Pichia pastoris engineering bacteria with high glucose oxidase production and preparation method and application thereof

Abstract

The present application provides a Pichia pastoris engineering bacterium and a kit and application thereof, a method for obtaining the Pichia pastoris engineering bacterium, a method for improving glucose oxidase yield of the Pichia pastoris, and a method for producing glucose oxidase, wherein the Pichia pastoris engineering bacterium carries a glucose oxidase gene and a pyruvate kinase gene. The Pichia pastoris engineering bacterium of the present application can realize high glucose oxidase yield and high enzyme activity, and lays a good foundation for large-scale production of glucose oxidase.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology. Specifically, the present application relates to a Pichia pastoris engineering bacterium with high glucose oxidase yield, and a preparation method and application thereof. BACKGROUND

[0002] Glucose oxidase (GOD, EC 1.1.3.4) has the advantages of high efficiency, specificity, no toxic side effects, etc., and can oxidize glucose to generate gluconic acid, thereby producing special effects such as sterilization, deoxidization, and glucose removal, and is widely used in medicine, food, feed, textiles and other fields.

[0003] At present, glucose oxidase on the domestic market is mainly prepared by large-scale fermentation of Aspergillus niger and Penicillium, and is often accompanied by the production of amylase, cellulase and other by-products, which brings great difficulty to the separation and purification process of the target protein in the later stage, greatly increasing the cost of process production.

[0004] Therefore, the method for producing glucose oxidase at present still needs to be researched. SUMMARY

[0005] The present application aims to at least partially solve the technical problems existing in the prior art, and for this purpose, the present application provides a Pichia pastoris engineering bacterium and a kit and application thereof, a method for obtaining the Pichia pastoris engineering bacterium, a method for improving the glucose oxidase yield of Pichia pastoris, and a method for producing glucose oxidase. The Pichia pastoris engineering bacterium of the present application can realize high glucose oxidase yield and high enzyme activity, laying a good foundation for large-scale production of glucose oxidase.

[0006] It should be noted that the present application is based on the following findings of the inventors:

[0007] Pichia pastoris has the advantages of rapid growth and reproduction, high-level expression of proteins, post-translational modification, and mature fermentation process, both prokaryotic and eukaryotic expression systems. Therefore, the inventors introduced the glucose oxidase gene into Pichia pastoris to express glucose oxidase. In order to further improve the yield of glucose oxidase, the inventors found that enhancing the central carbon metabolism pathway helps to express the glucose oxidase gene through in-depth research. Further, the inventors screened out a gene that can effectively enhance the central carbon metabolism pathway and does not hinder the expression of the glucose oxidase gene, i.e., the pyruvate kinase gene, through a large number of experiments. The co-expression of the glucose oxidase gene and the pyruvate kinase gene in Pichia pastoris helps Pichia pastoris to better secrete glucose oxidase, and the enzyme activity is high, laying a good foundation for large-scale production of glucose oxidase.

[0008] To this end, in one aspect of the present application, the present application provides a Pichia pastoris engineering bacteria. According to an embodiment of the present application, the Pichia pastoris engineering bacteria carries a glucose oxidase gene and a pyruvate kinase gene.

[0009] According to the Pichia pastoris engineering bacteria of the embodiment of the present application, the glucose oxidase gene and the pyruvate kinase gene are co-expressed, which helps the Pichia pastoris to better secrete glucose oxidase, and the enzyme activity is high, which lays a good foundation for the large-scale production of glucose oxidase.

[0010] In another aspect of the present application, the present application provides a kit. According to an embodiment of the present application, the kit comprises: the aforementioned Pichia pastoris engineering bacteria; or, the kit comprises: a first expression vector carrying a glucose oxidase gene; and a second expression vector carrying a pyruvate kinase gene.

[0011] In another aspect of the present application, the present application provides a method for obtaining the aforementioned Pichia pastoris engineering bacteria. According to an embodiment of the present application, the method comprises: introducing a first expression vector carrying a glucose oxidase gene into Pichia pastoris, culturing, and screening the strain with the highest glucose oxidase yield as the primary screening strain; introducing a second expression vector carrying a pyruvate kinase gene into the primary screening strain, culturing, and screening the strain with the highest glucose oxidase yield as the target Pichia pastoris engineering bacteria.

[0012] In another aspect of the present application, the present application provides a method for improving the glucose oxidase yield of Pichia pastoris. According to an embodiment of the present application, the method comprises: making Pichia pastoris carry a glucose oxidase gene and a pyruvate kinase gene.

[0013] In another aspect of the present application, the present application provides the aforementioned Pichia pastoris engineering bacteria or kit for use in the production of glucose oxidase.

[0014] In another aspect of the present application, the present application provides a method for producing glucose oxidase. According to an embodiment of the present application, the method comprises: culturing using the aforementioned Pichia pastoris engineering bacteria, collecting the bacterial cells in the obtained culture solution, and isolating glucose oxidase.

[0015] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0016] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description, taken in conjunction with the accompanying drawings, in which:

[0017] Figure 1 A codon usage profile according to Embodiment 1 of the application is shown;

[0018] Figure 2 A recombinant expression plasmid pGAPZ alpha A-GOX4opt structure diagram according to Embodiment 2 of the application is shown;

[0019] Figure 3 and Figure 4 A codon usage profile according to Embodiment 3 of the application is shown;

[0020] Figure 5 and Figure 6 A GC content distribution profile according to Embodiment 3 of the application is shown;

[0021] Figure 7 A recombinant expression plasmid pPIC9K-BTS-opt structure diagram according to Embodiment 3 of the application is shown;

[0022] Figure 8 A glucose oxidase enzyme activity analysis diagram according to Embodiment 4 of the application is shown;

[0023] Figure 9 A protein electrophoresis diagram according to Embodiment 9 of the application is shown. DETAILED DESCRIPTION

[0024] Embodiments of the application are described in detail below. The embodiments described below are exemplary only and are not to be construed as limiting the application.

[0025] The present application proposes a Pichia pastoris engineering bacterium and a kit and application thereof, a method for obtaining the Pichia pastoris engineering bacterium, a method for improving glucose oxidase yield of the Pichia pastoris, and a method for producing glucose oxidase, which will be described in detail below.

[0026] Pichia pastoris engineering bacterium

[0027] In an aspect of the present application, the present application proposes a Pichia pastoris engineering bacterium. According to an embodiment of the present application, the Pichia pastoris engineering bacterium carries a glucose oxidase gene and a pyruvate kinase gene.

[0028] In the Pichia pastoris engineering bacterium according to an embodiment of the present application, the glucose oxidase gene and the pyruvate kinase gene are co-expressed, which helps the Pichia pastoris to better secrete glucose oxidase, and the enzyme activity is high, which lays a good foundation for large-scale production of glucose oxidase.

[0029] According to an embodiment of the present application, the glucose oxidase gene is derived from Aspergillus niger.

[0030] According to an embodiment of the present application, the glucose oxidase gene is obtained by optimizing the wild-type Aspergillus niger glucose oxidase gene. In order to enable the glucose oxidase gene derived from Aspergillus niger to be expressed efficiently in Pichia pastoris, the inventors optimized the glucose oxidase gene without changing the amino acid sequence of the protein, considering various factors including but not limited to codon usage frequency, distribution of GC content in different gene regions, codon adaptation index CAI, etc., while eliminating the restriction enzyme cutting sites of EcoRI, Sail, Xbal, etc. for the convenience of subsequent genetic engineering operation.

[0031] According to an embodiment of the present application, the glucose oxidase gene has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence having at least 80% homology thereto. The glucose oxidase gene is obtained by optimizing the wild-type Aspergillus niger glucose oxidase gene (the encoded amino acid sequence is as shown in SEQ ID NO: 3), which can express the glucose oxidase gene efficiently in Pichia pastoris.

[0032]

[0033] According to an embodiment of the present application, the pyruvate kinase gene has a nucleotide sequence as shown in SEQ ID NO: 2 or a nucleotide sequence with at least 80% homology thereto.

[0034]

[0035] According to an embodiment of the present application, the first expression vector is selected from pGAPZ alpha A. The pGAPZ alpha A has a glyceraldehydes-3-phosphate dehydrogenase (GAP) promoter, which can express an exogenous protein that is not toxic to the cell while the cell is growing, compared to an inducible promoter, the required period of expression is short, and the production efficiency can be significantly improved. Moreover, it does not need to change the non-methanol carbon source during the fermentation process, thereby avoiding the cost and danger of storing and transporting a large amount of methanol.

[0036] According to an embodiment of the present application, the second expression vector is selected from pPIC9K. In this way, high copy of the BTS gene can be achieved, and the expression of the protein pyruvate kinase can be regulated by changing the carbon source.

[0037] According to an embodiment of the present application, the pPIC9K is pre-removed of the alpha-factor signal peptide sequence.

[0038] Kit

[0039] In another aspect of the present application, a kit is provided. According to an embodiment of the present application, the kit comprises: the Pichia pastoris engineering bacteria described above, and / or the kit comprises: a first expression vector carrying a glucose oxidase gene; and a second expression vector carrying a pyruvate kinase gene. The first expression vector and the second expression vector are introduced into Pichia pastoris to obtain the Pichia pastoris engineering bacteria described above, and high-yield glucose oxidase is achieved.

[0040] According to an embodiment of the present application, the substances contained in the kit can exist in the following three cases: 1) containing the Pichia pastoris engineering bacteria; 2) containing the first expression vector and the second expression vector; 3) containing the Pichia pastoris engineering bacteria, the first expression vector and the second expression vector.

[0041] According to an embodiment of the present application, the glucose oxidase gene is derived from Aspergillus niger.

[0042] According to an embodiment of the present application, the glucose oxidase gene is obtained by optimizing the wild-type Aspergillus niger glucose oxidase gene.

[0043] According to an embodiment of the present application, the pyruvate kinase gene is obtained by optimizing the wild-type Pichia pastoris pyruvate kinase gene.

[0044] According to an embodiment of the present application, the glucose oxidase gene has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence having at least 80% homology thereto.

[0045] According to an embodiment of the present application, the pyruvate kinase gene has a nucleotide sequence as shown in SEQ ID NO: 2 or a nucleotide sequence with at least 80% homology thereto.

[0046] According to an embodiment of the present application, the first expression vector is selected from pGAPZ alpha A.

[0047] According to an embodiment of the present application, the second expression vector is selected from pPIC9K.

[0048] According to an embodiment of the present application, the pPIC9K is pre-removed of an alpha-factor signal peptide sequence.

[0049] It should be noted that the features and advantages described above for the Pichia pastoris engineering bacteria also apply to the kit, and will not be repeated here.

[0050] Method for obtaining Pichia pastoris engineering bacteria

[0051] In another aspect of the present application, a method for obtaining the Pichia pastoris engineering bacteria described above is provided. According to an embodiment of the present application, the method comprises: introducing a first expression vector carrying a glucose oxidase gene into Pichia pastoris, culturing, and screening a strain with the highest glucose oxidase yield; introducing a second expression vector carrying a pyruvate kinase gene into the strain screened in the above step, culturing, and screening a strain with the highest glucose oxidase yield as the target Pichia pastoris engineering bacteria.

[0052] According to the method of the present application, the first expression vector carrying a glucose oxidase gene is introduced into Pichia pastoris, and the culture is performed in a plate medium containing different concentrations of antibiotics. The grown colonies are picked, and the glucose oxidase yield is determined. A strain with the highest glucose oxidase yield is screened as a primary screening strain. The second expression vector carrying a pyruvate kinase gene is introduced into the primary screening strain, and the culture is performed. A strain with the highest glucose oxidase yield is screened as the final target Pichia pastoris engineering bacteria.

[0053] It should be noted that the features and advantages described above for the Pichia pastoris engineering bacteria also apply to the method for obtaining the Pichia pastoris engineering bacteria, and will not be repeated here.

[0054] Method for improving glucose oxidase yield of Pichia pastoris

[0055] In yet another aspect of the present application, the present application provides a method for improving the yield of glucose oxidase of Pichia pastoris. According to an embodiment of the present application, the method comprises: making Pichia pastoris carry a glucose oxidase gene and a pyruvate kinase gene. As described above, the co-expression of the glucose oxidase gene and the pyruvate kinase gene helps Pichia pastoris to better secrete glucose oxidase, and the high enzyme activity lays a good foundation for the large-scale production of glucose oxidase.

[0056] According to an embodiment of the present application, the glucose oxidase gene is derived from Aspergillus niger.

[0057] According to an embodiment of the present application, the glucose oxidase gene is obtained by optimizing the wild-type Aspergillus niger glucose oxidase gene.

[0058] According to an embodiment of the present application, the pyruvate kinase gene is obtained by optimizing the wild-type Pichia pastoris pyruvate kinase gene.

[0059] According to an embodiment of the present application, the glucose oxidase gene has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence having at least 80% homology thereto.

[0060] According to an embodiment of the present application, the pyruvate kinase gene has a nucleotide sequence as shown in SEQ ID NO: 2 or a nucleotide sequence having at least 80% homology thereto.

[0061] According to an embodiment of the present application, the method comprises: introducing a first expression vector carrying a glucose oxidase gene into Pichia pastoris, culturing, screening the strain with the highest yield of glucose oxidase as the primary screening strain; introducing a second expression vector carrying a pyruvate kinase gene into the primary screening strain, culturing, screening the strain with the highest yield of glucose oxidase and collecting the glucose oxidase in the strain.

[0062] According to an embodiment of the present application, the first expression vector is selected from pGAPZαA.

[0063] According to an embodiment of the present application, the second expression vector is selected from pPIC9K.

[0064] According to an embodiment of the present application, the pPIC9K is pre-removed of the α-factor signal peptide sequence.

[0065] It should be noted that the features and advantages described above for the Pichia pastoris engineering bacteria also apply to the method for improving the yield of glucose oxidase of Pichia pastoris, which will not be described here.

[0066] Applications

[0067] In another aspect of the present application, the present application provides an application of the Pichia pastoris engineering bacteria or the kit in producing glucose oxidase. The Pichia pastoris engineering bacteria or the kit can be used to produce high-yield glucose oxidase with high enzyme activity, which lays a good foundation for large-scale production of glucose oxidase.

[0068] It should be noted that the features and advantages described above for the Pichia pastoris engineering bacteria and the kit also apply to the application, which will not be repeated here.

[0069] Method for producing glucose oxidase

[0070] In another aspect of the present application, the present application provides a method for producing glucose oxidase. According to an embodiment of the present application, the method comprises: culturing the Pichia pastoris engineering bacteria described above, collecting the bacteria in the obtained culture solution, and isolating glucose oxidase. Thus, the Pichia pastoris engineering bacteria described above can be used to produce high-yield glucose oxidase with high enzyme activity, which lays a good foundation for large-scale production of glucose oxidase.

[0071] It should be noted that the features and advantages described above for the Pichia pastoris engineering bacteria also apply to the method for producing glucose oxidase, which will not be repeated here.

[0072] The schemes of the present application will be explained below in combination with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained by purchase.

[0073] Example 1 Sequence optimization design of Aspergillus niger glucose oxidase gene

[0074] Taking the Aspergillus niger glucose oxidase gene GOX4 (the encoded amino acid sequence is shown as SEQ ID NO: 3) as a template, the glucose oxidase gene sequence was optimized by various strategies without changing its protein amino acid sequence, considering the codon usage frequency, the distribution of GC content in different gene regions, the codon adaptation index CAI, etc. At the same time, in order to facilitate subsequent genetic engineering operation, the restriction enzyme cutting sites such as EcoRI, Sail, Xbal were removed. The optimized GOX4 sequence is shown as SEQ ID NO: 1, which was synthesized by Beijing Qikexing Biological Technology Co., Ltd.

[0075] Example 2 Construction and screening of glucose oxidase recombinant Pichia pastoris strain

[0076] The recombinant vector pGAPZαA-GOX4opt was constructed using homologous recombination. This vector was chemically transformed into *E. coli* DH5α, and the transformed bacterial culture was plated on LB agar plates containing ampicillin (50 μg / mL) and incubated overnight at 37°C. This yielded the recombinant expression plasmid pGAPZαA-GOX4opt containing the GOX4opt gene. Figure 2 Verification was performed using double enzyme digestion.

[0077] The recombinant plasmid pGAPZαA-GOX4opt was digested with DarI and then transformed into Pichia pastoris X33 competent cells by electroporation. Gradient screening was performed on plates containing different concentrations of G418 antibiotic (0.25, 0.5, 2.0, 4.0, and 8.0 mg / ml, respectively). Combined with high-throughput screening, strains with high copies of the GOX4 gene that were positively correlated with glucose oxidase production were identified and named Pichia pastoris X33-pGAPZαA-GOX4opt.

[0078] Example 3 Construction of glucose oxidase secretion-enhancing strain

[0079] The pyruvate kinase gene BTS (name: PAS_chr2-1_0769, Gene ID: NC_012964.1, SEQ ID NO: 4) from Pichia pastoris was optimized according to the method in Example 1, that is, without changing its protein amino acid sequence, the frequency of codon usage was comprehensively considered. Figure 3 and Figure 4 Distribution of GC content in different gene regions Figure 5 and Figure 6 The glucose oxidase gene sequence was optimized using various strategies, including codon fitness index (CAI). To facilitate subsequent gene engineering operations, restriction endonuclease sites such as BamHI, DraI, EcoRI, NotI, and PshAI were removed. The optimized BTS gene sequence is shown in SEQ ID NO: 2 and was synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0080] The BTS memory clone was cloned into the intermediate vector pPIC9K (with the α-factor signal peptide sequence removed) using homologous recombination to obtain the recombinant expression plasmid pPIC9K-BTS-opt. Figure 7 The recombinant expression plasmid was transformed into the strain Pichia pastorisX33-pPGAPZαA-GOX4opt. After screening and identification, a recombinant strain Pichiapastoris X33-pPGAPαA-GOX4opt / pPIC9K-BTSopt with the highest glucose oxidase production was obtained.

[0081] Enzyme activity determination and protein electrophoresis of recombinant bacteria of Example 4

[0082] The fermentation strains were Aspergillus niger of Example 1, and the engineered Pichia pastoris strains constructed in Example 2 and Example 3. The fermentation conditions and methods were as follows:

[0083] Aspergillus niger and the engineered Pichia pastoris strain constructed in Example 2: single colonies were picked and cultured in YPD medium in a shake flask and subcultured twice to prepare primary seed liquid and secondary seed liquid. The culture conditions were 30°C, 180 r·min -1 , and 10% inoculation amount was inoculated into a 100 mL conical flask with a liquid volume of 20%, and the fermentation was carried out for 7 days. Glucose oxidase enzyme activity was detected every 24 h.

[0084] The engineered Pichia pastoris strain constructed in Example 3: single colonies were picked and inoculated into 10 mL liquid YPD medium, and cultured at 30°C, 180 r·min -1 , until OD600 was between 1.3 and 1.5. 5 mL of the bacterial liquid was transferred to 50 mL of BMGY medium and cultured for 24 h. 2 mL of the bacterial liquid was inoculated into a 100 mL conical flask with a liquid volume of 20 mL, and the fermentation medium was BMMY medium. The fermentation was carried out at 30°C, 180 r·min

[0085] Medium: YPD medium (1 L): glucose 20 g, peptone 20 g, yeast extract 10 g, and agar 20 g added to the slant medium. BMGY medium (1 L): tryptone 20 g, yeast extract 10 g, glycerol 10 mL, YNB 13.4 g, and 100 mM phosphate buffer (pH 6.0). The induction medium was BMMY medium (1 L): tryptone 20 g, yeast extract 10 g, methanol 5 mL, YNB 13.4 g, and 100 mM phosphate buffer (pH 6.0).

[0086] As can be seen from Figure 8 , the glucose oxidase enzyme activity of the Pichia pastoris X33-pPGAPZαA-GOX4opt strain was 6.93 U / mL, which was 10 times higher than that of the wild-type Aspergillus niger strain. The highest enzyme activity was produced by the Pichia pastoris X33-pPGAPαA-GOX4opt / pPIC9K-BTSopt strain, and the yield reached 11.12 U / mL, which was 1.6 times higher than that of the Pichia pastoris X33-pPGAPZαA-GOX4opt strain and 16 times higher than that of the wild-type Aspergillus niger.

[0087] The supernatant of the strain Pichia pastoris X33-pPGAPαA-GOX4opt / pPIC9K-BTSopt with the highest GOD enzyme activity was subjected to protein electrophoresis (SDS-PAGE), and a protein band with a molecular weight of about 70 kDa was obtained Figure 9 ), which was consistent with the theoretical value.

[0088] Although the embodiments of the present application have been shown and described above, it should be understood by those skilled in the art that the above embodiments are exemplary and cannot be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A Pichia pastoris engineering bacteria, characterized in that, The Pichia pastoris engineering bacteria carries a glucose oxidase gene and a pyruvate kinase gene; The nucleotide sequence of the glucose oxidase gene is shown in SEQ ID NO: 1; The nucleotide sequence of the pyruvate kinase gene is shown in SEQ ID NO:

2.

2. The engineered Pichia pastoris bacterium of claim 1, wherein, The glucose oxidase gene is derived from Aspergillus niger; The glucose oxidase gene is obtained by optimizing the wild-type Aspergillus niger glucose oxidase gene; The pyruvate kinase gene is obtained by optimizing the wild-type Pichia pastoris pyruvate kinase gene.

3. The engineered Pichia pastoris bacterium of claim 1, wherein, The glucose oxidase gene is introduced into Pichia pastoris by being connected to a first expression vector; and the pyruvate kinase gene is introduced into Pichia pastoris by being connected to a second expression vector; The first expression vector is selected from pGAPZαA; The second expression vector is selected from pPIC9K.

4. The Pichia pastoris engineered bacteria according to claim 3, characterized in that, The pPIC9K has been pre-removed of an α-factor signal peptide sequence.

5. A kit characterized in that, Comprise: The Pichia pastoris engineering bacteria according to any one of claims 1-4.

6. A method for increasing the production of glucose oxidase by Pichia pastoris, characterized by, Comprise: The Pichia pastoris carries a glucose oxidase gene and a pyruvate kinase gene; The nucleotide sequence of the glucose oxidase gene is shown in SEQ ID NO: 1; The nucleotide sequence of the pyruvate kinase gene is shown in SEQ ID NO:

2.

7. The method of claim 6, wherein, The glucose oxidase gene is derived from Aspergillus niger; The glucose oxidase gene is obtained by optimizing the wild-type Aspergillus niger glucose oxidase gene; The pyruvate kinase gene is obtained by optimizing the wild-type Pichia pastoris pyruvate kinase gene.

8. The method of claim 7, wherein, The method comprises: introducing a first expression vector carrying a glucose oxidase gene into Pichia pastoris, culturing, screening the strain with the highest glucose oxidase yield as the primary screening strain; introducing a second expression vector carrying a pyruvate kinase gene into the primary screening strain, culturing, screening the strain with the highest glucose oxidase yield and collecting the glucose oxidase in the strain.

9. The method of claim 8, wherein, The first expression vector is selected from pGAPZαA; The second expression vector is selected from pPIC9K.

10. The method of claim 9, wherein, The pPIC9K has been pre-removed of an α-factor signal peptide sequence.

11. The use of the Pichia pastoris engineering bacteria according to any one of claims 1-4 or the kit according to claim 5 in the production of glucose oxidase.

12. A method for producing glucose oxidase, characterized by, Comprise: Culturing the Pichia pastoris engineering bacteria according to any one of claims 1-4, collecting the bacterial cells in the obtained culture solution, and isolating glucose oxidase.

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