CN115369049A

CN115369049A - Genetically engineered bacterium secreting glucose oxidase, and construction method and application thereof

Info

Publication number: CN115369049A
Application number: CN202110535932.6A
Authority: CN
Inventors: 谭天伟; 陈浩
Original assignee: Beijing University of Chemical Technology
Current assignee: Beijing University of Chemical Technology
Priority date: 2021-05-17
Filing date: 2021-05-17
Publication date: 2022-11-22
Anticipated expiration: 2041-05-17
Also published as: CN115369049B

Abstract

The invention relates to a genetic engineering bacterium secreting glucose oxidase. The engineering bacterium is obtained by adopting pichia pastoris as a host cell, introducing a gene for coding Glucose Oxidase (GOX) or a gene for coding the Glucose Oxidase (GOX) through codon optimization, and constructing through strengthening expression of a pichia pastoris ubiquitination transport gene, and is a genetic engineering bacterium for efficiently secreting the glucose oxidase. The invention also relates to a construction method of the genetic engineering bacteria secreting the glucose oxidase. The method enhances the secretory expression level of the glucose oxidase by over-expressing the ubiquitination gene related to secretory expression, has simple operation, can be applied to the construction of high-yield glucose oxidase strains and the industrial preparation of the glucose oxidase, and has good market application prospect.

Description

Genetically engineered bacterium secreting glucose oxidase, and construction method and application thereof

Technical Field

The invention belongs to the technical field of gene recombination, and relates to a genetic engineering bacterium secreting glucose oxidase, and a construction method and application thereof.

Background

In 1928, muller first discovered glucose oxidase from the cell-free extract of Aspergillus niger. In the seventies of the 20 th century, the glucose oxidase is developed to a full application scene at home and abroad.

Glucose Oxidase (GOX) can be fixed on the surface of an electrode to detect glucose, is often used for detecting blood sugar in the field of medicine, and is rapid and convenient. Can be used in food field for removing oxygen to protect food from oxidation by oxygen in air, and removing glucose in food to avoid Maillard reaction.

In the wine industry, GOX is used for reducing the concentration of alcohol, inhibiting the growth of acetic acid bacteria and lactic acid bacteria which cause wine deterioration, and reducing the addition of preservatives in the wine. The glucose oxidase and the catalase can be co-immobilized and can be used for preparing sodium gluconate.

The glucose oxidase is fully applied to the feed for breeding animals, and is added into the chicken feed, so that the survival rate and the feed intake rate of the broiler are improved. GOX in the pig feed can reduce perinatal syndrome of sows and improve daily gain of weaned piglets. The GOX in the feed for cattle and sheep eliminates the decrease of appetite of the dairy cattle in the perinatal period and promotes the digestion and absorption of the feed.

The content of the glucose oxidase in animal and plant bodies is low, and huge requirements are difficult to meet through extraction means. Therefore, the glucose oxidase with high purity is obtained by production, separation, extraction and purification by means of genetic engineering, and the current industrial bacterial strains are aspergillus niger and penicillium.

The glucose oxidase is researched by utilizing a genetic engineering means, a certain effect is achieved by genetically modifying the structure of the enzyme, such as error-prone PCR (polymerase chain reaction), and although theoretical means and technical support are provided for the production of GOX, the effect is still not satisfactory.

In the existing method for enhancing the secretion efficiency of heterologous proteins by carrying out overexpression strengthening on pichia pastoris protein secretion pathways, the interaction mechanism of related protein secretion pathway factors and foreign proteins is unclear, trial and error is needed to judge which gene is overexpressed to promote the enzyme activity, the operation is complex, the workload is great, and the cost is high.

The heterologous protein is usually a strong promoter in pichia pastoris, so that the pressure of a transport pathway is easily overlarge, and a large amount of target protein cannot be smoothly secreted to the outside, and the method for enhancing the transport pathway has extremely important application value.

Therefore, the problem exists at present that the construction of a strain producing glucose oxidase is low in cost, rapid and efficient.

Disclosure of Invention

The invention aims to solve the problem of providing a genetically engineered bacterium for secreting glucose oxidase aiming at the defects in the prior art, the genetically engineered bacterium enhances the secretion of the glucose oxidase by strengthening a protein transport secretion pathway, and the secreted glucose oxidase has higher enzyme activity.

The invention also provides a construction method of the genetic engineering bacteria for secreting the glucose oxidase, and the method can strengthen protein transport and secretion pathways, thereby enhancing the secretion of the glucose oxidase, improving the enzyme activity of the glucose oxidase and obtaining the industrial bacterial strain for fermenting and synthesizing the glucose oxidase.

Therefore, the invention provides a genetically engineered bacterium secreting glucose oxidase in a first aspect, which is a recombinant pichia pastoris containing ubiquitination transport genes and genes encoding Glucose Oxidase (GOX) or genes encoding Glucose Oxidase (GOX) subjected to codon optimization.

According to the invention, the ubiquitination transporter gene comprises an endogenous pichia pastoris ubiquitination transporter gene, and optionally an exogenous ubiquitination transporter gene.

In some embodiments of the invention, the exogenous ubiquitination transporter is derived from saccharomyces cerevisiae.

In another embodiment of the invention, the gene encoding Glucose Oxidase (GOX) is derived from Penicillium sp (Penicillium Amagasakiense) with Genebank accession number AAD01493.1.

According to the invention, the ubiquitination transporter is preferably a pichia endogenous ubiquitination transporter.

In some embodiments of the invention, the pichia endogenous ubiquitination transporter comprises one or more of SEC12, SEC13, SEC23, SEC24 and SEC31 genes.

Specifically, the SEC12 gene has a Genebank accession number of AF216960.1;

and/or, the Genebank accession number of the SEC13 gene is AAB01155.2;

and/or, the Genebank accession number of the SEC23 gene is CAY67406.1;

and/or, the Genebank accession number of SEC24 gene is CAY71741.1;

and/or, the Genebank accession number of the SEC31 gene is CAY68066.1.

According to the invention, the genetic engineering bacteria are formed by overexpression of ubiquitination transport genes and Glucose Oxidase (GOX) genes or genes which are subjected to codon optimization and code Glucose Oxidase (GOX) in Pichia pastoris GS115 through vector plasmids.

In some embodiments of the invention, the gene encoding Glucose Oxidase (GOX) or the codon-optimized gene encoding Glucose Oxidase (GOX) is integrated into pichia pastoris through a PPIC9K plasmid.

In another embodiment of the invention, the ubiquitination transporter is integrated into pichia pastoris by the PGAPZB plasmid.

In the invention, the pichia pastoris is pichia pastoris GS115.

The second aspect of the present invention provides a method for constructing a genetically engineered bacterium that secretes glucose oxidase according to the first aspect of the present invention, comprising:

step A, integrating a gene for coding Glucose Oxidase (GOX) or a gene for coding Glucose Oxidase (GOX) subjected to codon optimization into pichia pastoris through a first carrier plasmid to obtain the pichia pastoris containing the gene for coding Glucose Oxidase (GOX) or the gene for coding Glucose Oxidase (GOX) subjected to codon optimization;

and step B, integrating the ubiquitination transport gene into pichia pastoris containing a Glucose Oxidase (GOX) gene or a gene which is subjected to codon optimization and encodes the Glucose Oxidase (GOX) through a second carrier plasmid to obtain the genetic engineering bacteria secreting the glucose oxidase.

In some embodiments of the invention, the first vector plasmid is a PPIC9K plasmid.

In other embodiments of the invention, the second vector plasmid is a PGAPZB plasmid.

In a third aspect, the invention provides the use of the genetically engineered bacterium according to the first aspect of the invention or the genetically engineered bacterium constructed by the method according to the second aspect of the invention in the production of Glucose Oxidase (GOX) by fermentation.

In some embodiments of the invention, the application comprises inoculating genetically engineered bacteria secreting glucose oxidase into a fermentation medium, and performing fermentation culture to obtain the glucose oxidase.

In some specific embodiments of the invention, the fermentation culture conditions are: the fermentation temperature is 28 ℃, the inoculation amount is 8%, the rotation speed is 500rpm, the ventilation amount is 1.5vvm, the pH value is 5.5, the culture medium is BMGY culture medium, methanol is added after the dissolved oxygen rises, the methanol concentration is controlled to be 5g/L, the fermentation time is not less than 240h, and the enzyme activity of the glucose oxidase is not less than 1638.96U/mL.

The invention has the beneficial effects that:

(1) The genetic engineering bacteria for secreting the glucose oxidase provided by the invention can enhance the secretion of the glucose oxidase by strengthening a protein transport secretion pathway, and the secreted glucose oxidase has higher enzyme activity.

(2) The construction method of the genetic engineering bacteria for secreting the glucose oxidase provided by the invention solves the problem of limited transportation and secretion passages of the glucose oxidase gene expression in pichia pastoris by intensively expressing the pichia pastoris transport gene.

(3) The invention only needs to express two genes, such as GOX gene and SEC12 gene, has simpler operation and lower application cost, improves the extracellular enzyme activity of the glucose oxidase, and can be applied to the construction and application of high-yield glucose oxidase strains.

Drawings

The invention is described in further detail below with reference to the attached drawing figures:

FIG. 1 shows the results of the effect of overexpression of a transporter gene on biomass of recombinant Pichia strains.

FIG. 2 shows the results of the effect of overexpression of the transporter on the heterologous expression of glucose oxidase by recombinant Pichia strains.

FIG. 3 shows the change in biomass, enzyme activity of PPG in 5L fermentation.

FIG. 4 shows the change in biomass, enzyme activity of PPG-SEC12 in 5L fermentation.

Detailed Description

In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to the appended drawings. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Term (I)

The term "genetically engineered bacterium" refers to a fungus, such as pichia pastoris, that is, a fungus that produces a desired protein by introducing a target gene into a host organism (i.e., a host cell or a fungus body) and expressing the target gene. The core technology of genetic engineering is the recombination technology of DNA, therefore, the genetically engineered bacteria are also called recombinant microorganisms in the invention.

The term "recombinant" as used herein refers to the construction of a transgenic organism that utilizes the genetic material of a donor organism or an artificially synthesized gene, which is cleaved with restriction enzymes in vitro or ex vivo and then ligated with a suitable vector to form a recombinant DNA molecule, which is then introduced into a recipient cell or a recipient organism to construct a transgenic organism that exhibits a certain property of another organism according to a human blueprint that has been previously designed.

Embodiments II

As mentioned above, in the prior art, the glucose oxidase is researched by utilizing a genetic engineering means, and the operation of improving the yield of the glucose oxidase by genetically modifying the structure of the enzyme is complex, the workload is great, and the cost is high; meanwhile, the existing genetic engineering bacteria for secreting glucose oxidase have the problem that the transport and secretion channel of glucose oxidase gene expression in pichia pastoris is limited. In view of this, the inventor of the present invention has made a great deal of research on the construction of genetically engineered bacteria secreting glucose oxidase, and has solved the problem of limited transport and secretion pathways of glucose oxidase gene expression in pichia by enhancing expression of pichia ubiquitination transport gene, and has successfully constructed a strain producing glucose oxidase with low cost, high speed and high efficiency.

Therefore, the invention provides a new approach for synthesizing glucose oxidase, which enhances the secretion of the glucose oxidase by strengthening a protein transport and secretion approach, improves the enzyme activity of the glucose oxidase and realizes the efficient synthesis of the glucose oxidase.

In order to realize the technical scheme, the invention firstly provides a genetic engineering bacterium capable of secreting glucose oxidase, which expresses a gene of a glucose oxidase synthetic pathway in an original or modified fungal cell to prepare a host capable of synthesizing the glucose oxidase; based on the host capable of synthesizing glucose oxidase, the invention strengthens ubiquitination transport genes, thereby constructing and obtaining the strain capable of quickly and efficiently producing the glucose oxidase.

In an embodiment of the first aspect of the invention, the invention provides a genetically engineered bacterium secreting glucose oxidase, which is a recombinant pichia pastoris containing a ubiquitinated transporter gene and a gene encoding Glucose Oxidase (GOX) or a codon-optimized gene encoding Glucose Oxidase (GOX).

In another embodiment of the invention, the gene encoding Glucose Oxidase (GOX) is derived from Penicillium (Penicillium Amagasakiense) with Genebank accession number AAD01493.1 and strain accession number ATCC 332245.

In some preferred embodiments of the present invention, the ubiquitination transporter is a pichia endogenous ubiquitination transporter comprising one or more of SEC12, SEC13, SEC23, SEC24 and SEC31 genes; preferably, the pichia endogenous ubiquitination transporter is selected from SEC12, SEC13, SEC23, SEC24 and SEC31 genes; further preferably, the pichia endogenous ubiquitination transporter is SEC12 gene.

In the invention, the nucleotide sequence of the SEC12 gene is shown as Genebank AF216960.1; the nucleotide sequence of the SEC13 gene is shown as Genebank: AAB01155.2; the nucleotide sequence of the SEC23 gene is shown as Genebank: CAY67406.1; the nucleotide sequence of the SEC24 gene is shown as Genebank: CAY71741.1; the nucleotide sequence of the SEC31 gene is shown in Genebank: CAY68066.1.

According to the invention, the genetic engineering bacteria are formed by overexpression of ubiquitination transport genes and Glucose Oxidase (GOX) genes or genes which are optimized by codons and code for the Glucose Oxidase (GOX) in pichia pastoris GS115 through carrier plasmids.

In some embodiments of the invention, the gene encoding Glucose Oxidase (GOX) or the codon-optimized gene encoding Glucose Oxidase (GOX) is integrated into pichia pastoris by a PPIC9K plasmid.

In another embodiment of the invention, the ubiquitination transporter is integrated into pichia pastoris by a PGAPZB plasmid.

In some particularly preferred embodiments of the present invention, the genetically engineered bacterium consists of a codon-optimized gene encoding Glucose Oxidase (GOX) integrated into and overexpressed in pichia pastoris through a PPIC9K plasmid, and a gene encoding Glucose Oxidase (GOX) integrated into and overexpressed in pichia pastoris through a PGAPZB plasmid.

In the invention, the nucleotide sequence of the gene which is subjected to codon optimization and codes for Glucose Oxidase (GOX) is shown as SEQ No. 1.

In the invention, the pichia is the pichia GS115, and the strain preservation number is ATCC20864 (Baiolaibo).

The second aspect of the present invention provides a method for constructing a genetically engineered bacterium secreting glucose oxidase according to the first aspect of the present invention, which can be understood as a method for enhancing secretion of glucose oxidase by enhancing a protein transport secretion pathway, comprising:

step A, integrating a gene for coding Glucose Oxidase (GOX) or a gene for coding Glucose Oxidase (GOX) subjected to codon optimization into pichia pastoris through a first carrier plasmid (PPIC 9K plasmid) to obtain the pichia pastoris containing the gene for coding Glucose Oxidase (GOX) or the gene for coding Glucose Oxidase (GOX) subjected to codon optimization;

and step B, integrating the ubiquitination transporter gene into the pichia pastoris containing a Glucose Oxidase (GOX) gene or a gene which is subjected to codon optimization and encodes the Glucose Oxidase (GOX) through a second carrier plasmid (PGAPZB plasmid) to obtain the genetic engineering bacteria for secreting the glucose oxidase.

In some specific embodiments of the invention, the method for enhancing the secretion of glucose oxidase by enhancing the expression of pichia pastoris transporter gene and constructing the genetically engineered bacterium with high efficiency for secreting glucose oxidase comprises the following steps:

(1) Transforming a gene which is subjected to codon optimization and encodes Glucose Oxidase (GOX) into pichia pastoris GS115 by adopting PPIC9K plasmid to obtain the pichia pastoris GS115 containing the Glucose Oxidase (GOX) gene;

(2) The gene recombination technology is adopted to clone the ubiquitination transport gene of the pichia pastoris onto a common carrier PGAPZB of the pichia pastoris, and the ubiquitination transport gene is co-expressed in pichia pastoris GS115 containing Glucose Oxidase (GOX) genes.

(3) Finally, a strain of high-secretion expression glucose oxidase is obtained through screening and identification, and the enzyme activity in a 5L fermentation tank is 1638.96U/mL.

Specifically, the invention adopts Gibson self-assembly technology for connection, firstly, primers are used for respectively carrying out PCR to obtain a gene and a vector, and the gene, the vector and the Gibson enzyme are subjected to gel running and gel recovery and then are subjected to gel separation according to a ratio of a:5-a:5 (microliter) is mixed and added, the mixture is transformed into a commercial competent cell trans10 at 50 ℃ for 45min, the colony PCR verifies and sequences, and after the sequencing is correct, the AVRII linearized plasmid is electrically transformed into a PPG competent cell; the strains and plasmids constructed by the invention are shown in Table 1, and the primers used by the plasmids constructed by the invention are shown in Table 2.

TABLE 1 construction of strains and plasmids of the invention

TABLE 2 primers used for plasmids constructed according to the invention

The method enhances the secretory expression level of the glucose oxidase by over-expressing the ubiquitination gene related to secretory expression, has simple operation, can be applied to the construction of high-yield glucose oxidase strains and the industrial preparation of the glucose oxidase, and has good market application prospect.

In some specific embodiments of the invention, the fermentation culture is performed in a 5L fermentor under the following conditions: the fermentation temperature is 28 ℃, the inoculum size is 8%, the rotation speed is 500rpm, the aeration volume is 1.5vvm, the pH is adjusted to 5.5 by 3mol/L phosphoric acid and 28% ammonia water (v/v), the culture medium is BMGY culture medium (40 g/L of glycerol), methanol is added after the dissolved oxygen rises, the methanol concentration is controlled to be 5g/L by an FC2002 type methanol detection flow addition controller, and the enzyme activity is measured after fermentation is carried out for 240 hours; preferably, the enzyme activity of the glucose oxidase is more than or equal to 1638.96U/mL.

In the invention, the shake flask culture medium for amplifying the biomass of the thallus is a BMGY culture medium, the shake flask culture medium for inducing and synthesizing glucose oxidase is a BMMY culture medium, the seed culture medium for loading the thallus into the tank is a YPD culture medium, and the fermentation culture medium is a BMGY culture medium.

YPD medium (1L) used in the fermentation Process of the present invention: 10g of yeast powder, 20g of peptone and 20g of glucose; BMGY medium (1L): 10g of yeast powder, 20g of peptone, 40g of glycerol, 13.4g of YNBC, 100mM phosphoric acid buffer solution (pH6.0).

The method for measuring the enzyme activity of the glucose oxidase comprises the following steps:

(1) Preparing 0.1mol/L sodium phosphate buffer solution with pH =6, 180g/L glucose solution, 2mol/L sulfuric acid solution, 100U/mL horseradish peroxidase solution, dissolving 1g o-dianisidine and diluting to volume in 100mL methanol to obtain o-dianisidine methanol solution, mixing 1mL o-dianisidine methanol solution and diluting to volume of 100mL pH =6, and forming o-dianisidine buffer solution by using 0.1mol/L sodium phosphate buffer solution.

(2) Measuring a standard curve, preparing 10 groups of glucose oxidase standard solutions with different concentrations, adding an o-dianisidine buffer solution, a glucose solution and a horseradish peroxidase solution into a 10mL centrifuge tube, finally mixing the glucose oxidase standard solutions with accurate volumes of 2.5mL, 0.3mL, 0.1mL and 0.1mL respectively, reacting for 3min, and measuring an absorbance OD (optical Density) by using an enzyme-labeling instrument after the sulfuric acid solution stops reacting ₅₀₀ And repeating the steps three times to obtain an average value to prepare a standard curve.

(3) And (3) corresponding the light absorption value OD500 corresponding to the supernatant to the enzyme activity concentration corresponding to the standard curve, and multiplying the corresponding dilution times to obtain the corresponding enzyme activity.

III example

In order that the invention may be more readily understood, reference will now be made in detail to the following description of the invention taken in conjunction with the accompanying drawings, which are given by way of illustration only, and not by way of limitation with regard to the scope of the invention. The reagents or materials used in the present invention may be commercially available or prepared by a conventional method unless otherwise specified, and the specific experimental methods not mentioned in the following examples are generally performed by a conventional experimental method.

Example 1: construction of recombinant strains

Glucose oxidase genes derived from Penicillium Amagashaense are subjected to codon optimization and then connected to a PPIC9K plasmid, 2.0mg/mL G418 antibiotics are adopted for screening to obtain a high-yield strain PPG as a chassis cell, endogenous genes of SEC12, SEC13, SEC23, SEC24 and SEC31 in Pichia pastoris are respectively connected to a plasmid pGAPZB by adopting a gene recombination technology, and ubiquitination genes SEC12, SEC13, SEC23, SEC24 and SEC31 are respectively integrated into the high-yield strain PPG by linearizing the constructed plasmid pGAPZB, so that PPG-SEC12, PPG-SEC13, PPG-SEC23, PPG-SEC24 and PPG-SEC31 recombinant strains are obtained.

Example 2: shake flask fermentation of different recombinant strains

The recombinant strain is cultured at 28 ℃ and 200rpm until OD600 is 1.8-2.0, the recombinant strain is transferred into 50mL BMGY natural culture medium, the recombinant strain is cultured at 28 ℃,200rpm for 24h,3000rpm,3min, the supernatant is centrifuged and poured at 4 ℃, the thalli are all transferred into 100mL BMMY culture medium, and 1mL of methanol is added every 24h to induce the synthesis of glucose oxidase. As shown in FIG. 1, the growth trends of PPG-SEC12, PPG-SEC13, PPG-SEC23, PPG-SEC24 and PPG-SEC31 strains are basically the same as that of a control strain PPG, and the biomass OD600 of final fermentation 168h, PPG-SEC12, PPG-SEC13, PPG-SEC23, PPG-SEC24 and PPG-SEC31 strains are respectively 37.9, 39.1, 40.9, 38.4, 40.2 and 39.5, which indicates that over-expression of the vesicle-related ubiquitination transport factors SEC12, SEC13, SEC23, SEC24 and SEC31 has no influence on the normal growth of recombinant Pichia pastoris cells. And as for the trend of the change of the enzyme activity, as shown in figure 2, when the strain is fermented for 168 hours, the enzyme activities of the PPG, the PPG-SEC12, the PPG-SEC13, the PPG-SEC23, the PPG-SEC24 and the PPG-SEC31 strains are 118.86U/mL, 154.23U/mL, 129.21U/mL, 137.14U/mL, 131.15U/mL and 128.17U/mL respectively. Compared with a control strain PPG, the enzyme activities of the PPG-SEC12, the PPG-SEC13, the PPG-SEC23, the PPG-SEC24 and the PPG-SEC31 strains are respectively improved by 29.76%, 8.71%, 15.4%, 10.3% and 7.8%, which shows that the overexpression and vesicle-associated transport factors SEC12, SEC13, SEC23, SEC24 and SEC31 can improve the enzyme activity of producing glucose oxidase by pichia pastoris, and the SEC12 ubiquitination transport factor is preferably selected when coexpression the vesicle-associated ubiquitination transport factor and the glucose oxidase gene.

Example 3: 5L tank fermentation of different recombinant strains

A method for producing glucose oxidase using the strain in a 5L fermentor comprising the steps of: the pichia pastoris recombinant strain is activated in a 4mL YPD test tube, the pichia pastoris recombinant strain is inoculated into a 100mL YPD liquid culture medium shake flask (two bottles) according to 1 percent, the mixture is mixed with 2.3L BMGY culture medium according to the inoculum size of 8 percent, the temperature is maintained at 28 ℃, the stirring speed is 500rpm, the ventilation volume is 1.5vvm, the pH is adjusted to 5.5 by 3mol/L phosphoric acid and 28 percent ammonia water, the strain is enriched by 40g/L glycerol in the early stage, methanol is added after the dissolved oxygen rises, and the methanol concentration is controlled to be 5g/L by a methanol detection fed-batch controller of FC2002 type. The change of PPG in fermentation biomass and enzyme activity of 5L is shown in figure 3, the change of PPG-SEC12 in fermentation biomass and enzyme activity of 5L is shown in figure 4, as can be seen from figures 3 and 4, PPG strain 5L tank fermentation, 24h, biomass is 58.5, then methanol induction, fermentation 240h, biomass is 291.8, enzyme activity is 1249.79U/mL, while PPG-SEC12 strain, 24h biomass is 58.4, then methanol induction recombination strain heterologously expresses glucose oxidase, fermentation 240h, biomass is 289.2, enzyme activity is 1638.96U/mL, compared with PPG strain, the biomass is basically unchanged after SEC12 is over-expressed, and the enzyme activity is increased by 31.1%.

It should be noted that the above-mentioned embodiments are only used for illustrating and explaining the present invention, and do not limit the present invention in any way. It is understood that the words which have been used in the examples are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined within the scope of the claims and modifications may be made without departing from the scope and spirit of the invention. And all such modifications are intended to be included within the scope of the following claims, and although the invention has been described in connection with particular methods, materials, and embodiments, it is not intended to be limited to the specific embodiments disclosed herein; rather, the invention extends to all other methods and uses having the same functionality.

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<213> (primer GAP-SEC 23-F)

<400> 12

agagtgttta ggttttagcc ttagacatga ctgttcc 37

<210> 13

<211> 42

<212> DNA

<213> (primer GAP-SEC 23-R)

<400> 13

gtcttggtcc atatagttgt tcaattgatt gaaataggga ca 42

<210> 14

<211> 40

<212> DNA

<213> (primer SEC 24-F)

<400> 14

ttgaacaact atatggaaac tacacactcc atgaatgctg 40

<210> 15

<211> 46

<212> DNA

<213> (primer SEC 24-R)

<400> 15

ctaaggctaa aactcagtga atgaatatgg ttaaggaagt catggt 46

<210> 16

<211> 38

<212> DNA

<213> (primer GAP-SEC 24-F)

<400> 16

tcattcactg agttttagcc ttagacatga ctgttcct 38

<210> 17

<211> 43

<212> DNA

<213> (primer GAP-SEC 24-R)

<400> 17

gtgtagtttc catatagttg ttcaattgat tgaaataggg aca 43

<210> 18

<211> 57

<212> DNA

<213> (primer SEC 31-F)

<400> 18

caattgaaca actatatggt gaaaataagt gaaataaaaa gtacttcaac atttgca 57

<210> 19

<211> 35

<212> DNA

<213> (primer SEC 31-R)

<400> 19

ggctaaaact tagctactca gagccgagga catct 35

<210> 20

<211> 41

<212> DNA

<213> (primer GAP-SEC 31-F)

<400> 20

ctctgagtag ctaagtttta gccttagaca tgactgttcc t 41

<210> 21

<211> 39

<212> DNA

<213> (primer GAP-SEC 31-R)

<400> 21

ttttcaccat atagttgttc aattgattga aatagggac 39

Claims

1. A genetic engineering bacterium for secreting glucose oxidase is a recombinant Pichia pastoris containing ubiquitination transport genes and genes for coding Glucose Oxidase (GOX) or genes for coding Glucose Oxidase (GOX) subjected to codon optimization.

2. The genetically engineered bacterium of claim 1, wherein the ubiquitination transporter gene comprises an endogenous pichia pastoris ubiquitination transporter gene, and optionally an exogenous ubiquitination transporter gene; preferably, the exogenous ubiquitination transporter is derived from Saccharomyces cerevisiae; and/or the gene coding for Glucose Oxidase (GOX) is derived from Penicillium (Penicillium Amagasakiense) with Genebank accession number AAD01493.1.

3. The genetically engineered bacterium of claim 2, wherein the ubiquitination transporter is preferably a pichia endogenous ubiquitination transporter; preferably, the pichia endogenous ubiquitination transport gene comprises one or more of SEC12, SEC13, SEC23, SEC24 and SEC31 genes; further preferably, the SEC12 gene has Genebank accession number AF216960.1;

and/or, the Genebank accession number of the SEC13 gene is AAB01155.2;

and/or, the Genebank accession number of the SEC23 gene is CAY67406.1;

and/or, the Genebank accession number of SEC24 gene is CAY71741.1;

and/or, the Genebank accession number of the SEC31 gene is CAY68066.1.

4. The genetically engineered bacterium of any one of claims 1 to 3, wherein the genetically engineered bacterium is formed by overexpression of a ubiquitination transporter gene and a Glucose Oxidase (GOX) gene or a codon-optimized gene encoding Glucose Oxidase (GOX) in Pichia pastoris GS115 by a vector plasmid.

5. The genetically engineered bacterium of claim 4, wherein the gene encoding Glucose Oxidase (GOX) or the codon-optimized gene encoding Glucose Oxidase (GOX) is integrated into Pichia pastoris by means of a PPIC9K plasmid; and/or, the ubiquitination transporter is integrated into pichia pastoris through a PGAPZB plasmid.

6. The genetically engineered bacterium of any one of claims 1 to 5, wherein the Pichia pastoris is Pichia pastoris GS115.

7. The method for constructing genetically engineered bacteria secreting glucose oxidase according to any of claims 1 to 6, comprising:

8. The method of claim 7, wherein the first vector plasmid is a PPIC9K plasmid; and/or, the second carrier plasmid is PGAPZB plasmid.

9. Use of the genetically engineered bacterium of any one of claims 1 to 6 or constructed according to the method of claim 7 or 8 in the fermentative production of Glucose Oxidase (GOX); preferably, the application comprises inoculating the genetic engineering bacteria secreting the glucose oxidase into a fermentation culture medium, and performing fermentation culture to obtain the glucose oxidase.

10. The use according to claim 9, wherein the fermentation culture conditions are: the fermentation temperature is 28 ℃, the inoculation amount is 8%, the rotation speed is 500rpm, the ventilation amount is 1.5vvm, the pH value is 5.5, the culture medium is BMGY culture medium, methanol is added after the dissolved oxygen rises, the methanol concentration is controlled to be 5g/L, the fermentation time is not less than 240h, and the enzyme activity of the glucose oxidase is not less than 1638.96U/mL.