CN114410662A

CN114410662A - Method for improving expression efficiency of glucose oxidase gene in pichia pastoris

Info

Publication number: CN114410662A
Application number: CN202210153176.5A
Authority: CN
Inventors: 曹云鹤; 董冰; 黄菲; 王春林
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2022-02-18
Filing date: 2022-02-18
Publication date: 2022-04-29
Anticipated expiration: 2042-02-18
Also published as: CN114410662B

Abstract

The invention relates to the technical field of genetic engineering, in particular to a method for improving the expression efficiency of a glucose oxidase gene in pichia pastoris. The invention obtains the gene sequence shown in SEQ ID NO.2 by optimizing the original gene sequence of the Aspergillus niger Glucose Oxidase (GOD) and manually adjusting partial basic groups. The method provided by the invention constructs the gene sequence shown in SEQ ID NO.2 and the Flavin Adenine Dinucleotide (FAD) synthetase gene sequence shown in SEQ ID NO.3 in recombinant bacteria at the same time; the fermentation activity of glucose oxidase generated by the strain co-expressing the FAD synthetase gene and the GOD gene reaches 2.2 times of that of the strain singly expressing the GOD gene.

Description

Method for improving expression efficiency of glucose oxidase gene in pichia pastoris

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a high-efficiency expression method of a glucose oxidase gene in pichia pastoris.

Background

The system of Glucose Oxidase (GOD) is named as beta-D-glucose oxidoreductase (EC 1.1.3.4). GOD was first discovered by Muller in Aspergillus niger (Aspergillus niger) in 1928 and catalyzes the formation of gluconic acid and hydrogen peroxide from beta-D-glucose using molecular oxygen as an electron acceptor. In addition, GOD also has a slower oxidation of mannose, galactose and xylose. The enzyme has been widely used in the food industry and in medical diagnostics and research. The glucose oxidase is an important novel enzyme preparation for feed, can ensure the intestinal health of livestock and poultry, improve the digestion performance, promote the growth of animals and relieve the mycotoxin poisoning symptom to a certain extent.

GOD is a homodimeric enzyme, two subunits joined by two disulfide bonds, each containing two distinct regions: a region that binds Flavin Adenine Dinucleotide (FAD) in a non-covalent bond, the region being predominantly beta-sheet; the other region binds the substrate β -D-glucose, which is supported by four α -helices in an antiparallel β -sheet. Studies have shown that the dimeric form is more stable than the monomer; when FAD is cleaved from the monomeric enzyme, the stability of the monomer becomes worse, indicating that FAD plays an important role in either GOD activity or stability.

The process of oxidizing beta-D-glucose by glucose oxidase is divided into two half reactions, as shown in figure 1, in the reduction half reaction, the glucose oxidase oxidizes the beta-D-glucose to generate D-glucose-delta-lactone, the D-glucose-delta-lactone is spontaneously degraded to gluconic acid, and then the prosthetic group FAD on the glucose oxidase is reduced to FADH₂(ii) a In the oxidation half-reaction, FADH₂Quilt O₂Oxidation to FAD and H₂O₂. Since synthesis of FAD in cells is catalyzed by FAD Synthetase (FADs), overexpression of FAD synthetase can increase the amount of FAD synthesized and thus the amount of glucose oxidase expressed.

Disclosure of Invention

The invention aims to provide a method for highly expressing glucose oxidase with stable activity.

In the first aspect, the invention provides an optimized glucose oxidase gene sequence, wherein pichia pastoris high-frequency codons are used for replacing low-frequency codons, and restriction enzyme cutting sites such as EcoRI, Xba I, Sac I and the like are removed. In addition, the content of A, T bases in the sequence is adjusted through manual adjustment, so that a plurality of continuous A, T bases are avoided, and a glucose oxidase gene sequence which can be co-expressed with FAD synthetase in the same recombinant bacterium is obtained.

Specifically, the gene sequence of the glucose oxidase provided by the invention is shown in SEQ ID NO. 2.

In a second aspect, the invention claims expression vectors containing the glucose oxidase gene sequence shown in SEQ ID NO. 2.

In a third aspect, the invention claims recombinant bacteria containing a glucose oxidase gene sequence shown as SEQ ID NO. 2.

The recombinant strain provided by the invention simultaneously expresses a glucose oxidase gene sequence shown by SEQ ID NO.2 and a flavin adenine dinucleotide synthetase gene sequence shown by SEQ ID NO. 3.

The invention also provides a method for improving the activity and the expression efficiency of the glucose oxidase, and the recombinant bacterium co-expressed by the glucose oxidase gene sequence shown in SEQ ID NO.2 and the flavin adenine dinucleotide synthetase gene sequence shown in SEQ ID NO.3 is constructed.

In the method provided by the invention, a recombinant expression vector pPIC-GOD-opt containing a glucose oxidase gene sequence shown in SEQ ID No.2 is constructed by adopting a vector pPICz alpha A; an expression vector pGAP-FAD containing a flavin adenine dinucleotide synthetase gene sequence shown in SEQ ID NO.3 is constructed by using the vector pGAPkA.

Specifically, the method provided by the invention comprises the following steps:

(1) optimizing the gene sequence shown in SEQ ID NO.1 to obtain a glucose oxidase gene sequence GOD-opt shown in SEQ ID NO.2, and introducing EcoRI and Xba I enzyme cutting sites at the 5 'and 3' ends of the GOD-opt;

(2) carrying out enzyme digestion on the GOD-opt and the pPICz alpha A vector by EcoRI and Xba I respectively to obtain a recombinant expression vector pPIC-GOD-opt;

(3) the pPIC-GOD-opt is linearized by Sac I restriction endonuclease, and the linearized pPIC-GOD-opt is transferred into a pichia pastoris competent cell to obtain a recombinant strain X33/GOD;

(4) carrying out double enzyme digestion on a gene sequence shown in SEQ ID NO.3 and an expression vector pGAPkA to obtain a recombinant expression vector pGAP-FAD;

(5) and transferring the recombinant expression vector pGAP-FAD into a recombinant bacterium X33/GOD to obtain a strain X33/GOD-FAD with the co-expression of FAD synthetase genes and GOD genes.

In the method provided by the invention, the primer sequence used for synthesizing the gene sequence shown by SEQ ID NO.3 is shown as SEQ ID NO. 6-7.

According to the understanding of the technical personnel in the field, the invention claims the application of the glucose oxidase gene sequence shown as SEQ ID NO.2 or the recombinant bacteria in improving the expression quantity of the glucose oxidase.

And the glucose oxidase gene sequence shown in SEQ ID NO.2 or the application of the recombinant bacterium in improving the expression activity of the glucose oxidase.

The invention has the beneficial effects that the expression efficiency of GOD is improved through the co-expression of FAD synthetase.

According to the invention, a pichia pastoris high-frequency codon is replaced by a low-frequency codon, restriction enzyme cutting sites such as EcoRI, Xba I and Sac I are removed, manual adjustment is carried out, and the content of A, T bases in the sequence is adjusted, so that a glucose oxidase gene sequence which can be co-expressed with FAD synthetase in the same recombinant bacterium is obtained and is shown as SEQ ID No. 2.

The gene sequence shown in SEQ ID NO.2 and the Flavin Adenine Dinucleotide (FAD) synthetase gene sequence shown in SEQ ID NO.3 are simultaneously constructed in recombinant bacteria; the fermentation activity of glucose oxidase generated by the FAD synthetase gene and GOD gene coexpression strain is 2.2 times of that of GOD gene single expression strain; and the expression quantity of glucose oxidase generated by the strain co-expressing the FAD synthetase gene and the GOD gene is obviously improved.

Drawings

FIG. 1 shows the result of PCR amplification of FAD synthetase gene according to the present invention. Wherein, lane 1 is DNA Marker; lane 2 shows FAD synthetase gene.

FIG. 2 is the relative catalytic activity of shake flask fermented GOD.

FIG. 3 is an SDS-PAGE electrophoresis of fermentation supernatants. Wherein M is a protein molecular weight standard; lane 1 is X33/GOD fermentation supernatant; lane 2 is X33/GOD-FAD-1 fermentation supernatant; lane 3 is X33/GOD-FAD-2 fermentation supernatant; lane 4 is X33/GOD-FAD-3 fermentation supernatant; lane 5 is X33/GOD-FAD-4 fermentation supernatant.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

In the examples of the present invention, pichia pastoris expression vector pPICz α a and strain X33 were purchased from Invitrogen. The pGAPkA expression vector was constructed and stored in this laboratory. Coli Top10 competent cells were purchased from Biotechnology Ltd of New Engineers of Kyoto, Beijing.

The BCA protein quantitative analysis kit used in the embodiment of the invention is a product of Pierce company of America; PR1910 protein molecular weight standard and horseradish peroxidase, the manufacturer is a product of Solebao company in China; antibiotic Zeocin, manufactured by Invitrogen, usa; glucose oxidase standard, o-dianisidine, Sigma-Aldrich in USA; restriction enzyme, Phusion DNA polymerase, the manufacturer is American NEB company; g418, Producer Gibco USA.

Example 1 optimized synthesis of Aspergillus niger GOD Gene and construction of expression Strain

(1) Sequence optimization design and expression vector construction

An Aspergillus niger GOD gene sequence SEQ ID NO.1 (accession number: MH593586.1) disclosed by GenBank is optimally designed, a pichia pastoris high-frequency codon is used for replacing a low-frequency codon, enzyme cutting sites such as EcoRI, Xba I and Sac I are removed, the content of AT base in the sequence is manually adjusted, and a plurality of continuous A, T base is avoided. Then, the whole gene synthesis is carried out (the sequence is shown in SEQ ID NO. 2). The synthetic sequence was named GOD-opt, and EcoRI and Xba I cleavage sites were introduced at the 5 'and 3' ends of the GOD-opt, respectively. The GOD-opt and pPICz alpha A vectors are respectively cut by EcoRI and Xba I, and the specific reaction system is shown in Table 1.

TABLE 1 GOD-opt and pPICzaA restriction enzyme digestion systems

The temperature is kept at 37 ℃ for 5 h. Then, the DNA was purified using a DNA column purification kit (product of Omega Co.), and ligation was performed, and the reaction system was as shown in Table 2.

TABLE 2 ligation reaction System

The cells were incubated at 16 ℃ for 12h, transformed into E.coli Top10 competent cells (purchased from New Provisions Ltd. of Beijing Ongchon), and coated with low-salt LB plates (10g/L tryptone, 5g/L yeast extract, 5g/L NaCl, 15g/L agar, 25mg/L zeocin) containing the antibiotic zeocin. Cultured overnight at 37 ℃, and single colonies are picked and streaked on low-salt LB plates. And extracting plasmids, sequencing by using primers AOX-F and AOX-R, and confirming that the sequence connection is correct. The recombinant expression vector was named pPIC-GOD-opt. The primer sequences are shown in Table 3.

TABLE 3 primer sequences used for sequencing

(2) Constructing a recombinant strain:

the pPIC-GOD-opt is linearized by Sac I restriction endonuclease, cut at 37 ℃ for more than 12h, then the cut product is purified by a DNA column purification kit and dissolved in 10 mu L of sterilized ddH₂And (4) in O. The specific reaction system is shown in Table 4.

TABLE 4 linearization of recombinant expression vectors

A single colony of Pichia pastoris X33 was inoculated into 5mL of YPD medium (1% yeast extract, 2% peptone, 2% glucose) and cultured overnight at 30 ℃ under shaking at 220 rpm. Transferring 50mLYPD medium at 1%, and shaking at 30 deg.C and 220rpm until OD₆₀₀The cells are collected by centrifugation at 5000rpm for 5min at 4 ℃ and about 1.0, washed with ice-cold sterilized water for 2-3 times, and dissolved in 1mL of 1M sorbitol to obtain competent cells. mu.L of linearized pPIC-GOD-opt was added to 100. mu.L of competent cells, mixed well, added to a cuvette, shocked at 2000V for 5mS, immediately added to 1mL of ice-cold 1M sorbitol solution, and then re-incubated at 30 ℃ for 3 h. YPDS plates (1% yeast extract, 2% peptone, 2% glucose, 1.5% agar, 1M sorbitol) containing 100. mu.g/mL zeocin were spread and cultured at 30 ℃ for 3-4 days until colonies were clear. The recombinant strain was named X33/GOD.

(3) Identification of recombinant strains

The recombinant strain X33/GOD was inoculated into 5mL of YPD medium and cultured overnight at 30 ℃ with shaking at 220 rpm. mu.L of each was inoculated into 10mL of BMGY medium, and shaking culture was continued at 30 ℃ and 220rpm for 24 hours. The cells were centrifuged at 5000rpm at 4 ℃ for 5min to collect the cells, and the genomic DNA was extracted using a fungal DNA extraction kit (product of Omega). PCR amplification was performed. The amplification system is shown in Table 5. Amplification conditions: 30 cycles of 94 ℃ for 30s, 55 ℃ for 20s, and 72 ℃ for 2 min. Detecting the amplification product by agarose gel electrophoresis.

TABLE 5 PCR amplification System

Example 2 cloning of Pichia pastoris FAD synthetase Gene and construction of expression vector

(1) Cloning of FAD synthetase gene:

primers were designed based on the genomic sequence of Pichia pastoris (accession No. LT962476) published in GenBank, and the FAD synthetase sequence (SEQ ID NO.3) was amplified, with the primer sequences shown in Table 6. Inoculating Pichia pastoris X33 into YPD liquid culture medium, shake culturing at 30 deg.C for 18h, centrifuging at 5,000rpm for 5min, and collecting thallus. Genomic DNA was extracted using a fungal DNA extraction kit (product of Omega).

TABLE 6 primer sequences for cloning FAD synthetase genes

And amplifying FAD synthetase gene by PCR. The amplification system is shown in Table 7. Amplification conditions: 30 cycles of 94 ℃ for 30s, 58 ℃ for 20s, and 72 ℃ for 1 min. Detecting the amplification product by agarose gel electrophoresis. 1 band of about 800bp was amplified.

TABLE 7 FAD synthetase gene PCR amplification System

(2) Construction of an expression vector:

the double digestion reaction system of the PCR product and the expression vector pGAPkA is shown in Table 8.

TABLE 8 double digestion of FAD synthetase gene with expression vector pGAPkA

The temperature is kept at 37 ℃ for 5 h. Then, the DNA was purified using a DNA column purification kit (product of Omega Co.), and ligation was performed, and the reaction system was as shown in Table 9.

TABLE 9 ligation reaction System

Ligation was carried out at 16 ℃ for 12 h. E.coli Top10 competent cells were transformed. Low salt LB plates containing the antibiotic zeocin were coated. Cultured overnight at 37 ℃, and single colonies are picked and streaked on low-salt LB plates. And extracting plasmids, sequencing by using a primer AOX-R, and confirming that the sequence connection is correct. The recombinant expression vector was named pGAP-FAD.

Example 3 construction of a Strain in which FAD synthetase Gene and GOD Gene are coexpressed

A single X33/GOD colony was inoculated into 5mL YPD medium and cultured overnight at 30 ℃ with shaking at 220 rpm. Transferring 50mL YPD medium at 1%, shaking at 30 deg.C and 220rpm until OD₆₀₀The cells were collected by centrifugation at 4 ℃ and 5,000rpm for 5min at about 1.0, washed with ice-cold sterile water 2-3 times, and dissolved in 1mL of 1M sorbitol to give competent cells.

The recombinant plasmid pGAP-FAD was linearized with the restriction enzyme Bgl II, the digestion system is shown in Table 10. The temperature is kept at 37 ℃ for 5 h. Then, the DNA was purified by using a DNA column purification kit (product of Omega Co.), and the DNA was dissolved in 10. mu.L of ddH₂O。

TABLE 10 linearization of pGAP-FAD

10 μ L of linearized pGAP-FAD were shock-transformed into X33/GOD competent cells at 2000V, 5mS, plated with YPDS plates (1% yeast extract, 2% peptone, 2% glucose, 1.5% agar, 1M sorbitol) containing 100 μ G/mL antibiotic G418, and cultured at 30 ℃ for 3-4 days until colonies were clear. The recombinant strain was named X33/GOD-FAD.

Example 4 Shake flask fermentation

(1) And (3) shake flask fermentation culture:

recombinant Pichia pastoris strains X33/GOD, X33/GOD-FAD-1, X33/GOD-FAD-2, X33/GOD-FAD-3 and X33/GOD-FAD-4 (all randomly selected clones) are picked and inoculated into YPD liquid culture medium respectively, and shaking culture is carried out at 220rpm at 30 ℃ overnight. 1% was transferred to 20mL BMGY (1% yeast extract, 2% peptone, 1.34% YNB, 4X 10-5% biotin, 1% glycerol, 100mM phosphate buffer pH 6.0) in a 250mL shake flask and shake-cultured at 30 ℃ and 220rpm to OD₆₀₀The value is 2-6, and the thalli are collected by centrifugation at 5000rpm and 4 ℃ for 5 min. The cells were incubated with 20mL BMMY medium (1% yeast extract, 2% peptone, 0.1mol/L phosphate buffer pH 6.0, 1.34 percent YNB, 4 × 10-5 percent biotin), shaking culture at 30 ℃ and 220rpm, adding absolute methanol with the final concentration of 0.5 percent (V/V) for shake flask induction culture, adding methanol once every 24h, centrifuging to take supernatant, and storing at 4 ℃ for testing.

(2) And (3) enzyme activity analysis:

diluting a glucose oxidase standard substance into 0.4, 0.8, 1.2, 1.6, 2.0 and 2.4U/mL, adding 500 mu L of o-dianisidine buffer solution, 60 mu L of 1M glucose and 20 mu L of 100U/mL horseradish peroxidase solution into a 1.5mL centrifuge tube, preserving the temperature for 5min at the determination temperature, adding 20 mu L of diluted glucose oxidase standard substance, adding 100 mu L of 2mol/L sulfuric acid to terminate the reaction after reacting for 3min, adding 200 mu L of reaction liquid into a 96-well plate, determining the light absorption value of the reaction liquid at 460nm by using a microplate reader, and making an enzyme activity-light absorption value curve, namely the standard curve under the reaction condition.

Diluting the crude enzyme solution by corresponding times, replacing the standard substance in the same step, measuring the light absorption value, adding the reaction termination solution and the enzyme solution as a blank control, and calculating the enzyme activity under the reaction condition according to the standard yeast. The results are shown in FIG. 2, and the fermentation activities of the strains X33/GOD are 100%, X33/GOD-FAD-1 is 220%, X33/GOD-FAD-2 is 228%, X33/GOD-FAD-3 is 235%, and X33/GOD-FAD-4 is 216%. The transfer of the FAD synthetase is proved, and the fermentation activity of the GOD is obviously improved.

(3) SDS-PAGE electrophoresis of supernatant proteins:

a12.5% SDS-PAGE gel was prepared, and 10. mu.L of each of the fermentation supernatants was sampled to compare the brightness of the protein bands. As shown in FIG. 3, the brightness of the protein band of the co-expressed FAD synthetase is obviously higher than that of X33/GOD, which indicates that the transfer of FAD synthetase improves the GOD protein expression level. Quantitative analysis of the expressed protein by using a BSA quantitative kit shows that the GOD expression level of the strain co-expressing FAD is improved by about 1 time (see Table 11).

TABLE 11 recombinant strain fermentation supernatant protein quantification and fermentation viability results

Strain numbering	Protein content (mg/mL)	Fermentation vigor (U/mL)
			X33/GOD	1.32	145
X33/GOD-FAD-1	2.90	320
			X33/GOD-FAD-2	3.01	330
X33/GOD-FAD-3	3.10	340
			X33/GOD-FAD-4	2.85	313

Comparative example 1

The sequence of the GOD gene which is optimized only by software and is not adjusted manually is shown as SEQ ID NO.8 in the comparative example.

The procedure for obtaining SEQ ID NO.8 in this comparative example is the same as the sequence optimization procedure of example 1, except that the GOD gene sequence of SEQ ID NO.8 used in this comparative example was completely generated by software without manual adjustment.

When a co-expression strain is constructed by adopting a gene sequence shown in SEQ ID NO.8, the fermentation activity of glucose oxidase generated by the strain with the co-expression of FAD synthetase gene and GOD gene is found to be 140U/mL, and is basically similar to that of a strain with single expression of GOD gene. And the fermentation activity of the glucose oxidase obtained by the comparative example is obviously lower than the GOD fermentation activity of the co-expression strain obtained by the example 3.

When a co-expression strain is constructed by adopting a gene sequence shown in SEQ ID NO.8, the GOD expression quantity of the strain co-expressing the FAD synthetase gene and the GOD gene is 1.30mg/mL and is basically similar to the GOD expression quantity of the strain singly expressing the GOD gene.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> university of agriculture in China

<120> method for improving expression efficiency of glucose oxidase gene in pichia pastoris

<130> KHP221110429.2

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1761

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tacatcagga gcaatggcat tgaagccagc ctcctgactg accccaagga tgttaccggc 60

cgcactgtcg actacatcat cgctggtgga ggtctgactg gactcactac tgctgcccgt 120

ctgacggaga accccaatat cactgtgctt gtcatcgaaa gtggctccta cgagtctgac 180

agaggtccta tcattgagga cctgaacgct tacggtgaca tttttggcag cagtgtggac 240

cacgcctacg agactgtcga gctcgccacc aacaatcaga ctgcgctaat ccgctccgga 300

aatggtctcg gtggctctac cctcgtcaac ggtggcacct ggactcgccc ccacaaggca 360

caagttgact cgtgggagac cgtcttcgga aatgagggct ggaactggga cagcgtggcc 420

gcctactccc tccaggctga gcgtgctcgc gcaccaaatg ccaaacagat tgctgctggc 480

cactacttta atgcatcctg ccatggtctc aatggtactg tccacgccgg accccgcgat 540

accggtgatg actactcccc catcgtcaag gctctcatga gcgttgtcga agacaggggc 600

gtccccacca agaaggactt gggatgcggt gacccccatg gtgtgtccat gttccccaac 660

accttgcacg aagaccaagt gcgctctgat gccgctcgcg aatggctcct ccccaactac 720

cagcggccca acctgcaagt cctcactgga cagtatgttg gaaaggttct gctcagccag 780

aacgctacca cacctcgtgc cgttggcgtg gaattcggca cccacaaggg caacacccac 840

aacgtctacg ctaagcacga ggtcctccta gccgctggat ccgcggtctc tcccaccatc 900

ctcgaatatt ccggtatcgg aatgaagtcc attctggagc ctcttggaat tgacaccgtc 960

gttgacctgc ccgtcggtct caaccttcag gaccagacca cctctaccgt ccgctcacgc 1020

attacctccg ccggtgccgg acagggacag gccgcttggt tcgctacctt caacgagacc 1080

tttggcgact acaccgaaaa ggcccacgag ctgctcaaca ccaagctgga gcagtgggcc 1140

gaagaggccg tcgcccgtgg cggattccac aacaccaccg ctttgctcat ccagtacgag 1200

aactaccgcg actggatcgt caaggacaat gtcgcatact cggaactctt cctcgacacg 1260

gccggagtgg ccagtttcga tgtgtgggat cttctgccct tcactagagg atacgtccac 1320

atcctcgaca aggaccccta cctccgccat ttcgcctacg accctcagta cttcctcaac 1380

gagcttgacc tgctcggcca ggctgccgcc actcagctgg cccgcaacat ctccaactcc 1440

ggtgccatgc aaacttattt cgctggagag actattcccg gtgacaacct cgcgtatgat 1500

gccgacttga gcgcctgggt tgagtatatc ccgtacaact tccgccctaa ctaccatggt 1560

gtgggtactt gctccatgat gccgaaggag atgggcggtg ttgtcgacaa tgctgcccgt 1620

gtgtatggtg tgcagggact gcgagtcatc gatggttcta ttccccctac gcaaatgtcg 1680

tcccatgtta tgacggtctt ttatgccatg gccttgaaga ttgcggatgc cgtcttggcg 1740

gattatgctt ccatgcagtg a 1761

<210> 2

<211> 1773

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gaattctata ttagatccaa cggaatagaa gcatcacttc ttaccgatcc taaagacgtt 60

accggaagga ccgtcgacta tattattgct ggtggaggtc ttactggatt aactaccgct 120

gccaggttga cagagaatcc aaatatcact gtactggtta ttgaatcagg cagttatgaa 180

agtgataggg gtccaataat tgaggacctt aacgcttacg gtgacatctt tggatcttca 240

gttgaccatg cctatgaaac agttgagctt gccactaaca atcagactgc acttattaga 300

tccggaaacg gtcttggtgg atctactctt gttaatggtg gtacttggac caggcctcat 360

aaagcccaag tcgattcatg ggagactgtt ttcggaaatg agggctggaa ctgggattcc 420

gtcgcagctt acagtcttca agctgaaaga gccagagcac caaatgctaa gcaaattgct 480

gccggacact acttcaatgc atcctgtcac ggacttaatg gaactgtaca tgctggtcct 540

cgagatacgg gagatgatta ttctccaatc gttaaggctt taatgtcagt cgtagaagat 600

agaggtgttc ctactaagaa agatttgggt tgtggtgacc cccatggtgt ttccatgttt 660

ccaaacactc tgcatgaaga tcaagtacgt tctgacgccg ctagagaatg gctattacct 720

aactaccaga ggcccaattt gcaagtgctg actggacaat atgttggaaa ggtattactg 780

tcacagaacg ccactacccc aagggccgtt ggtgtagagt tcggtactca taaaggaaac 840

acacacaacg tttacgctaa acatgaagtc cttcttgccg caggttctgc tgtatcaccc 900

accatactgg aatactccgg tattggcatg aagtctattc tggaaccact gggtattgat 960

actgtagtcg acttacctgt tggcttgaac ttgcaagatc aaaccacttc taccgtcagg 1020

tccagaatca cttccgcagg tgcaggacaa ggtcaagccg cctggtttgc tacctttaac 1080

gaaacctttg gcgattatac agagaaagct cacgaactgc ttaacactaa gctagagcaa 1140

tgggctgaag aagctgtggc tcgtggtggt ttccataaca ccactgcttt attgatacaa 1200

tacgaaaact atagggattg gatcgttaag gataacgtcg cttattccga gctgttttta 1260

gataccgccg gagttgcttc ttttgatgtt tgggatttac tacccttcac acgtggatac 1320

gtccatattt tagacaagga tccttacttg agacattttg cttatgaccc tcaatacttc 1380

ctgaacgaat tggacttgtt gggccaagcc gccgcaactc agcttgctcg aaatatttca 1440

aactctggag caatgcaaac ctattttgct ggagaaacta tccctggtga taatcttgca 1500

tatgacgccg atctttctgc ctgggtagag tacatacctt acaacttcag acctaactac 1560

catggagtag gaacttgctc aatgatgcca aaagaaatgg gcggagttgt ggacaacgct 1620

gctagagtat acggtgtgca aggtcttcga gttatcgacg gaagtattcc acctactcag 1680

atgtcctccc acgtgatgac agtattttat gctatggctc ttaaaattgc tgatgctgtg 1740

cttgctgact acgctagtat gcagtaatct aga 1773

<210> 3

<211> 807

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gaattcatgg taactgttct ctccaatagt agtaagcaga ccacacagat tcagcacaat 60

cacaatatga taaaagactc atttctctca gtgtgtgaac acagctataa ccttatcact 120

gattttttga atgagtcaga tcctgaaaac gaacttttga catcgaccca ggaacaggtc 180

agaaaatcct atgacatact ctttgaatct ttgaacttgt acaaattaga tgaactatca 240

atatcatata acggcggaaa agactgtctt gtactgctga tcatttactt agcagtcatt 300

tacgataaat atcaaaacga gtcgatacca agggattaca aattaaacgc actatacatc 360

aaaaatgagt caatgttcca agaacaggac gatttcataa aaaaatcggc ggcaaaatac 420

cagctaaatc tacagccatt tagagacact atgaagagag ctctccacaa atatttggta 480

caaaacccat gcataaaagc cgtcattata ggcatcagaa gatgtgatcc atatggtaag 540

gatttacatt atttacaaga aacagatcca agctggcccc ggctaatgcg ggtcaatccg 600

attctggaat gggattacca ccaaatttgg ttcttcctcc gtcatttaaa aatagaatac 660

tgcaagctct atgatatggg ctatacttct ctgggtggta ccaacaacac tattaagaac 720

cctgacctgc aggacggtga tggctatctt cctgcgtact tgctggatga ttccgaaaag 780

gaaagatcca gtagattatg atctaga 807

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gactggttcc aattgacaag c 21

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gcaaatggca ttctgacatc c 21

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggaattcatg gtaactgttc tctcc 25

<210> 7

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gctctagatc ataatctact ggatct 26

<210> 8

<211> 1761

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tatattcgtt caaacggcat cgaggcttct ctgttaaccg atcccaaaga cgtcaccggt 60

agaactgtgg attatattat tgccggaggt ggtctgacag gtttgacaac cgccgctcgt 120

ttaactgaga accctaatat tacggttttg gttatcgaaa gtggttcata tgagtccgat 180

agaggcccaa taattgaaga tttgaatgct tacggtgata ttttcggttc cagtgtagac 240

catgcttacg agactgttga acttgctacg aataaccaaa ccgccttaat tagatcagga 300

aatggcttag gtggttccac attagttaat ggtggcacat ggacaagacc acacaaggct 360

caggtcgatt catgggaaac tgtttttgga aacgaaggat ggaattggga ttctgtcgca 420

gcatattcct tacaagctga acgtgctagg gccccaaacg ctaaacaaat tgctgcagga 480

cattatttca acgcctcttg tcatggacta aacggaaccg tacatgcagg acctagagac 540

actggtgatg attactctcc aattgttaaa gcactgatgt ccgttgttga agatagaggt 600

gtcccaacaa agaaagattt aggttgtggt gaccctcatg gcgtttctat gtttccaaac 660

actttgcacg aagatcaggt caggtccgac gcagctcgtg agtggctttt accaaactac 720

caaagaccaa acttgcaagt cttgaccggt caatacgtag gaaaggttct attgagtcaa 780

aacgccacta caccaagagc cgtgggtgtt gagtttggaa cccataaagg taatacgcac 840

aacgtttacg caaagcacga agtcctactt gctgcaggtt ccgctgtttc tccaacaatt 900

cttgagtaca gtggaatcgg catgaaatcc atattagaac cacttggaat tgacaccgtg 960

gttgacttgc ctgtcggact gaacttgcag gatcaaacga cctcaaccgt tagaagtaga 1020

atcacttctg ctggcgcagg tcaaggtcaa gcagcttggt tcgctacgtt caatgaaact 1080

tttggtgact ataccgaaaa agctcacgaa ttattgaata ccaaattgga gcaatgggct 1140

gaggaagctg tagctagagg aggatttcac aacaccacag cacttttaat tcagtatgag 1200

aactacagag actggatcgt gaaggataat gtggcctatt ctgaactgtt cttggatacc 1260

gccggtgtcg catccttcga tgtgtgggac ttgttgcctt ttactagagg ttacgtccac 1320

attttggata aagacccata cttaagacac tttgcttacg accctcaata ttttctgaac 1380

gaattggact tgctgggaca ggctgccgca acacagcttg cacgtaatat atccaactcc 1440

ggtgctatgc agacttattt tgctggcgaa actatcccag gagacaacct tgcttatgac 1500

gctgatttgt cagcatgggt tgagtatatt ccatacaact ttcgtcctaa ttaccatggt 1560

gtgggaactt gttctatgat gccaaaggaa atgggtggtg tagttgacaa tgctgcccga 1620

gtctacggtg ttcaaggttt gagagttatc gacggctcca ttccccctac tcaaatgtca 1680

agtcacgtca tgacagtttt ctacgctatg gctcttaaga ttgctgatgc tgttttggct 1740

gattatgcca gtatgcaata a 1761

Claims

1. The glucose oxidase gene sequence is characterized in that the glucose oxidase gene sequence is shown as SEQ ID No. 2.

2. An expression vector comprising the glucose oxidase gene sequence of claim 1.

3. A recombinant bacterium comprising the glucose oxidase gene sequence of claim 1.

4. The recombinant strain as claimed in claim 3, wherein the recombinant strain simultaneously expresses a glucose oxidase gene sequence shown by SEQ ID No.2 and a flavin adenine dinucleotide synthetase gene sequence shown by SEQ ID No. 3.

5. A method for improving activity and expression efficiency of glucose oxidase is characterized in that a recombinant bacterium co-expressed by a glucose oxidase gene sequence shown in SEQ ID No.2 and a flavin adenine dinucleotide synthetase gene sequence shown in SEQ ID No.3 is constructed.

6. The method according to claim 5, characterized in that the vector pPICz α A is used to construct a recombinant expression vector pPIC-GOD-opt containing the glucose oxidase gene sequence shown in SEQ ID No. 2; an expression vector pGAP-FAD containing a flavin adenine dinucleotide synthetase gene sequence shown in SEQ ID NO.3 is constructed by using the vector pGAPkA.

7. The method of claim 5, comprising:

(1) optimizing the gene sequence shown in SEQ ID NO.1 to obtain a glucose oxidase gene sequence GOD-opt shown in SEQ ID NO.2, and respectively introducing EcoRI enzyme cutting sites and Xba I enzyme cutting sites at the 5 'end and the 3' end of the GOD-opt;

(3) linearizing the pPIC-GOD-opt by using Sac I restriction endonuclease, and transferring the linearized pPIC-GOD-opt into a pichia pastoris competent cell to obtain a recombinant strain X33/GOD;

8. The method according to claim 7, wherein the primer sequence used for synthesizing the gene sequence shown in SEQ ID No.3 is shown in SEQ ID No. 6-7.

9. Use of the glucose oxidase gene sequence of claim 1 or the recombinant bacterium of any one of claims 3-4 for increasing the expression level of glucose oxidase.

10. Use of the glucose oxidase gene sequence of claim 1 or the recombinant bacterium of any one of claims 3-4 for increasing the expression activity of glucose oxidase.