K vector. And (3) taking pUC57-GOD as a template, introducing EcoR I at the 5 'end and Not I at the 3' end of the GOD sequence through PCR, performing enzyme digestion, and connecting with pPIC9K subjected to the same enzyme digestion to construct a GOD secretion expression vector pPIC9K-GOD, wherein the construction flow chart is shown in figure 1. More specifically, plasmid pUC57-GOD (a GOD nucleic acid sequence is linked to pUC 57) is used as a template, primers GODF and GODR are used, a target fragment GOD (1749bp, see fig. 2) is prepared by PCR, two digestion sites of EcoR Ι and Not Ι are simultaneously introduced, an amplification product and a pPIC9K vector are respectively linearized by using restriction enzymes, then a ligation reaction is carried out, and finally, the plasmid is transformed into escherichia coli DH5 α competent cells by heat shock and coated on an Ampicillin-resistant LB culture dish.

And (3) selecting a positive clone of the recombinant escherichia coli from an Ampicillin resistant plate, carrying out amplification culture, extracting a plasmid, carrying out double enzyme digestion verification by using BamHI and SalI, wherein as shown in a figure 3, the size of a band generated by carrying out enzyme digestion on pPIC9K-GOD is consistent with the expected size, sending the band to a sequencing company for sequencing, and comparing to obtain a sequencing result which is consistent with the expected result of the target gene. The plasmid pPIC9K-GOD was successfully constructed.

The plasmid pPIC9K-GOD with correct restriction enzyme digestion verification is linearized by restriction enzyme SalI and purified, and is transformed into a pichia pastoris GS115 competent cell through electric shock, and is integrated on a genome of GS115 through a homologous recombination method, the recombinant bacteria are screened on an MD solid medium culture dish by utilizing the histidine defect type characteristic of GS115 and the principle that the recombinant bacteria can grow on a culture dish without histidine, a single colony on the MD culture dish is picked, a primer GODF/GODR is used for colony PCR verification of the recombinant bacteria (figure 4), and the construction success of the recombinant bacteria G/GOD is verified through sequencing.

The recombinant bacteria in the MD culture dish are collected by using sterile water washing, and are spread on YPD culture dishes containing different antibiotic concentrations of G418(0, 0.25, 0.5, 0.75, 1.0, 1.5, 1.75, 2.0, 3.0 and 4.0mg/mL), and high-copy recombinant bacteria G/GODM are obtained by screening.

2. Construction of recombinant bacteria G/GMS3 and G/GMM1

Based on recombinant bacteria G/GODM, the inventor selects RH (PAS _ chr-4_0191) and DDKC (PAS _ chr-4_0192) on P.pastoris GS115 as homologous recombination sites, and integrates sol3, mdh1, zwf1 and gdh3 on a P.pastoris genome in a homologous recombination mode, wherein the recombination principle is shown in figure 6, and X represents sol3, mdh1, zwf1 or gdh 3.

Constructing a co-expression strain, using pAOX-SSN as an initial vector, and inserting sol3 or mdh1 into SacI and NotI to obtain pAOX-zwf1, pAOX-sol3, pAOX-gdh3 and pAOX-mdh 1. Plasmids pAOX-zwf1, pAOX-sol3, pAOX-gdh3 and pAOX-mdh1 (FIG. 5) were used directly, transformed into E.coli, cultured, and plasmids were extracted, digested with XhoI and NotI, and used for transformation of recombinant strain G/GODM (FIG. 7) after electrophoresis verification.

The expression vectors pAOX-sol3, pAOX-mdh1, pAOX-zwf1 and pAOX-gdh3 are subjected to Xho I/EcoR I double enzyme digestion, fragments for homologous recombination are obtained by separation and purification, recombinant bacterium G/GODM competent cells are transformed by electric shock, transformants are picked from YPD plates containing Zeocin resistance, RHF/pAOXR and CYCTTF2/DDKCR2 are used as primers, positive recombinant bacteria (shown in figure 8) are verified by colony PCR, sequencing is carried out, and the results of nucleic acid electrophoresis and sequencing show that the recombinant bacteria G/GMS3, G/GMM1, G/GMZ1 and G/GMG3 are successfully constructed.

Example 3 investigation of recombinant Strain Performance

Transformants G/GMS3 and G/GMM1, which were confirmed to be correct, were selected, inoculated into 3mL YPG medium in a test tube, cultured for about 18 hours, transferred into a 250mL shake flask containing 25mL BMGY medium, and enrichment-cultured to OD₆₀₀4-6, collecting thallus, inoculating to 500mL shake flask containing 50mL BMMY culture medium, culturing, and starting OD ₆₀₀1% methanol solution was added every 24 hours while sampling to determine OD₆₀₀And the growth influence, the enzyme activity change and the like of the recombinant bacteria caused by coexpression of zwf1, sol3, gdh3 and mdh1 are examined.

1. Growth of bacteria

As can be seen from FIG. 9, the recombinant bacteria G/GMZ1, G/GMS3, G/GMG3 and G/GMM1 grewThe trend is very close to that of the starting bacterium G/GODM, and the average specific growth rate mu is 0.039h^-1On the left and right, it is shown that the expression of the co-expressed genes sol3 and mdh1 does not affect the growth of the thallus.

2. Enzyme activity

As shown in FIG. 10A, the enzyme activities per cell of G/GMS3 and G/GMM1 were 6209.25U/g.DCW and 6521.2U/g.DCW, respectively, which were 6.3% and 11.6% higher than those of G/GODM, respectively, at 120h of induction. However, G/GMZ1 and G/GMG3 did not show higher enzyme activity than G/GODM.

From the intracellular expression quantity of the GOD (figure 10B), when the induction is carried out for 96 hours, the intracellular unit bacterial enzyme activity reaches the maximum value, but the intracellular expression levels of the recombinant bacteria G/GMS3, G/GMM1 and G/GODM are not obviously different, and the highest intracellular expression level is 1850-2300U/g.DCW.

The calculated volume expression level shows that the extracellular unit volume enzyme activities of the recombinant bacteria G/GMS3 and G/GMM1 are 55.21U/mL and 60.50U/mL (figure 11), which are respectively improved by 7.3% and 17.5% compared with G/GODM. In the two strains, the GOD secretion rate is 75.4 percent and 75.8 percent, and is improved to a certain extent compared with that of G/GODM (74.6 percent). However, G/GMZ1 and G/GMG3 showed no increase in extracellular enzyme activity per unit volume, but rather decreased.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> university of east China's college of science

<120> method for carrying out glucose oxidase secretion expression based on metabolic engineering optimization, recombinant bacterium and application thereof

<130> 176037

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1005

<212> DNA

<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> primers (Primer)

<400> 4

agagaggctg aagcttacgt agaattctct aatggtattg aagcatcat 49

<210> 5

<211> 44

<212> DNA

<213> primers (Primer)

<400> 5

taaggcgaat taattcgcgg ccgcttgcat tgaagcgtag tctt 44

<210> 6

<211> 25

<212> DNA

<213> primers (Primer)

<400> 6

gctggaaagg tttgaggagg atctg 25

<210> 7

<211> 24

<212> DNA

<213> primers (Primer)

<400> 7

gtaatgcgga gcttgttgca ttcg 24

<210> 8

<211> 24

<212> DNA

<213> primers (Primer)

<400> 8

ccgctctaac cgaaaaggaa ggag 24

<210> 9

<211> 26

<212> DNA

<213> primers (Primer)

<400> 9

gaacccaaca aattcagaga tgcctc 26

Claims

1. A method of expressing glucose oxidase using a yeast cell, the method comprising:

(1) introducing exogenous glucose oxidase coding gene into yeast cells;

(2) introducing into the yeast cells of step (1) an exogenous at least one gene selected from the group consisting of: a malate dehydrogenase 1 encoding gene, a 6-phosphogluconolactonase encoding gene; the nucleotide sequence of the malate dehydrogenase 1 encoding gene is shown as SEQ ID NO. 1, and the nucleotide sequence of the 6-phosphogluconolactonase encoding gene is shown as SEQ ID NO. 2;

(3) and (3) culturing the yeast cells in the step (2) to express glucose oxidase, wherein the glucose oxidase is high-enzyme-activity glucose oxidase, and the amino acid sequence of the glucose oxidase is shown as SEQ ID NO. 3.

2. The method of claim 1, wherein the glucose oxidase-encoding gene, the malate dehydrogenase 1-encoding gene or the 6-phosphogluconolactonase-encoding gene is integrated into the genome of the yeast cell by means of homologous recombination.

3. The method of claim 1, wherein the method comprises:

4. The method of claim 1, wherein the yeast cell is a pichia pastoris cell.

5. The method of any one of claims 1 to 4, characterized in thatAOX1The promoter drives the expression of exogenous glucose oxidase coding gene toAOX1The promoter drives the expression of the exogenous 6-phosphogluconolactonase coding gene.

6. A method of producing a high enzyme activity glucose oxidase expressing cell, comprising:

(1) introducing exogenous glucose oxidase coding gene into yeast cells, wherein the amino acid sequence of the glucose oxidase is shown as SEQ ID NO. 3;

7. An expression cell of high-enzyme-activity glucose oxidase, which is a yeast cell having integrated in its genome:

an exogenous glucose oxidase coding gene, wherein the amino acid sequence of the glucose oxidase is shown as SEQ ID NO. 3; and

exogenous at least one gene selected from the group consisting of: a malate dehydrogenase 1 encoding gene, a 6-phosphogluconolactonase encoding gene; the nucleotide sequence of the malate dehydrogenase 1 encoding gene is shown as SEQ ID NO. 1, and the nucleotide sequence of the 6-phosphogluconolactonase encoding gene is shown as SEQ ID NO. 2.

8. The cell of claim 7, wherein the exogenous glucose oxidase-encoding gene, exogenous malate dehydrogenase 1-encoding gene, or exogenous gluconolactonase 6-phosphate is integrated into the genome of the yeast cell by homologous recombination.

9. The cell of claim 7, which is prepared by the method of claim 6.

10. A kit for producing glucose oxidase with high enzyme activity is characterized in that the amino acid sequence of the glucose oxidase is shown as SEQ ID NO. 3, and the kit comprises: the yeast cell according to any one of claims 7 to 9.