Method for improving yield of extracellular glucose oxidase of pichia pastoris and application thereof

Abstract

The application provides a method for improving the yield of Pichia pastoris extracellular glucose oxidase and application. The method comprises the following steps: (1) introducing an exogenous (a) Pichia pastoris signal peptide-glucose oxidase expression cassette and (b) Ees expression cassette into Pichia pastoris; (2) culturing the Pichia pastoris in step (1) to express glucose oxidase. Optionally, the (b) further comprises introducing the expression cassette of Sec22 and / or Bet1. The recombinant expression system constructed by the application is used for expression, and the expressed protein is close to nature. The expression system and method constructed by the application can efficiently express and secrete glucose oxidase.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioengineering, and more particularly, the present application relates to a method for improving the yield of extracellular glucose oxidase of Pichia pastoris and application thereof. BACKGROUND

[0002] Glucose oxidase (GOD; EC 1.1.3.4) is an oxidoreductase with high catalytic specificity for β-D-glucose, which catalyzes β-D-glucose to generate gluconolactone, and the gluconolactone is further hydrolyzed to generate gluconic acid.

[0003] GOD has a wide range of applications and has been applied in food, textile, medical, biosensor and biofuel cell industries. In the food industry, it can be used as a food preservative and color stabilizer, and also can be used to produce gluconic acid. In the textile industry, it can be used to produce hydrogen peroxide for textile bleaching. In the medical industry, it can be used to detect blood glucose concentration of diabetic patients. GOD is also applied in the biofuel cell industry. The catalytic reaction of GOD is an oxidation-reduction reaction, so the electrons can be transferred to the carbon electrode on the other end, thereby forming a green and environmentally friendly biofuel cell, which can provide a continuous energy source for biosensors and artificial organs. In the oral cavity, the presence of streptococci usually increases the probability of tooth decay. Adding GOD to toothpaste can react with residual glucose in the oral cavity to produce hydrogen peroxide, which can inhibit the growth and reproduction of harmful bacteria, thereby reducing the probability of oral diseases.

[0004] However, the yield of commercial GOD is low, which is a major limiting factor for its large-scale industrial application. Currently, GOD is produced by microbial fermentation in the art. However, the production of GOD by Aspergillus niger or Penicillium fermentation still needs to be improved in terms of enzyme yield and separation method. The production of GOD by Escherichia coli cannot be post-translationally processed, and the synthesized GOD is inactive. The skilled person in the art also produces GOD by Pichia pastoris, but the yield and enzyme activity still need to be improved, and the production process of GOD needs to be further optimized.

[0005] Pichia pastoris is a eukaryotic system widely used for secretory expression of foreign proteins, which has many advantages: simple operation at the molecular genetic level, easy to construct engineering bacteria; can express or secretory express recombinant proteins in the cell; post-translational modification of proteins is closer to higher eukaryotes, such as protein glycosylation, disulfide bond formation, etc., and recombinant proteins are easy to fold correctly; recombinant proteins can be produced in large quantities by fermentation, and factors affecting the yield and quality of recombinant proteins are easy to control. Although its acceptance is increasingly high and its application is increasingly successful, Pichia pastoris still has many limitations in expressing foreign proteins, among which the protein secretion process is one of the most common bottlenecks. SUMMARY

[0006] The purpose of the present application is to provide a method for improving the yield of Pichia pastoris extracellular glucose oxidase and its application.

[0007] In the first aspect of the present application, a method for improving the yield of Pichia pastoris extracellular glucose oxidase is provided, comprising: (1) introducing into Pichia pastoris an expression cassette of (a) Pichia pastoris signal peptide-glucose oxidase, and (b) an expression cassette of a protein of the amino acid sequence shown in SEQ ID NO: 1 (Ees); (2) culturing the Pichia pastoris of step (1) to express glucose oxidase.

[0008] In one or more embodiments, the improvement is a significant improvement, preferably, the extracellular expression / total expression of glucose oxidase is improved by more than 10%, more than 20%, more than 30%, more than 50%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 300%, more than 400%, more than 500% or more compared with Pichia pastoris that does not introduce the expression of the protein of the amino acid sequence shown in SEQ ID NO: 1 to express glucose oxidase.

[0009] In one or more embodiments, “-” means that the Pichia pastoris signal peptide is operatively linked to the glucose oxidase.

[0010] In one or more embodiments, in (b), the expression cassette of a protein selected from the group consisting of a protein of the amino acid sequence shown in SEQ ID NO: 2 (Sec22) or a protein of the amino acid sequence shown in SEQ ID NO: 3 (Bet1) is also introduced.

[0011] In one or more embodiments, the Pichia pastoris signal peptide comprises a signal peptide selected from the group consisting of: a signal peptide of the amino acid sequence set forth in SEQ ID NO: 4 (GAS1); a signal peptide of the amino acid sequence set forth in SEQ ID NO: 5 (FRE2); a signal peptide of the amino acid sequence set forth in SEQ ID NO: 6 (DAN4); a signal peptide of the amino acid sequence set forth in SEQ ID NO: 7 (MSB2); or a signal peptide of the amino acid sequence set forth in SEQ ID NO: 8 (DSE4).

[0012] In one or more embodiments, the Pichia pastoris signal peptide is a signal peptide of the amino acid sequence set forth in SEQ ID NO: 4 (GAS1).

[0013] In one or more embodiments, in (a), the Pichia pastoris signal peptide-glucose oxidase is 1-15 copies, preferably 2-12 copies, more preferably 3-10 copies, more preferably 5-9 copies (e.g., 5, 6, 7, or 8 copies).

[0014] In one or more embodiments, in (a), the glucose oxidase is a mutant glucose oxidase, wherein the 20th amino acid is mutated from Val to Trp, and the 30th amino acid is mutated from Thr to Val.

[0015] In one or more embodiments, the wild-type GOD gene encodes an amino acid sequence as set forth in Uniprot: P13006 (without the first 22 amino acids which are signal peptide sequence).

[0016] In one or more embodiments, the exogenously introduced gene is integrated into the genome of the Pichia pastoris by homologous recombination.

[0017] In one or more embodiments, the expression cassette further comprises: a promoter and a terminator suitable for expression in Pichia pastoris; preferably, the promoter comprises: an AOX promoter, a GAP promoter, a DAS1 promoter, a FDH1 promoter; and preferably, the terminator comprises an AOXTT, an RPS3tt.

[0018] In another aspect of the present application, there is provided a recombinant Pichia pastoris producing high level of glucose oxidase, comprising exogenously introduced: (a) a Pichia pastoris signal peptide-glucose oxidase expression cassette, and (b) an expression cassette of a protein of the amino acid sequence set forth in SEQ ID NO: 1 (Ees).

[0019] In one or more embodiments, in (b), the expression cassette of a protein of the amino acid sequence set forth in SEQ ID NO: 2, or the amino acid sequence set forth in SEQ ID NO: 3 is further introduced.

[0020] In one or more embodiments, the Pichia pastoris signal peptide comprises a signal peptide selected from the group consisting of: the signal peptide of the amino acid sequence set forth in SEQ ID NO: 4 (GAS1); the signal peptide of the amino acid sequence set forth in SEQ ID NO: 5 (FRE2); the signal peptide of the amino acid sequence set forth in SEQ ID NO: 6 (DAN4); the signal peptide of the amino acid sequence set forth in SEQ ID NO: 7 (MSB2); or the signal peptide of the amino acid sequence set forth in SEQ ID NO: 8 (DSE4); preferably, the Pichia pastoris signal peptide is the signal peptide of the amino acid sequence set forth in SEQ ID NO: 4 (GAS1).

[0021] In one or more embodiments, the Pichia pastoris signal peptide-glucose oxidase is 1-15 copies, preferably 2-12 copies, more preferably 3-10 copies, more preferably 5-9 copies (e.g., 5, 6, 7, or 8 copies).

[0022] In one or more embodiments, the glucose oxidase is a mutant glucose oxidase, wherein the 20th amino acid is mutated from Val to Trp, and the 30th amino acid is mutated from Thr to Val.

[0023] In another aspect of the present application, there is provided a use of the recombinant Pichia pastoris for producing glucose oxidase.

[0024] In another aspect of the present application, there is provided a kit for producing glucose oxidase, wherein the kit comprises the recombinant Pichia pastoris.

[0025] Other aspects of the present application will be apparent to those skilled in the art from consideration of the disclosure herein. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Construction of MGOD expression vectors with different signal peptides. pX represents vector pGAMu1, pFRMu1, pDAMu1, pDSMu1 and pMSMu1, respectively.

[0027] Figure 2 Construction of co-expression plasmid of secretory components by enzyme digestion and ligation. X represents BET1, EES, SAR1, SEC22 and YKT6, respectively; pUX represents pUBE1, pUEE, pUSA1, pUSE22 and pUYK6, respectively.

[0028] Figure 3 Construction of co-expression plasmid of secretory components by homologous exchange.

[0029] Figure 4 Construction of co-expression plasmid pUEE / SE22.

[0030] Figure 5 Construction of recombinant bacteria G1Be1, G1Ees, G1Sa1, G1Se13, G1Sec, G1Yk6(A) and G1EeSe(B). X represents the secretion-promoting components BET1, EES, SAR1, SEC13, SEC22 and YKT6; G1x represents the corresponding recombinant bacteria G1Be1, G1Ees, G1Sa1, G1Se13, G1Sec and G1Yk6. CYCTT is the terminator, and Hph is the hygromycin B phosphotransferase gene from Klebsiella pneumoniae driven by the TEF1 promoter.

[0031] Figure 6 PCR verification of MGOD expression plasmids containing different signal peptides.

[0032] Figure 7 Plate assay of recombinant bacteria with different signal peptides.

[0033] Figure 8 Intracellular and extracellular unit enzyme activity of recombinant bacteria M1, GM1, 5GM1 and 8GM1 after 144h of shake flask induction culture.

[0034] Figure 9 , 6 PCR product electrophoretogram of a target gene (A) and EES expression unit (B). Lane M1 and M2: DL2000 DNA Marker; Lane M3: DL10000 DNA Maker; Lane 1: BET1; Lane 2: YKT6; Lane 3: SEC22; Lane 4: SEC13; Lane 5: EES; Lane 6: SAR1; Lane 7: EES expression unit with AOX as the promoter and AOXTT as the terminator.

[0035] Figure 10 DCW (A) and extracellular GOD yield (B) of each recombinant bacteria after 144h of shake flask induction culture. Control is the starting bacteria 8GM1.

[0036] Figure 11 Growth curve (A) and intracellular and extracellular GOD unit biomass yield (B) of recombinant bacteria 8GM1, G1Sec, G1Ees and G1EeSe after shake flask induction culture.

[0037] Figure 12 , 5 GOD production by recombinant bacteria G1EeSe at the L reactor level. DETAILED DESCRIPTION

[0038] In the field of biotechnology, the recombinant expression of heterologous proteins is an important research topic. In order to improve the expression rate of recombinant proteins, a large amount of laboratory work is required, and multiple experiments, analysis, summary and retesting efforts are required to achieve the final success. There are many factors that can affect the expression rate of recombinant proteins; and if the secretion expression of the protein is considered, more factors need to be considered.

[0039] The present inventors are committed to improving the production of glucose oxidase (GOD) by yeast cells and the secretion expression yield thereof, and through in-depth research and a large number of screening, a method for effectively improving the yield of extracellular glucose oxidase of Pichia pastoris is developed, which co-expresses the protein (Ees) of the amino acid sequence shown in SEQ ID NO: 1 and GOD in the yeast strain, preferably also co-expresses the protein (Sec22) of the amino acid sequence shown in SEQ ID NO: 2 or the protein (Bet1) of the amino acid sequence shown in SEQ ID NO: 3. At the same time, the present application also provides a recombinant bacterium with high-efficiency GOD secretion constructed by using Pichia pastoris as a host. The same functional variants with high homology to the proteins of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 can also be applied in the present application. At the same time, the present inventors have also optimized the expression strategy of GOD secretion expression, and effectively improved the yield of GOD by setting the appropriate copy number.

[0040] Terms

[0041] As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences from different sources, or refers to the relationship between a protein / gene and a host cell. For example, although the host cell itself can also contain the corresponding gene or produce the corresponding protein, the synthetic / recombinant established gene / protein is "exogenous" or "heterologous" to the host cell when it is introduced into the host cell by genetic engineering methods.

[0042] As used herein, "operably linked" or "operatively linked" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed in a specific position relative to the nucleic acid sequence of the target gene, so that the transcription of the nucleic acid sequence is guided by the promoter region, and thus the promoter region is "operably linked" to the nucleic acid sequence.

[0043] As used herein, the "recombinant expression cassette" refers to a gene expression system comprising all necessary elements required for the expression of a target protein, which usually includes the following elements: a promoter, a gene sequence encoding a protein, a terminator; in addition, a signal peptide coding sequence can be optionally included. These elements are operatively linked.

[0044] As used herein, the term "construct" or "expression construct" refers to a recombinant DNA molecule comprising a nucleic acid coding sequence of interest, which can comprise one or more gene expression cassettes. The "construct" is typically contained in an expression vector.

[0045] The term "promoter" refers to a nucleic acid sequence, usually upstream (5' to) of a coding sequence of interest, capable of directing RNA sequence transcription into mRNA. Generally, a promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription.

[0046] As used herein, the term "signal peptide" refers to a peptide segment consisting of a stretch of amino acid residues at the N-terminus of a nascent peptide chain of a secreted protein, usually 20-30 amino acid residues in length. The signal peptide can direct the proper targeting of the secreted protein, and this peptide segment can be cleaved off.

[0047] Promoter

[0048] After a large number of research screening and experimental work, the present inventors determined an optimized scheme to greatly improve the production of GOD, including up-regulating the expression or activity of Ees, Sec22 or Betl in host cells (Pichia pastoris cells). Preferably, Ees, Sec22 or Betl is recombinantly expressed (overexpressed) in the yeast cells to promote the expression of MOD.

[0049] As a preferred mode of the present application, Ees is derived from Pichia pastoris and has the sequence shown in SEQ ID NO: 1; Sec22 is derived from Pichia pastoris and has the sequence shown in SEQ ID NO: 2; and Betl is derived from Pichia pastoris and has the sequence shown in SEQ ID NO: 3.

[0050] It should be understood that, after knowing the function of the Ees, Sec22 or Betl disclosed in the present application or the function of the Ees, Sec22 or Betl in the signal pathway in which it is involved (preferably, also including its upstream and downstream genes), various methods well known to those skilled in the art can be used to regulate the expression or activity of the Ees, Sec22 or Betl or to regulate the related upstream or downstream genes of Ees, Sec22 or Betl. For example, various methods well known to those skilled in the art can be used to overexpress Ees, Sec22 or Betl or its upstream or downstream genes.

[0051] In the present application, up-regulation of the expression of the Ees, Sec22 or Betl protein or its encoding gene, its upstream or downstream protein or its encoding gene includes the use of an up-regulator of the Ees, Sec22 or Betl protein or its encoding gene. The up-regulator can include a promoter, an agonist, an activator. The "up-regulation" or "promotion" includes the "up-regulation" or "promotion" of the activity of the protein or the "up-regulation" or "promotion" of the expression of the protein, and they are statistically significant "up-regulation" or "promotion". Any substance that can increase the activity of the Ees, Sec22 or Betl or the signal pathway protein containing the same (including its upstream and downstream proteins), increase the stability of the Ees, Sec22 or Betl or the signal pathway protein containing the same, up-regulate the expression of the Ees, Sec22 or Betl or the signal pathway gene containing the same, increase the effective action time of the Ees, Sec22 or Betl or the signal pathway protein containing the same, increase the phosphorylation / activation level of each protein, can be used in the present application as a substance useful for up-regulating the Ees, Sec22 or Betl or the signal pathway. They can be a compound, a small chemical molecule, a biological molecule. The biological molecule can be at the nucleic acid level (including DNA, RNA) or at the protein level.

[0052] The present application also provides a method for up-regulating the expression of Ees, Sec22 or Betl in a cell, which comprises: introducing the encoding gene of Ees, Sec22 or Betl or an expression construct or a vector containing the encoding gene into the cell. In addition, a gain-of-function mutation can also be made to Ees, Sec22 or Betl or its encoding gene; an enhanced promoter or a tissue-specific promoter can be used to promote the expression of the encoding gene of Ees, Sec22 or Betl; or an enhancer can be used to promote the expression of the encoding gene of Ees, Sec22 or Betl. It should be understood that other methods for up-regulating the expression of Ees, Sec22 or Betl in a cell should also be included in the present application.

[0053] The nucleotide full-length sequence of the encoding gene of Ees, Sec22 or Betl of the present application or a fragment thereof can generally be obtained by PCR amplification method, recombination method or artificial synthesis method. For the PCR amplification method, primers can be designed according to the nucleotide sequence disclosed in the present application, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared according to the conventional method known to those skilled in the art is used as a template for amplification to obtain the relevant sequence.

[0054] Signal peptide

[0055] Signal peptide is important for the expression of secretory recombinant protein. Suitable signal peptide can make the expression amount of target protein, especially the secretory expression amount, doubled. Pichia pastoris secretes less endogenous protein, so the secretion of exogenous protein guided by signal peptide sequence can facilitate the purification operation. The commonly used signal peptides include Saccharomyces cerevisiae α mating factor (α-MF) and Pichia pastoris acid phosphatase (PHO1) signal peptides, and the former is more widely used. Although α-MF signal peptide is a strong signal peptide, recombinant protein can obtain high expression under the guidance of the signal peptide. However, the secretion expression of some exogenous proteins using α-MF signal peptide can make the amino terminal of recombinant protein extended (Liu PT et al. Protein Expr Purif, 2001, 22: 381-387). For example, the secretion expression of human α-1 interferon using α-MF signal peptide can see that the N-terminus of part of the recombinant protein has 9-11 amino acid residues of α-MF signal peptide, which causes great difficulty in the purification process. Similar problems are also encountered in the expression of GOD.

[0056] The inventors have found that the signal peptide of the amino acid sequence shown in SEQ ID NO: 4 (GAS1), the signal peptide of the amino acid sequence shown in SEQ ID NO: 5 (FRE2), the signal peptide of the amino acid sequence shown in SEQ ID NO: 6 (DAN4), the signal peptide of the amino acid sequence shown in SEQ ID NO: 7 (MSB2), or the signal peptide of the amino acid sequence shown in SEQ ID NO: 8 (DSE4) has good expression efficiency when applied to the secretion expression of GOD in Pichia pastoris, and the GOD protein fused therewith can be correctly processed and guided to promote the secretion expression. Most preferably, the signal peptide is the signal peptide of the amino acid sequence shown in SEQ ID NO: 4 (GAS1).

[0057] Expression cassette, recombinant expression vector and recombinant cell

[0058] The expression cassette of GOD guided by the Pichia pastoris signal peptide of the application (Pichia pastoris signal peptide-GOD) is constructed in the application, which is used for transfection into yeast cells for expression. The Pichia pastoris signal peptide is selected from GAS1, FRE2, DAN4, MSB2 and DSE4, and preferably GAS1.

[0059] In the application, the expression cassette of Ees, Sec22 and / or Bet1 is also constructed, which is used for expression with GOD to promote the expression of GOD.

[0060] As a preferred mode of the application, the yeast promoter used is preferably AOX1 promoter, and can also be GAP promoter or other yeast promoter.

[0061] As a preferred mode of the present application, the expression cassette of the GOD of the present application comprises, from 5' to 3', a promoter sequence, the Pichia pastoris signal peptide coding sequence of the present application, the GOD mature peptide coding sequence, and a translation terminator sequence, which are operatively linked.

[0062] The cell used for recombinant expression of the present application is a yeast cell, such as Pichia, Hansenula, Candida, Torulopsis, etc. As a preferred mode of the present application, the yeast cell is a Pichia cell. Pichia has no natural plasmid in its body, so the expression vector needs to be homologously recombined with the host chromosome to integrate the foreign gene expression frame into the chromosome to realize the expression of the foreign gene. The foreign gene expression frame comprises a promoter, a foreign gene cloning site, a signal peptide, a foreign gene expression cassette, a termination sequence, a screening marker, etc. The expression plasmid can be stably integrated in the form of single copy or multiple copies at a specific site of the genome; since Pichia can use methanol as the sole carbon source and energy source, most microorganisms cannot use methanol as the carbon source, so the contamination of miscellaneous bacteria can be reduced during the fermentation process, and the large-scale industrial fermentation technology is relatively mature. The culture medium, fermentation method, etc. have been thoroughly studied, so that the reproducibility and automation degree of the fermentation are extremely good.

[0063] As a preferred mode of the present application, the "Pichia signal peptide-GOD" is 1-15 copies, preferably 2-12 copies, more preferably 3-10 copies, and more preferably 5-9 copies (such as 5, 6, 7, or 8 copies).

[0064] In the method of the present application, the method for culturing Pichia and the culture medium are not particularly limited, and the method and the culture medium commonly used in the art can be used. After the recombinant cell is cultured to express and secrete GOD, a step of separating GOD from the culture product (culture medium or fermentation broth) can be further included. The separation or purification of GOD from the culture product can be performed by using the techniques well known to those skilled in the art. For example, ammonium sulfate precipitation, DEAE-Sepharose ion exchange, gel filtration purification, molecular sieve, or affinity chromatography purification can be used.

[0065] The present application also provides a kit comprising the recombinant expression vector constructed by the present application and the yeast cell; or a kit comprising the recombinant yeast cell constructed by the present application.

[0066] Other reagents commonly used for transgenic operation can also be included in the kit to facilitate the use of those skilled in the art.

[0067] In addition, the kit can further comprise an instruction manual for guiding the operation of those skilled in the art.

[0068] Expression methods and applications

[0069] GOD has a wide range of applications, and the low production of commercial GOD is the main limiting factor for its large-scale industrial application. The Pichia pastoris expression system is a widely used eukaryotic system for secretory expression of foreign proteins. When expressing foreign proteins, the protein secretion process is one of the most common bottlenecks. The present application provides an optimized expression strategy for GOD.

[0070] Based on the optimization strategy of the present inventors, the present application provides a method for improving the production of extracellular glucose oxidase of Pichia pastoris, comprising: (1) introducing into Pichia pastoris an exogenous (a) Pichia pastoris signal peptide-glucose oxidase expression cassette, and (b) an expression cassette of the following proteins: a protein of the amino acid sequence shown in SEQ ID NO: 1 (Ees); (2) culturing the Pichia pastoris of step (1) to express glucose oxidase. Preferably, in (b), an expression cassette of a protein selected from the group consisting of a protein of the amino acid sequence shown in SEQ ID NO: 2 (Sec22) or a protein of the amino acid sequence shown in SEQ ID NO: 3 (Bet1) is introduced.

[0071] In a preferred embodiment of the present application, in order to improve the production of GOD secreted by Pichia pastoris, the present inventors optimized the signal peptide and the GOD gene dose, and obtained a recombinant bacterium 8GM1 with high GOD production. On this basis, a plurality of genes were co-expressed, including BET1, EES, SEC22, YKT6, SEC13 and SAR1, etc. Among them, the corresponding recombinant bacteria G1Ees, G1Sec and G1Be1 co-expressing EES, SEC22 and BET1, respectively, were induced in a shake flask for 144 h, and the extracellular unit bacterial GOD production was increased by 1.86, 1.14 and 1.07 times, respectively, compared with the control bacterium 8GM1, i.e. co-expression of EES, SEC22 and BET1 can effectively promote the secretory expression of GOD by the recombinant bacteria.

[0072] In the preferred embodiment of the present application, for the genes EES and SEC22 with greater promoting effect, co-expression of EES increases the intracellular and total GOD production of recombinant bacteria G1Ees, and co-expression of SEC22 increases the GOD secretion rate of recombinant bacteria G1Sec. Co-expression of EES and SEC22 in combination, recombinant bacteria G1EeSe, increases the secretion rate compared with co-expression of EES alone, recombinant bacteria G1Ee, and further increases the extracellular unit bacterial GOD production to 1.14 times of G1Ee, which is 2.15 times of the starting bacteria 8GM1, that is, the effects of co-expression of EES and SEC22 are relatively independent, and co-expression of the two in combination will achieve a synergistic expression effect. On the 5-L reactor level, the highest GOD volumetric enzyme production of recombinant bacteria G1EeSe reaches 7223.0 U / mL, which is a very high level in the field for GOD expression.

[0073] The recombinant expression system constructed by the present application is used for expression, and the expressed protein is close to nature. The expression system and method constructed by the present application can realize high-density culture in a simple synthetic medium, and the operation is simple, can be expressed by large-scale fermentation equipment, and the production cost is low.

[0074] The present application will be further described below in combination with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods not specified in the following examples are generally carried out according to the conventional conditions, such as the conditions described in J. Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or the conditions suggested by the manufacturer.

[0075] Materials and methods

[0076] 1. Candidate expression-assisting genes and signal peptides

[0077] The present inventors have screened a large number of candidate genes to find preferred genes suitable for promoting the expression of glucose oxidase, and part of the information of the genes is shown in Table 1. The sequences of some signal peptides are shown in Table 1.

[0078] Table 1

[0079]

[0080] 2. Plasmids and strains

[0081] The plasmids and strains used and constructed by the present application are shown in Table 2 and Table 3 below.

[0082] Table 2, plasmids

[0083]

[0084] Table 3, strains

[0085]

[0086]

[0087] 3. Medium

[0088] (1) LB medium

[0089] Yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L.

[0090] (2) YPD medium

[0091] Tryptone 20 g / L, glucose 20 g / L, yeast extract 10 g / L.

[0092] (3) BMGY growth medium

[0093] Tryptone 20 g / L, yeast extract 10 g / L, YNB 13.4 g / L, glycerol 10 g / L, 0.1 M K2HPO4 / KH2PO4 buffer pH 6.0.

[0094] (4) BMMY induction medium

[0095] Tryptone 20 g / L, yeast extract 10 g / L, YNB 13.4 g / L, 0.1 M K2HPO4 / KH2PO4 buffer pH 6.0. 1% methanol / 24h for induction of expression.

[0096] (5) YPD medium

[0097] Tryptone 20 g / L, glycerol 10 g / L, yeast extract 10 g / L.

[0098] (6) BSM medium

[0099] Glycerol 40 g / L, potassium sulfate 18 g / L, potassium hydroxide 4.13 g / L, magnesium sulfate 14.88 g / L, phosphoric acid 27 mL, calcium sulfate 0.93 g / L (1 / 1000 antifoam added at sterilization, 4.13 mL PTM1 added at inoculation of fermentation).

[0100] (7) PTM1 trace elements

[0101] Potassium iodide 0.09 g, copper sulfate pentahydrate 6 g, manganese sulfate monohydrate 3 g, sodium molybdate dihydrate 0.2 g, boric acid 0.02 g, zinc chloride 20 g, cobalt chloride 0.5 g, ferrous sulfate heptahydrate 65 g, biotin 0.2 g, concentrated sulfuric acid 5 mL.

[0102] (8) Glycerol feed

[0103] 50% (w / v) glycerol, supplemented with 1.2% PTM1 at the time of use.

[0104] (9) Methanol feed

[0105] 100% methanol, supplemented with 1.2% PTM1 at the time of use.

[0106] 4. Reagents and primers

[0107] The primers are shown in Table 4. The GOD standard was purchased from Sigma, and the horseradish peroxidase was purchased from Shanghai Sangon Biological Co., Ltd. o-Phthalaldehyde was purchased from Shanghai Mayre Chemical Technology Co., Ltd. Hygromycin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Other conventional reagents were imported or domestic analytical pure. Super-Fidelity DNA Polymerase, ClonExpress II One Step Cloning Kit were purchased from Nanjing Novizen Biological Technology Co., Ltd. Ligation Mix, yeast RNA extraction kit, reverse transcription kit, restriction endonuclease were purchased from Dalian Baobio Engineering Co., Ltd. Plasmid extraction kit, PCR purification kit, gel recovery kit were purchased from Axygen.

[0108] Table 4, primers

[0109]

[0110]

[0111] 5. Strain culture

[0112] Escherichia coli culture: a single colony was inoculated into a test tube containing LB and incubated at 37°C, 220 rpm overnight.

[0113] Pichia pastoris well plate screening investigation: a single colony was selected from the transformation plate and inoculated into a 96-well plate containing 300 μL of BMGY medium, 250 rpm, 30°C for 24 h as seed liquid. Then, the seed liquid was aspirated and inoculated into a 48-deep well plate containing 900 μL of BMMY induction medium per well, 220 rpm, 30°C for 24 h. The culture liquid was collected to determine the bacterial concentration and extracellular enzyme activity.

[0114] Pichia pastoris seed culture: a single colony was selected from the plate and inoculated into a test tube containing 3 mL of YPD and incubated at 30°C, 220 rpm for 18-24 h.

[0115] Pichia pastoris induction culture: the above Pichia pastoris seed liquid was aspirated and inoculated into a 250 mL flask containing 25 mL of BMGY medium, and incubated at 30°C, 220 rpm until the OD600 =4~6, centrifuge 5 min to collect all bacteria, resuspend the bacteria in BMMY medium, and adjust OD 600 =1, then transfer to a 500 mL flask containing 50 mL of BMMY medium for induction culture, sample every 24 h, and supplement 1% methanol.

[0116] Pichia pastoris 5L reactor fermentation experiment, the whole fermentation is divided into three stages: batch fermentation stage, glycerol feeding stage, methanol induction stage; in the batch fermentation process, the aeration rate is 1.5 VVM, the stirring speed is 600 rpm, when the batch fermentation is over and the DO starts to rise, start to supplement glycerol, control the glycerol flow rate to maintain the DO at about 20%, to OD 600 near 400, stop adding glycerol, wait for the DO to rise, starve for 30-60 min, then slowly supplement methanol, control the DO at about 10% by adjusting the methanol flow rate, stirring rate, etc. The whole fermentation process maintains the pH at 5.5 by supplementing ammonia water, and the fermentation temperature is 29.5°C. During the induction stage, sample every certain period of time to determine the cell concentration and extracellular enzyme activity.

[0117] Determination of dry weight of bacteria: take the fermentation broth, dilute to a certain multiple, and measure the OD 600 of the bacteria, calculate the DCW according to the relationship between OD 600 and dry weight: DCW (g / L) = 0.24 x OD 600 + 1.23 (R 2 = 0.994).

[0118] 6. GOD enzyme activity determination

[0119] Take 2.5 mL of o-dianisidine solution (0.21 mM o-dianisidine, 0.1 M Na2HPO4-citric acid, pH = 6), 0.3 mL of glucose solution (18%), and 0.1 mL of horseradish peroxidase solution (90 U / mL), mix well, and place in a 37°C water bath for 5 min. Take a certain volume of the sample to be tested, dilute it to an appropriate multiple, and add it to the above system. After 3 min of reaction, add 2 mol / L sulfuric acid to terminate the reaction. Take the reaction system without the sample to be tested as the control, and measure OD 500 . Take the pure glucose oxidase (sigma) solution as the standard curve.

[0120] GOD enzyme activity definition: the amount of enzyme that catalyzes the generation of 1 μmol of β-D-glucose to gluconic acid per minute at 37°C is one enzyme unit U.

[0121] Unit enzyme activity (specific enzyme activity) = volume enzyme activity (U / mL) / dry weight of bacteria (g / mL).

[0122] 7. Intracellular GOD detection

[0123] After centrifuging a certain volume of sample and discarding the supernatant, add Breaking Buffer and mix well. Wash the cells, centrifuge at 5000 rpm for 5 min, and discard the supernatant. Then, resuspend the cells in a certain volume of Breaking Buffer and add an equal volume of 0.5 mm acid-washed glass beads. Next, use a cryogenic homogenizer (JXFSTPRP-CL, Shanghai Jingxin) to disrupt the cells, centrifuge at 12000 rpm for 10 min, and transfer the supernatant to a new 1.5 mL centrifuge tube for determining intracellular GOD enzyme activity (see Invitrogen Pichia pastoris manual).

[0124] 8. Construction of MGOD expression vectors containing different signal peptides

[0125] like Figure 1 As shown in Table 4, the synthesized GAS1-F sequence (containing the forward sequence of the GAS1 signal peptide and the BamHI restriction site) and the GAS1-R sequence (containing the reverse complementary sequence of GAS1 and the EcoRI restriction site) were annealed to obtain a GAS1 signal peptide sequence containing BamHI and EcoRI restriction sites at both ends. This sequence was then digested and ligated to the BamHI / EcoRI double-digested pαMu1 fragment. The resulting fragment was heat-shocked and transformed into competent E. coli DH5α cells. Transformants were screened on plates containing Amp antibiotic to construct the vector pGAMu1. Using the same method, sequence pairs FRE2-F / FRE2-R, DAN4-F / DAN4-R, DSE4-F / DSE4-R, and MSB2-F / MSB2-R were annealed, and then ligated with the pαMu1 fragment digested with BamHI / EcoRI. After transformation and screening, vectors pFRMu1, pDAMu1, pDSMu1, and pMSMu1 were constructed, respectively.

[0126] 9. Construction of co-expression plasmids

[0127] like Figure 2 As shown, primers BET1-F / R, EES-F / R, SAR1-F / R, SEC22-F / R, and YKT6-F / R were used, with GS115 cDNA as a template, to obtain the corresponding BET1, EES, SAR1, SEC22, and YKT6 fragments by PCR. Then, the corresponding fragments and plasmid pAOX-UH were linearized by double digestion with NotⅠ and Xba I, respectively. After purification, the purified fragments were ligated into linearized vectors to construct the corresponding plasmids pUBE1, pUEE, pUSA1, pUSE22, and pUYK6.

[0128] like Figure 3 Using primers SEC13-F / R and GS115 cDNA as a template, PCR was performed to obtain the SEC13 fragment. Then, the corresponding fragment was seamlessly ligated (using homologous sequence exchange) with the plasmid pAOX-UH, which had been linearized with the enzyme SalI, to construct the plasmid pUSE13.

[0129] like Figure 4 Using primers EES / SEC22-F / R and plasmid pUEE as a template, PCR was performed to obtain the corresponding fragments containing the AOX promoter and AOXTT terminator. After gel purification, the fragments were seamlessly ligated (using homologous sequence exchange) to plasmid pUSE22 linearized with restriction endonuclease Spe I to construct plasmid pUEE / SE22.

[0130] 10. Construction of recombinant bacteria with different signal peptides MGOD and multi-copy recombinant bacteria 8GM1

[0131] The MGOD expression vectors pGAMu1, pFRMu1, pDAMu1, pDSMu1 and pMSMu1 containing different signal peptides were linearized with the enzyme Sal I and then electroporated into Pichia pastoris GS115 competent cells. The cells were then screened by MD plates to construct recombinant strains GM1, FM1, DAM1, DSM1 and MM1.

[0132] The high-copy recombinant strain 8GM1 was obtained by electroporating the linearized Sal I-modified vector pGAMu1 into Pichia pastoris GS115 competent cells and culturing on MD agar plates. After colony growth, the recombinant bacteria on the MD plates were collected by washing with sterile water and spread onto YPD plates containing different concentrations of the antibiotic G418 (0, 0.25, 0.5, 0.75, 1.0, 1.5, 1.75, and 2.0 mg / mL). High-copy recombinant strain 8GM1 was obtained through screening. Based on the principles of gene transformation, this high-copy strain can be repeatedly prepared.

[0133] 11. Determination of gene copy number

[0134] According to the manufacturer's manual, use Dr. GenTLE TM Genomic DNA was extracted using the High Recovery (TAKARA) method from Yeast. The copy number of the GOD gene was determined according to the method reported by Abad et al. (Abad S et al. Real-time PCR-based determination of gene copy numbers in Pichia pastoris. Biotechnol J. 2010, 5(4): 413-420).

[0135] 12. Construction of co-expression strains

[0136] The co-expression vectors (pUBE1, pUEE, pUSA1, pUSE13, pUSE22 and pUYK6) of each of the components of the secretion-promoting elements and the vector pUEE / SE22 co-expressing EES and SEC22 were digested with Xho I and Avr II, and then transformed into the competent cells of the recombinant Pichia pastoris 8GM1 by electroporation. The positive transformants were screened by culturing on YPD plates containing hygromycin (100 μg / mL) for 48-72 h. The co-expression recombinant strains G1Be1, G1Ees, G1Sa1, G1Se13, G1Sec, G1Yk6 and G1EeSe were obtained. The linearized fragments containing the expression units of the target genes were integrated into the genome of the recombinant strain 8GM1 by homologous recombination (U1-up / U1-dn: PAS_chr1-4_0695 / PAS_chr1-4_0164), as shown in the following reaction scheme: Figure 5 wherein X represents the components of the secretion-promoting elements BET1, EES, SAR1, SEC13, SEC22 and YKT6 (G1x represents the corresponding recombinant strains G1Be1, G1Ees, G1Sa1, G1Se13, G1Sec and G1Yk6).

[0137] Example 1. Construction and investigation of recombinant strains of MGOD with different signal peptides

[0138] The sequences GAS1-F / GAS1-R, FRE2-F / FRE2-R, DAN4-F / DAN4-R, DSE4-F / DSE4-R and MSB2-F / MSB2-R were subjected to annealing reaction, and then ligated with the pαMu1 fragment digested with BamH I / EcoR I. The ligation mixture was then transformed into the competent cells of E. coli DH5α by heat shock, and the transformants were screened on plates containing Amp. The MGOD expression vectors pGAMu1, pFRMu1, pDAMu1, pDSMu1 and pMSMu1 containing different signal peptides were constructed. The constructed plasmids were verified by colony PCR using the primers AOX-F / GOD-R. The results are shown in the following table: Figure 6 The MGOD expression vectors pGAMu1, pFRMu1, pDAMu1, pDSMu1 and pMSMu1 containing different signal peptides were linearized with Sal I, and then transformed into the competent cells of Pichia pastoris GS115 by electroporation. The recombinant strains GM1, FM1, DAM1, DSM1 and MM1 were constructed by screening on MD plates.

[0139] More than 20 different transformants were selected from MD plates after transformation by various recombinant bacteria and inoculated into 96-well plates for preliminary investigation. The MGOM recombinant bacterium M1 containing the α-MF signal peptide was used as a control. The effects of five different signal peptides on MGOD expression were preliminarily investigated. Results are shown below. Figure 7 .

[0140] like Figure 7 As shown, the secretory expression level of MGOD varied with different signal peptides. The relative enzyme activity of extracellular GOD in recombinant bacteria using signal peptides GAS1, FRE2, DAN4, DSE4, and MSB2 was higher than that of the control bacteria M1 using the α-MF signal peptide. Among them, the extracellular unit cell enzyme activity of recombinant bacteria GM1 using the signal peptide GAS1 was the highest, which was 5.3 times that of the control bacteria M1 (at the well plate level). After 144 h of shake-flask induction culture, the extracellular unit cell enzyme activity of recombinant bacteria GM1 was 8617.4 U / g DCW, which was 2.7 times that of the control bacteria M1. Figure 8 ).

[0141] Example 2: Construction and Investigation of High-Copy Recombinant Bacterium 8GM1

[0142] Based on the recombinant strain GM1, the gene dosage of MGOD was further optimized to improve the secretory expression of MGOD in the recombinant strain. The vector pGAMu1, linearized with the enzyme Sal I, was electroporated into Pichia pastoris GS115 competent cells and cultured on MD solid medium plates. After colony growth, the recombinant bacteria on the MD plates were collected by washing with sterile water and spread on YPD plates containing different concentrations of the antibiotic G418 (0, 0.25, 0.5, 0.75, 1.0, 1.5, 1.75 and 2.0 mg / mL) for G418 antibiotic gradient screening.

[0143] like Figure 8 As shown, two recombinant bacteria, 5GM1 and 8GM1, with significantly increased extracellular enzyme production were obtained through screening. After 144 hours of shake-flask induction culture, their extracellular unit cell enzyme activities were 22422.8 and 22769.5 U / g DCW, respectively, which are 2.6 times that of GM1 (8617.41 U / g DCW). The copy numbers of the GOD gene in recombinant bacteria 5GM1 and 8GM1 were determined to be 5 and 8, respectively. Of these two recombinant bacteria, 8GM1 had a slightly higher extracellular unit cell enzyme activity than 5GM1, and also exhibited a higher GOD gene dose and intracellular enzyme production. Modification of its protein secretion process suggests a more effective way to improve GOD secretion expression; therefore, 8GM1 was selected for further modification.

[0144] Example 3: Construction of co-expression plasmids and corresponding recombinant bacteria

[0145] The cDNA of G / GS115 was used as a template to amplify the six target genes by PCR, BET1 (421 bp), YKT6 (625 bp), SEC22 (667 bp), SEC13 (910 bp), EES (655 bp) and SAR1 (595 bp), as shown in Figure 9 A, the fragment length was as expected. The six target gene fragments were ligated with the linearized vector pAOX-UH to obtain the corresponding six co-expression plasmids, named pUBE1, pUYK6, pUSE22, pUSE13, pUEE and pUSA1, respectively. The sequencing results showed that the six co-expression plasmids were successfully constructed. Similarly, the EES expression unit (2047 bp) with AOX promoter and AOXTT terminator was obtained by PCR amplification using the plasmid pUEE as a template, as shown in Figure 9 B, the fragment size was as expected. The target fragment was ligated with the linearized vector pUSE22 to obtain the co-expression plasmid pUEE / SE22 of EES and SEC22, and the sequencing results showed that the plasmid was successfully constructed. Then, each recombinant plasmid was transformed into the competent cells of the recombinant strain 8GM1 by electroporation, and positive transformants were obtained by screening.

[0146] Example 4, Effect of co-expression of secretory components on production of recombinant strain GOD

[0147] To investigate the effect of co-expression of the six secretory components on the secretion and expression of GOD by the recombinant strain, the recombinant strains G1Be1, G1Ees, G1Sa1, G1Se13, G1Sec and G1Yk6 were induced for culture. As shown in Figure 10 A, after 144 h of induction, the cell dry weight (DCW) of each co-expression recombinant strain was similar to that of the control strain 8GM1, and the co-expression of each secretory component had little effect on growth. Figure 10 B, the extracellular unit of the recombinant strain co-expressing EES, SEC22 and BET1 produced GOD at a yield of 1.89, 1.14 and 1.07 times that of the control strain (22769.5 U / g DCW), respectively, i.e., 43095.1, 26031.2 and 24255.1 U / g DCW. It can be seen that co-expression of EES, SEC22 and BET1 helps the recombinant strain to secrete and express GOD.

[0148] Meanwhile, for the genes EES and SEC22 with greater promoting effect, as shown in Table 5, after 144 h of shake flask induction culture, the total intracellular and extracellular GOD production of the recombinant bacteria co-expressing EES is increased, and the secretion rate is decreased to 0.65 compared with the control (0.70); while the total GOD production of the recombinant bacteria co-expressing SEC22 is slightly decreased compared with the control, but the secretion rate is significantly increased to 0.82, that is, co-expression of EES increases the total intracellular and extracellular GOD production of the recombinant bacteria G1Ees, and co-expression of SEC22 increases the GOD secretion rate of the recombinant bacteria G1Sec. It can be seen that when the recombinant bacteria secrete and express GOD, the secretion process may become a bottleneck of the secretion and expression of GOD, which may limit the secretion of GOD and also affect the production of GOD. Co-expression of EES can promote the production and secretion of GOD by the recombinant bacteria, and co-expression of SEC22 can promote the secretion of GOD by the recombinant bacteria.

[0149] Table 5, GOD production and secretion rate

[0150]

[0151] In addition, in view of the above results, co-expression of EES increases the total GOD production of the recombinant bacteria, and co-expression of SEC22 increases the GOD secretion rate of the recombinant bacteria. According to the experimental results, the present inventors consider that EES and SEC22 may have different functions in the secretion process, and co-expression of EES and SEC22 may increase the secretion rate of GOD by the recombinant bacteria, promote the secretion of intracellular GOD to the extracellular, and further increase the extracellular production of GOD compared with co-expression of EES alone. Therefore, the present inventors further co-express EES and SEC22 in the starting bacteria 8GM1 to construct the recombinant bacteria G1EeSe, and investigate the influence of co-expression of EES and SEC22 on the secretion and expression of GOD by the recombinant bacteria through shake flask induction culture.

[0152] As Figure 11 A, the growth of the recombinant bacteria G1EeSe co-expressing EES and SEC22 is similar to that of the recombinant bacteria G1Ees or G1Sec co-expressing EES or SEC22 alone and the starting bacteria 8GM1, wherein the growth of the recombinant bacteria G1EeSe and G1Ees is slightly lower than that of G1Sec and 8GM1, and in combination with Figure 11 B, it can be seen that the extracellular and total GOD production of the recombinant bacteria G1EeSe and G1Ees is significantly higher than that of the recombinant bacteria G1Sec and 8GM1, and the production of G1EeSe is particularly increased. Therefore, the growth of the recombinant bacteria G1EeSe and G1Ees is slightly lower than that of G1Sec and 8GM1, which may be due to more carbon source and energy source flowing to the generation of GOD. However, the slight decrease in growth is basically negligible, and the increase in the production of GOD formed thereby is more significant.

[0153] As Figure 11B and Table 5, the recombinant bacteria G1EeSe co-expressing EES and SEC22 has higher extracellular unit cell GOD production than G1Ees co-expressing EES alone, lower intracellular, slightly higher total and higher secretion rate than G1Ees. Compared with G1Sec co-expressing SEC22 alone, G1EeSe has higher intracellular, extracellular and total unit cell GOD production and lower secretion rate. After 144h induction, the extracellular unit cell GOD production, total production and secretion rate of recombinant bacteria 8GM1, G1Sec, G1Ees and G1EeSe are respectively: 22769.5, 26031.2, 43095.1, 49065.2U / g DCW (extracellular); 32550.4, 31826.6, 66172.3, 67682.9 (total); 0.70, 0.82, 0.65, 0.72 (secretion rate). Thus, co-expression of EES and SEC22 has independent effect on recombinant bacteria expressing GOD, and the combined co-expression of the two has synergistic effect.

[0154] In addition, after 144h induction of combined co-expression of EES and SEC22, the recombinant bacteria G1EeSe has further improved extracellular unit cell enzyme production to 1.14 times of G1Ee and 2.15 times of 8GM1.

[0155] Example 5, Investigation of GOD production by GOD high-yield bacteria G1EeSe in 5-L reactor

[0156] In order to more comprehensively investigate the characteristics of recombinant bacteria G1EeSe22 and verify its industrial value, the growth and enzyme production process of recombinant bacteria G1EeSe22 were investigated in a 5-L reactor.

[0157] A single colony of recombinant bacteria G1EeSe22 was picked into a test tube containing 3mL YPD medium and cultured at 250rpm and 30°C for 20-24h. Then the above bacterial solution was taken and inoculated into a 500mL flask containing 50mL YPG medium at a 10% inoculation amount, and cultured at 250rpm and 30°C for 18-20h. Then the bacterial solution was taken and inoculated into a fermenter containing 2.5L BSM medium at a 10% inoculation amount for fermentation investigation.

[0158] The fermentation of recombinant bacteria was divided into three stages: batch culture stage, glycerol feeding stage and methanol induction stage. As shown in Table 6, the fermentation process of recombinant bacteria G1EeSe22 in a 5-L reactor is as follows: Figure 12, glycerol feeding phase, the recombinant bacteria grew rapidly, and the bacterial concentration increased rapidly. After about 29 h, the DCW reached 95.2 g / L. In the methanol induction phase, the specific growth rate was relatively low. During the induction of 0-120 h, the bacterial concentration slowly increased, and the DCW increased from 95.2 to 108.8 g / L, and then began to decrease, and the biomass decreased to 87.9 g / L at the end of the fermentation (312 h). In the early induction stage (0-48 h), the enzyme production rate was low. In this stage, the recombinant bacteria were constantly adapting to methanol culture; the enzyme production rate rapidly increased after 48 h of induction, and reached the highest level during 96-120 h, and then began to gradually decrease, and the supernatant enzyme activity reached the highest level of 7223.0 U / mL at 288 h, which exceeded the highest level expressed by other systems. The extracellular protein was mainly GOD, and the concentration could reach 30.7 g / L.

[0159] CONCLUSION

[0160] Based on the recombinant bacteria 8GM1 containing 8 copies of the exogenous protein GOD gene, the genes BET1, EES, SEC22, YKT6, SEC13 and SAR1 are co-expressed, wherein the corresponding recombinant bacteria G1Ees, G1Sec and G1Be1 co-express EES, SEC22 and BET1, and the extracellular unit enzyme yield of the recombinant bacteria is increased by 1.86, 1.14 and 1.07 times, respectively, compared with the control bacteria 8GM1 after 144 h of shake flask induction culture, that is, co-expression of EES, SEC22 and BET1 helps the recombinant bacteria to secrete and express GOD.

[0161] Meanwhile, for the recombinant bacteria G1Ees and G1Sec with higher extracellular enzyme yield, the intracellular enzyme yield is determined, and the GOD secretion and expression characteristics are analyzed: compared with the control bacteria 8GM1, the intracellular and extracellular (total) GOD yield of the recombinant bacteria G1Ees is increased, and the secretion rate is decreased to 0.65 (the control is 0.70); the intracellular GOD yield of the recombinant bacteria G1Sec is decreased, the total GOD yield is slightly decreased, but the secretion rate is significantly increased to 0.82, that is, co-expression of EES increases the intracellular and extracellular total GOD yield of the recombinant bacteria G1Ees, and co-expression of SEC22 increases the GOD secretion rate of the recombinant bacteria G1Sec.

[0162] Further co-expressing EES and SEC22 in the starting strain 8GM1, the shake flask induction culture, compared with the recombinant strain G1Ees, the extracellular unit cell enzyme yield of G1EeSe is increased, the intracellular is decreased, the total yield is slightly increased, and the secretion rate is increased; compared with the recombinant strain G1Sec, the intracellular and extracellular and total unit cell enzyme yield of G1EeSe is increased, and the secretion rate is decreased. After 144h of induction culture, the extracellular unit cell GOD yield, total yield and secretion rate of the recombinant strains 8GM1, G1Sec, G1Ees and G1EeSe are respectively: 22769.5, 26031.2, 43095.1, 49065.2U / g DCW (extracellular); 32550.4, 31826.6, 66172.3, 67682.9 (total); 0.70, 0.82, 0.65, 0.72 (secretion rate). That is, co-expressing SEC22 can promote the recombinant strain to secrete GOD, co-expressing EES can promote the recombinant strain to produce and secrete GOD, the two effects are relatively independent, and the combined co-expression of the two has an additive effect.

[0163] In addition, the recombinant strain G1EeSe co-expressing EES and SEC22 has a further increased extracellular unit cell GOD yield of 1.14 times that of the recombinant strain G1Ee co-expressing EES alone and 2.15 times that of the starting strain 8GM1, and the highest volumetric enzyme yield reaches 7223.0U / mL at the 5-L reactor level.

[0164] The above-described embodiments only express several embodiments of the present application, which are described in a more specific and detailed manner, but cannot be understood as limiting the scope of the patent of the present application. It should be noted that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims. Meanwhile, all the documents mentioned in the present application are cited as references in the present application, just as each document is cited as a reference individually. SEQUENCE LISTING <110> East China University of Technology <120> Method for improving extracellular glucose oxidase yield of Pichia pastoris and application <130> 21A098 <160> 32 <170> SIPOSequenceListing 1.0 <210> 1 <211> 210 <212> PRT <213> Pichia pastoris <400> 1 Met Lys Phe Gly Val Ser Val Phe Leu Thr Ile Ile Ser Leu Cys Gln 1 5 10 15 Leu Ala Leu Gly Ser Gln Val Thr Phe Val Leu Gly Ala Gln Asp Arg 20 25 30 Glu Cys Tyr Tyr Val Phe Asn Asn Lys Pro Gly Ser Asn Ile Gly Tyr 35 40 45 Tyr Phe Ala Val Gln Ser Gly Gly Ser Phe Asp Val Asp Tyr Gln Ile 50 55 60 Lys Ala Pro Asn Gly Lys Ile Ile Val Lys Glu Asn Lys Gln Arg Gln 65 70 75 80 Gly Asp Trp Val Phe Asn Ala Asp Gln Asn Gly Glu Tyr Glu Phe Cys 85 90 95 Phe Ser Asn Gly Met Ser Thr Phe Ala Glu Lys Val Val Asp Phe Glu 100 105 110 Ile Lys Leu Glu Asp Asp Asp Phe Arg Ala Ala Leu Pro Asn Ala Pro 115 120 125 Ser Gln Gln Val Ala Thr Asp Glu Ile His Arg Thr Ile Asn Ser Leu 130 135 140 Glu Glu Lys Leu Gln Thr Leu Thr Arg Asp Tyr Gln Tyr Tyr Lys Thr 145 150 155 160 Arg Asn Asn Arg Asn Gin Ser Thr Val Lys Ser Thr Glu Ser Arg Ile 165 170 175 Phe Tyr Phe Ser Ile Phe Asp Val Leu Leu Met Cys Gly Met Ala Gly 180 185 190 Phe Gin Val Ala Val Val Gin Leu Phe Phe Lys Gly Ser Arg Lys Gin 195 200 205 Leu Val 210 <210> 2 <211> 214 <212> PRT <213> Pichia pastoris <400> 2 Met Val Lys Ser Thr Leu Ile Phe Arg Asn Asp Gly Leu Pro Leu Ser 1 5 10 15 Ala Thr Val Asp Asp Asp Gin Thr Gin Asn Leu Thr Glu Gin Lys Asn 20 25 30 Gln Ala Lys Ala Ile Val Asn Lys Val Asn Ser Asn Ser Ala Thr Glu 35 40 45 Ala Ser Ile Arg Ser Gly Asn Tyr Thr Ile His Tyr Leu Leu Arg Glu 50 55 60 Ser Ile Val Phe Leu Val Ile Val Asp Lys Ser Phe Ser Arg Asn Leu 65 70 75 80 Ala Phe Ala Tyr Leu Gin Gin Ile Ala Asn Gin Phe Ile Asn Ser His 85 90 95 Gly Asn Ala Ala Leu Arg Ser Asp Thr Arg Pro Tyr Gin Phe Val Ser 100 105 110 Phe Asp Ser Phe Met Ser Lys Thr Lys Lys Leu Tyr Gin Asp Ser Arg 115 120 125 Thr Gin Ser Asn Leu Asp His Leu Asn Asn Gin Leu Ser Asp Val Lys 130 135 140 Arg Ile Met Thr Lys Asn Ile Gin Asp Leu Leu Tyr Arg Gly Glu Ser 145 150 155 160 Leu Asp Asp Met Ser Asp Leu Ser Tyr Asn Leu Arg Ala Gin Ser Lys 165 170 175 Lys Tyr Arg Lys Ala Ala Arg Arg Ile Asn Leu Gin Ala Leu Ile Lys 180 185 190 Gln Tyr Val Pro Val Ala Met Val Gly Ile Phe Phe Val Phe Ile Ile 195 200 205 Trp Trp Ile Phe Leu Arg 210 <210> 3 <211> 132 <212> PRT <213> Pichia pastoris <400> 3 Met Ser Ser Arg Tyr Ser Ser Asn Leu His Gin Arg Asp Asn Arg Ser 1 5 10 15 Ser Leu Phe Glu Thr Arg Ser Ala Ser Pro Tyr Asp Asp Ala Pro Ser 20 25 30 Lys Lys Asp Tyr Lys Ala Ser Leu Leu Ser Gin Leu Glu Ser Gin Asn 35 40 45 Glu Asp Glu Leu Ser Ser Met Ser Glu Lys Val Leu Met Leu Lys Asn 50 55 60 Leu Gly Ser Arg Met Gly Asp Glu Ile Lys Asn Ser Gin Leu Asn Ile 65 70 75 80 Asp Asp Leu His Ser Thr Met Thr Asn Thr Gin Thr Arg Leu Lys Asn 85 90 95 Thr Phe Lys Arg Met Met Val Met Ala Lys Lys Thr Gly Ile Ser Trp 100 105 110 Lys Leu Trp Leu Leu Phe Phe Phe Leu Val Trp Leu Trp Phe Phe Phe 115 120 125 Val Trp Leu Arg 130 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <400> 4 Met Leu Ser lie Leu Ser Ala Leu Thr Leu Leu Gly Leu Ser Cys Ala 1 5 10 15 <210> 5 <211> 21 <212> PRT <213> Artificial Sequence <400> 5 Met Arg Asn His Leu Asn Asp Leu Val Val Leu Phe Leu Leu Leu Thr 1 5 10 15 Val Ala Ala Gln Ala 20 <210> 6 <211> 18 <212> PRT <213> Artificial Sequence <400> 6 Met Phe Leu Lys Ser Leu Leu Ser Phe Ala Ser lie Leu Thr Leu Cys 1 5 10 15 Lys Ala <210> 7 <211> 20 <212> PRT <213> Artificial Sequence <400> 7 Met lie Asn Leu Asn Ser Phe Leu lie Leu Thr Val Thr Leu Leu Ser 1 5 10 15 Pro Ala Leu Ala 20 <210> 8 <211> 23 <212> PRT <213> Artificial Sequence <400> 8 Met Ser Phe Ser Ser Asn Val Pro Gin Leu Phe Leu Leu Leu Val Leu 1 5 10 15 Leu Thr Asn Ile Val Ser Gly 20 <210> 9 <211> 60 <212> DNA <213> Artificial Sequence <400> 9 gatccatgtt gtccatttta agtgcattaa ctctgctggg cctgtcttgt gcttacgtag 60 <210> 10 <211> 60 <212> DNA <213> Artificial Sequence <400> 10 aattctacgt aagcacaaga caggcccagc agagttaatg cacttaaaat ggacaacatg 60 <210> 11 <211> 75 <212> DNA <213> Artificial Sequence <400> 11 gatccatgag aaaccaccta aatgatctag tggtattgtt tttgcttctc acagtagcag 60 ctcaggccta cgtag 75 <210> 12 <211> 75 <212> DNA <213> Artificial Sequence <400> 12 aattctacgt aggcctgagc tgctactgtg agaagcaaaa acaataccac tagatcattt 60 aggtggtttc tcatg 75 <210> 13 <211> 81 <212> DNA <213> Artificial Sequence <400> 13 gatccatgtc attctcttcc aacgtgccac aacttttctt gttgttggtt ctgttgacca 60 atatagtcag tggatacgta g 81 <210> 14 <211> 81 <212> DNA <213> Artificial Sequence <400> 14 aattctacgt atccactgac tatattggtc aacagaacca acaacaagaa aagttgtggc 60 acgttggaag agaatgacat g 81 <210> 15 <211> 72 <212> DNA <213> Artificial Sequence <400> 15 gatccatgat taatttaaac tcctttctta tacttacagt aacactgtta tctccagctt 60 tggcatacgt ag 72 <210> 16 <211> 72 <212> DNA <213> Artificial Sequence <400> 16 aattctacgt atgccaaagc tggagataac agtgttactg taagtataag aaaggagttt 60 aaattaatca tg 72 <210> 17 <211> 66 <212> DNA <213> Artificial Sequence <400> 17 gatccatgtt cctcaaaagt ctccttagtt ttgcgtctat cctaacgctt tgcaaggcct 60 acgtag 66 <210> 18 <211> 66 <212> DNA <213> Artificial Sequence <400> 18 aattctacgt aggccttgca aagcgttagg atagacgcaa aactaaggag acttttgagg 60 aacatg 66 <210> 19 <211> 40 <212> DNA <213> Artificial Sequence <400> 19 ttgcggccgc aaatgtcaag tcgctattcg tcaaacttac 40 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <400> 20 gctctagagc tcatctaagc cacacaaaaa agaacc 36 <210> 21 <211> 36 <212> DNA <213> Artificial Sequence <400> 21 ttgcggccgc aaatgaagtt tggggtttcc gtattt 36 <210> 22 <211> 35 <212> DNA <213> Artificial Sequence <400> 22 gctctagagc ctacaccaat tgttttctgg aaccc 35 <210> 23 <211> 38 <212> DNA <213> Artificial Sequence <400> 23 ttgcggccgc aaatgtgggt actaaactgg ttccagga 38 <210> 24 <211> 40 <212> DNA <213> Artificial Sequence <400> 24 gctctagagc ttaaatgtac tgagagagcc atctgatacc 40 <210> 25 <211> 42 <212> DNA <213> Artificial Sequence <400> 25 ttgcggccgc aaatggtaaa gtccaccttg atctttagaa ac 42 <210> 26 <211> 35 <212> DNA <213> Artificial Sequence <400> 26 gctctagagc tcagcgcaag aatatccacc atatt 35 <210> 27 <211> 44 <212> DNA <213> Artificial Sequence <400> 27 ttgcggccgc aaatgaaact gtattattta ggagtgatca agac 44 <210> 28 <211> 41 <212> DNA <213> Artificial Sequence <400> 28 gctctagagc ctacataatt aaacagcaag agtttgtctt c 41 <210> 29 <211> 46 <212> DNA <213> Artificial Sequence <400> 29 atctgaatag cgccgtcgac atggttacaa ttggaaacgc acatga 46 <210> 30 <211> 42 <212> DNA <213> Artificial Sequence <400> 30 atgatgatga tgatggtcga ttattgatcg acttcgccag cg 42 <210> 31 <211> 32 <212> DNA <213> Artificial Sequence <400> 31 actttaaaga gtacgtagca ctagtaacat cc 32 <210> 32 <211> 51 <212> DNA <213> Artificial Sequence <400> 32 tcgtctttgg atgttactag gaagatcttc atccgcacaa acgaaggtct c 51

Claims

1. A method for increasing the production of extracellular glucose oxidase by Pichia pastoris, characterized by, The method comprises: (1) introducing into Pichia pastoris an exogenous (a) Pichia pastoris signal peptide-glucose oxidase expression cassette, and (b) an expression cassette of a protein of an amino acid sequence shown in SEQ ID NO: 1; (2) culturing the Pichia pastoris of step (1) to express glucose oxidase.

2. The method of claim 1, wherein, In (b), an expression cassette of a protein selected from the group consisting of a protein of an amino acid sequence shown in SEQ ID NO: 2 or a protein of an amino acid sequence shown in SEQ ID NO: 3 is further introduced.

3. The method of claim 1 or 2, wherein, The Pichia pastoris signal peptide comprises a signal peptide selected from the group consisting of a signal peptide of an amino acid sequence shown in SEQ ID NO: 4, a signal peptide of an amino acid sequence shown in SEQ ID NO: 5, a signal peptide of an amino acid sequence shown in SEQ ID NO: 6, a signal peptide of an amino acid sequence shown in SEQ ID NO: 7, or a signal peptide of an amino acid sequence shown in SEQ ID NO:

8.

4. The method of claim 1 or 2, wherein, The Pichia pastoris signal peptide is a signal peptide of an amino acid sequence shown in SEQ ID NO:

4.

5. The method of claim 3, wherein, In (a), the Pichia pastoris signal peptide-glucose oxidase is 1-15 copies.

6. The method of claim 5, wherein, In (a), the Pichia pastoris signal peptide-glucose oxidase is 2-12 copies.

7. The method of claim 6, wherein, In (a), the Pichia pastoris signal peptide-glucose oxidase is 3-10 copies.

8. The method of claim 7, wherein, In (a), the Pichia pastoris signal peptide-glucose oxidase is 5-9 copies.

9. The method of claim 1, wherein, In (a), the glucose oxidase is a mutant glucose oxidase, which has an amino acid sequence shown in Uniprot: P13006 and is mutated from Val to Trp at the 20th position and from Thr to Val at the 30th position.

10. The method of claim 1, wherein, The expression cassette further comprises a promoter and a terminator suitable for Pichia pastoris expression.

11. The method of claim 10, wherein, The promoter comprises an AOX promoter, a GAP promoter, a DAS1 promoter, or a FDH1 promoter.

12. The method of claim 10, wherein, The terminator comprises an AOXTT or an RPS3tt.

13. A recombinant Pichia pastoris producing glucose oxidase at a high yield, characterized in that, The Pichia pastoris signal peptide comprises a signal peptide selected from the group consisting of a signal peptide of an amino acid sequence shown in SEQ ID NO: 4, a signal peptide of an amino acid sequence shown in SEQ ID NO: 5, a signal peptide of an amino acid sequence shown in SEQ ID NO: 6, a signal peptide of an amino acid sequence shown in SEQ ID NO: 7, or a signal peptide of an amino acid sequence shown in SEQ ID NO:

8.

14. The recombinant Pichia of claim 13, wherein, The Pichia pastoris signal peptide is a signal peptide of an amino acid sequence shown in SEQ ID NO:

4.

15. The recombinant Pichia pastoris of claim 13, wherein, The Pichia pastoris signal peptide-glucose oxidase is 1-15 copies.

16. The recombinant Pichia of claim 15, wherein, The Pichia pastoris signal peptide-glucose oxidase is 2-12 copies.

17. The recombinant Pichia of claim 13, wherein The Pichia pastoris signal peptide-glucose oxidase is 3-10 copies.

18. The recombinant Pichia of claim 17, wherein, ​ 19. The recombinant Pichia of claim 18, wherein, ​ 20. The recombinant Pichia of claim 19, wherein, The Pichia pastoris signal peptide-glucose oxidase is 5-9 copies.

21. The recombinant Pichia of claim 13, wherein, The glucose oxidase is a mutant glucose oxidase, the amino acid sequence of which is Uniprot: P13006, and the 20th amino acid is mutated from Val to Trp and the 30th amino acid is mutated from Thr to Val.

22. Use of the recombinant Pichia pastoris according to any one of claims 13-21 for producing glucose oxidase.

23. A kit for producing glucose oxidase, the kit comprising the recombinant Pichia pastoris according to any one of claims 13-21.

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