Construction and application of highly efficient nuclease-producing yeast

Abstract

The present invention provides the construction and application of an efficient nuclease-producing yeast engineering bacterium. Specifically, the present invention provides a variant nuclease derived from Bacillus nuclease, which has an amino acid sequence shown in SEQ ID NO:2.

Description

Technical Field

[0001] The invention relates to the fields of genetic engineering and protein engineering, and in particular to the construction and application of a yeast engineering bacterium for producing high-efficiency nuclease. Background Art

[0002] With the development of biomedicine, recombinant proteins are increasingly used in the pharmaceutical industry. They can not only be used directly for treatment, but are also key biological reagents and core raw materials for the development of innovative drugs. Using microorganisms to produce recombinant proteins is an economical and efficient way, but there are still many difficulties in the process of microbial production of recombinant proteins. For example: in the process of recombinant expression of target proteins in cells, nucleic acids are a common type of impurity molecules, which not only increase the viscosity of the lysate and affect the downstream purification chromatography; excessive nucleic acids will also lead to nucleic acid contamination of purified products. From the perspective of biopharmaceuticals, residual host DNA fragments of a certain length have a carcinogenic risk. Therefore, in the process of recombinant protein production, it is crucial to control the content of nucleic acids.

[0003] Commercial nucleases (such as Benzonase nucleases) derived from Bacillus fasciatus are important nucleic acid pollution control enzymes in the field of biochemistry and pharmaceutical preparations due to their high catalytic efficiency. At present, the recombinant expression of nucleases is mostly carried out in prokaryotic systems, but the intracellular recombinant expression of nucleases often has high toxicity to host cells, which makes it difficult for the host to grow or express in the form of inactive inclusion bodies. Some studies (such as CN113234704A) show that the inclusion body problem can be solved by adding soluble tags, but such operations will introduce label proteins, resulting in low specific enzyme activity of recombinant proteins. Other studies (such as US5173418A) attempt to use Escherichia coli for secretory expression, but due to the low secretion efficiency, the problem of difficult harvesting and purification of intracellular products cannot be effectively solved. In addition, the endotoxins introduced by Escherichia coli also increase the difficulty of subsequent protein purification operations. There are also studies (such as CN109852633A) showing that Bacillus can be used for secretory expression of nucleases, but due to the potential risk of contamination, it is not easy to scale up production. Therefore, there are great technical barriers to expressing nucleases using prokaryotic systems.

[0004] There is still a need for an improved nuclease production system that can overcome the problems of high host cell toxicity, low nuclease specific activity, and cumbersome subsequent purification steps of traditional expression systems, and provide efficient nuclease production. Summary of the invention

[0005] After long-term research, the inventors discovered and constructed a highly efficient nuclease-producing yeast strain. The yeast strain can overcome the toxicity of nuclease expression to host cells, produce highly efficient nucleases with high secretion efficiency, and is easy to purify, which has broad application prospects.

[0006] In one aspect, the present invention provides a variant nuclease derived from Bacillus nuclease, which has an amino acid sequence shown in SEQ ID NO:2.

[0007] In another aspect, provided herein is a polynucleotide encoding the variant nuclease.

[0008] In another aspect, provided herein is a yeast expression vector comprising a polynucleotide encoding a variant nuclease described herein.

[0009] In another aspect, the present invention provides a host cell selected from the group consisting of:

[0010] (a) a cell having a polynucleotide encoding the variant nuclease integrated into its chromosome; and

[0011] (b) A cell containing the yeast expression vector.

[0012] In another aspect, the present invention provides an engineered yeast comprising a polynucleotide encoding the variant nuclease.

[0013] In another aspect, the present invention provides a method for constructing an engineered yeast, comprising: introducing a polynucleotide encoding the variant nuclease into the yeast.

[0014] In another aspect, the present invention provides a method for preparing the variant nuclease, comprising:

[0015] (i) culturing the yeast engineered bacteria described herein under conditions suitable for expression, so that the yeast engineered bacteria express the variant nuclease; and,

[0016] (ii) purifying the variant nuclease from the culture of the engineered yeast.

[0017] In another aspect, provided herein is a use of the variant nuclease for controlling nucleic acid contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described below in conjunction with the accompanying drawings, wherein these drawings are only for illustrating the embodiments of the present invention rather than for limiting the scope of the present invention.

[0019] Figure 1 The electrophoresis gel image of the pJAG-s1-M-NucA plasmid according to the illustrative embodiment of the present invention after linearization by Sac I is shown. The lanes from left to right are: molecular ladder, plasmid pJAG-s1-M-NucA, and linearized fragment of plasmid pJAG-s1-M-NucA digested by Sac I.

[0020] Figure 2The SDS-PAGE electrophoresis diagram of the variant nuclease expressed in the illustrative embodiment of the present invention is shown. The lanes are respectively from left to right: molecular weight ladder, fermentation supernatant of recombinant yeast clone 1 expressing variant nuclease, fermentation supernatant of recombinant yeast clone 2 expressing variant nuclease, fermentation supernatant of recombinant yeast clone 3 expressing variant nuclease, fermentation supernatant of recombinant yeast clone 4 expressing variant nuclease, and fermentation supernatant of recombinant yeast clone 5 expressing variant nuclease.

[0021] Figure 3 The SDS-PAGE electrophoresis diagram of the purified variant nuclease protein expressed in the illustrative embodiment of the present invention is shown. The lanes from left to right are: molecular weight ladder, unpurified variant nuclease, purified variant nuclease.

[0022] Figure 4 Glycosylation analysis of variant nuclease proteins expressed according to an illustrative example of the present invention is shown. The lanes from left to right are: molecular weight ladder, purified variant nuclease, variant nuclease after PNGase F enzyme treatment, PNGase F enzyme. DETAILED DESCRIPTION

[0023] The meaning of scientific and technological terms in this application is consistent with the general understanding of those skilled in the art, unless otherwise specified. In this application, "one" or its combination with various quantifiers includes both singular and plural meanings, unless otherwise specified. In this application, for the same parameter or variable, when multiple numerical values, numerical ranges, or combinations thereof are given for description, it is equivalent to specifically revealing these numerical values, range ends, and numerical ranges formed by any combination thereof. In this application, any numerical value, whether or not it carries a modifier such as "about", covers an approximate range that can be understood by those skilled in the art, such as plus or minus 10%, 5%, etc. In this article, each "implementation method" refers to and covers the implementation methods of various methods and systems of this application equally. In this application, one or more technical features in any implementation method can be freely combined with one or more technical features in any one or more other implementation methods, and the implementation methods obtained thereby also belong to the content disclosed in this application.

[0024] Nucleases are a class of enzymes that use nucleic acids as substrates and catalyze the hydrolysis of phosphodiester bonds. Generally, nucleases can be divided into three categories: DNA enzymes, RNA enzymes, and nonspecific nucleases. Among them, nonspecific nucleases have low specificity and can nonspecifically degrade almost all forms of nucleic acids, including single-stranded, double-stranded, linear, circular, and supercoiled DNA and RNA, and have no requirements for the sequence of nucleic acids. Nonspecific nucleases are widely present in a variety of organisms such as viruses, bacteria, fungi, and animal cells. Nonspecific nucleases play an important role in many biochemical processes, including DNA replication and repair; DNA / RNA recombination, maturation, and editing; host resistance to foreign nucleic acid molecules, etc. Nonspecific nucleases are mainly used in industry to remove exogenous nucleic acids in biological products to improve safety. At the same time, nonspecific nucleases can also be used in the development of new biological disinfectants, which can lyse the nucleic acid substances of bacteria and viruses, thereby achieving the purpose of disinfection and sterilization. Compared with chemical disinfectants, nonspecific nucleases are an ideal green and environmentally friendly disinfectant that does not pollute the environment and has no toxic side effects on the body. At present, a variety of non-specific nucleases have been reported, such as bovine islet DNase I, Staphylococcus aureus nuclease and Serratia marcescen (also known as Bacillus) extracellular nuclease. So far, the most studied non-specific nuclease is Bacillus nuclease. Currently, Bacillus nuclease has commercial products launched, including, for example, the nuclease sold by Merck under the trade name "Benzonase", which is used as a tool in industrial production to remove nucleic acid contamination in biological products.

[0025] Currently, the recombinant expression of Bacillus nuclease is carried out in prokaryotic systems, but its intracellular recombinant expression is often highly toxic to host cells, making it difficult for the host to grow or expressing in the form of inactive inclusion bodies. People have tried to improve the prokaryotic expression system, but they usually introduce new problems such as low recombinant protein specific enzyme activity, difficulty in harvesting and purifying the product, or potential contamination risks.

[0026] After long-term research, the inventor discovered and constructed a highly efficient nuclease eukaryotic expression system through engineering modification of Bacillus nuclease and exploration and screening of expression hosts. In addition to the advantages of easy cultivation, fast reproduction, convenient genetic engineering operation and high-density fermentation, the host used in this expression system also has an efficient protein modification system and protein secretion system, which can not only overcome the high toxicity of nuclease expression to the host (such as a prokaryotic host), but also produce nuclease variants with high secretion efficiency. The produced nuclease variants have high specific enzyme activity and are easy to purify. This expression system can be suitable for large-scale production, and can effectively simplify the purification process, improve purification efficiency, and reduce the risk of contamination of intracellular impurities.

[0027] In one aspect, a nuclease is provided herein, which is a variant of the nuclease of Bacillus spp., also referred to herein as a "variant nuclease". Herein, the term "variant" refers to a "variant protein" meaning a protein that does not exist in nature, which is obtained by genetic engineering or random mutation, i.e., an engineered protein. The variant nuclease is a non-specific nuclease.

[0028] The amino acid sequence of the variant nuclease (SEQ ID NO: 2) is generated by amino acid mutation based on the amino acid sequence of Bacillus nuclease (SEQ ID NO: 1). Specifically, the amino acid mutation includes: replacing asparagine at position 22 of the original amino acid sequence with alanine (N22A), and replacing asparagine at position 34 of the original amino acid sequence with alanine (N34A), and the amino acid residue position numbering is based on the amino acid sequence of Bacillus nuclease (SEQ ID NO: 1). In some embodiments, the variant nuclease has the amino acid sequence shown in SEQ ID NO: 2.

[0029] Amino acid sequence of Bacillus nuclease:

[0030] MADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGANAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAW ARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN(SEQ ID NO:1).

[0031] Amino acid sequence of variant nuclease:

[0032] MADTLESIDNCAVGCPTGGSS A VSIVRHAYTLN ANSTTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGANAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAWARLEDQERKLIDRADIS SEQ ID NO:2).

[0033] It should be understood that variant nucleases can be obtained based on the amino acid sequence of Bacillus nuclease by genetic modification methods known in the art. In some embodiments, variant nucleases can be obtained by site-directed mutagenesis of Bacillus nuclease to introduce desired amino acid substitutions. In an exemplary embodiment, site-directed mutagenesis may include: introducing corresponding codons of desired amino acid substitutions (e.g., including N22A and N34A) into the coding polynucleotide of Bacillus nuclease by methods such as polymerase chain reaction (PCR) to obtain a modified coding polynucleotide, and expressing the modified coding polynucleotide to obtain. In other embodiments, variant nucleases can also be obtained by artificially synthesizing a variant nuclease coding polynucleotide containing a desired amino acid substitution codon, and expressing the variant nuclease coding polynucleotide.

[0034] In some embodiments, the variant nuclease is obtained by expressing in yeast. In some embodiments, the variant nuclease is obtained by expressing in a yeast selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, and any combination thereof.

[0035] In some embodiments, the variant nuclease has a 6 U / mg, at least 2×10 6 U / mg, at least 2.2×10 6 U / mg, at least 2.5×10 6 U / mg, or at least 2.8×10 6 U / mg specific enzyme activity. For the "specific enzyme activity" described herein, the amount of enzyme used to change the ΔA260 absorbance by 1.0 in 2.625 mL of a reaction system within 30 minutes at 37°C and pH 8.0 is defined as one activity unit U. For example, salmon sperm DNA can be used as a substrate.

[0036] In some embodiments, the specific enzymatic activity of the variant nuclease is increased by at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, or at least 180% compared to the specific enzymatic activity of a commercially available Benzonase nuclease (e.g., a Benzonase nuclease commercially available from Merck).

[0037] In another aspect, provided herein is a polynucleotide encoding the variant nuclease.

[0038] In another aspect, provided herein is a yeast expression vector comprising a polynucleotide encoding a variant nuclease described herein.

[0039] In some embodiments, the yeast expression vector includes, but is not limited to, a Saccharomyces cerevisiae expression vector, a Pichia pastoris expression vector, and a Hansenula polymorpha expression vector. In some embodiments, the yeast expression vector is a Pichia pastoris expression vector.

[0040] In some embodiments, the yeast expression vector includes but is not limited to: pJAG-s1 vector, pPICZ vector, pPICZα vector, pPIC9k vector, pPIC9k-His vector, pGAPZA vector, pGAPZαA vector, pGAPZαA vector, pAO815 vector, pPIC9 vector, pPIC6A vector, pPIC6αA vector, pAUR123 vector, pRS303TEF vector, pRS304 vector, pRS305 vector, pRS306 vector, pY13TEF vector, pY14TEF vector, pY15TEF vector, pY16TEF vector and pYES2 vector, etc. In some exemplary embodiments, the yeast expression vector is a pJAG-s1 vector containing a polynucleotide encoding the variant nuclease.

[0041] In some embodiments, the yeast expression vector is obtained by cloning the encoding polynucleotide of the variant nuclease into a blank vector. In some embodiments, the encoding polynucleotide of the variant nuclease is integrated downstream of the promoter. In some embodiments, examples of suitable promoters include but are not limited to: AOX1 promoter, GAP promoter, ADH promoter, etc. In some embodiments, the yeast expression vector comprises an α-factor signal peptide encoding sequence.

[0042] In another aspect, the present invention provides a host cell selected from the group consisting of:

[0043] (a) a cell having a polynucleotide encoding the variant nuclease integrated into its chromosome; and

[0044] (b) A cell containing the yeast expression vector.

[0045] In some embodiments, the host cell is selected from the group consisting of: prokaryotic cells, eukaryotic cells, and combinations thereof. In some embodiments, the host cell is an Escherichia coli cell. In other embodiments, the host cell is a yeast cell. In some embodiments, the yeast cell includes, but is not limited to, a Saccharomyces cerevisiae cell, a Pichia pastoris cell, and a Hansenula polymorpha cell.

[0046] In another aspect, the present invention provides an engineered yeast comprising a polynucleotide encoding the variant nuclease.

[0047] In some embodiments, the yeast engineered bacteria expresses the variant nuclease. In some embodiments, the yeast engineered bacteria expresses and secretes the variant nuclease.

[0048] In another aspect, the present invention provides a method for constructing an engineered yeast, comprising: introducing a polynucleotide encoding the variant nuclease into the yeast.

[0049] In some embodiments, the yeast may include but is not limited to: Saccharomyces cerevisiae, Pichia pastoris and Hansenula polymorpha. In some embodiments, the yeast is Pichia pastoris BG10.

[0050] In some embodiments, the method for constructing an engineered yeast may include: transfecting, transforming or transducing the polynucleotide encoding the variant nuclease into the yeast.

[0051] In another aspect, the present invention provides a method for preparing the variant nuclease, comprising:

[0052] (i) culturing the yeast engineered bacteria described herein under conditions suitable for expression, so that the yeast engineered bacteria express the variant nuclease; and,

[0053] (ii) purifying the variant nuclease from the culture of the engineered yeast.

[0054] In some embodiments, the variant nuclease is transcribed and expressed under the control of the AOX1 promoter. In some embodiments, the variant nuclease is secreted and expressed in the culture medium under the action of the α-factor signal peptide. In some embodiments, the variant nuclease is expressed under methanol induction.

[0055] In some embodiments, the purification in step (ii) comprises collecting the supernatant from the culture of the engineered yeast.

[0056] In some embodiments, the purification comprises performing one or more operations of filtration, ultrafiltration, diafiltration, and ion exchange on the supernatant collected in step (ii).

[0057] In some embodiments, step (ii) of the method for preparing the variant nuclease does not include one or more operations of collecting bacteria, washing bacteria, cell lysis, and clarifying. In some embodiments, step (ii) of the method for preparing the variant nuclease does not include any of the operations of collecting bacteria, washing bacteria, cell lysis, and clarifying. The term "clarification" as used herein means a solid-liquid separation operation, for example, an operation to separate solids (including cells and cell debris, etc.) from liquids (e.g., supernatant) in a cell culture.

[0058] Since the yeast engineered bacteria of the present invention can express and secrete the variant nuclease into the culture medium, the purification of the variant nuclease only requires three operations: collecting the culture supernatant, fine purification, and ultrafiltration / diafiltration, and the purity can reach more than 90%. In comparison, the purification of nucleases using a prokaryotic expression system requires at least seven operations: collecting bacteria, washing bacteria, cell lysis, clarification, coarse separation, fine purification, and ultrafiltration / diafiltration. The use of the yeast engineered bacteria of the present application to prepare variant nucleases greatly reduces the steps of downstream purification operations.

[0059] In another aspect, provided herein is a use of the variant nuclease for controlling nucleic acid contamination.

[0060] In some embodiments, the nucleic acid contamination is nucleic acid contamination in a product or an environment.

[0061] Example

[0062] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make appropriate modifications and changes to the present invention, and these modifications and changes are within the scope of the present invention.

[0063] 1. Construction of plasmids expressing variant nuclease genes and construction of recombinant yeast expressing variant nucleases

[0064] Obtain the non-specific nuclease sequence NucA of Bacillus fasciculatus and perform site-directed mutagenesis to produce the sequence M-NucA (the original NucA sequence is shown in SEQ ID NO: 1, and the amino acid sequence after mutation is shown in SEQ ID NO: 2). The mutated M-NucA gene is cloned into the pJAG-s1 vector and transformed into Escherichia coli. The recombinant Escherichia coli is cultured in a medium containing LB + 100 μg / mL Amp to amplify the pJAG-s1-M-NucA plasmid. Subsequently, the plasmid is linearized using the SacI endonuclease.

[0065] Figure 1The electrophoresis gel image shows the linearized fragment of pJAG–s1-M-NucA after digestion with Sac I. The lanes from left to right are: molecular ladder, plasmid pJAG–s1-M-NucA, and linearized fragment of plasmid pJAG–s1-M-NucA digested with Sac I.

[0066] After purification, the linearized plasmid was electrotransformed into Pichia pastoris BG10, and then the positive recombinant yeast BG10 / pJAG-s1-M-NucA was screened using YPD+1.5g / LG418 resistance plates.

[0067] 2. Yeast Inducible Expression of Variant Nuclease

[0068] The positive recombinant yeast BG10 / pJAG-s1-M-NucA single clone was inoculated into YPD medium and cultured at 30°C, 250 rpm for 24 h. 600 =0.005 inoculation ratio was inoculated into BMGY medium until OD 600 =2~6. According to OD 600 =1 inoculation ratio was inoculated into BMMY medium and cultured at 30°C and 250rpm. Every 24h, 0.8% methanol was added to the culture system to induce the expression of the target protein. After 96h of induction, the culture was terminated and the supernatant was collected.

[0069] The variant nuclease is expressed under the control of the promoter AOX1 and secreted into the culture medium under the action of the α-factor signal peptide.

[0070] Figure 2 The SDS-PAGE electrophoresis diagram of the expressed variant nuclease is shown, and the supernatant protein size is detected to be 30Kda. The lanes from left to right are: molecular weight ladder, fermentation supernatant of recombinant yeast clone 1 expressing variant nuclease, fermentation supernatant of recombinant yeast clone 2 expressing variant nuclease, fermentation supernatant of recombinant yeast clone 3 expressing variant nuclease, fermentation supernatant of recombinant yeast clone 4 expressing variant nuclease, and fermentation supernatant of recombinant yeast clone 5 expressing variant nuclease.

[0071] 3. Purification of variant nuclease samples

[0072] The fermentation supernatant was collected and filtered using a 0.22 μM filter membrane. The fermentation supernatant was replaced with 50 mM Tris-HCl, pH = 8.2 using a 10 KDa ultrafiltration tube. The commercial anion exchange filler was then treated, and after balancing three times with 50 mM Tris-HCl, pH 8.2, the protein sample was added, and after incubation at room temperature for 60 minutes, the supernatant protein was removed by centrifugation. 50 mM Tris-HCl, pH 8.2 was added for washing three times, 50 mM Tris-HCl, 25 mM NaCl pH 8.2 was added for washing three times, and 50 mMTris-HCl, 100 mM NaCl was used for elution. The eluted protein was subjected to SDS-PAGE detection. According to the grayscale analysis, the protein purity can reach 92% ( Figure 3 ).

[0073] Figure 3 The SDS-PAGE electrophoresis of the expressed variant nuclease protein after purification is shown. The lanes from left to right are: molecular weight ladder, unpurified variant nuclease, and purified variant nuclease.

[0074] Compared with the traditional expression of nucleases using prokaryotic expression systems (such as Escherichia coli), the subsequent purification steps are reduced from the traditional 7 steps of collecting bacteria, washing bacteria, cell lysis, clarification, crude separation, fine purification, and ultrafiltration / diafiltration to 3 steps of collecting supernatant, fine purification, and ultrafiltration / diafiltration, greatly reducing the downstream purification operation steps.

[0075] 4. Analysis of the activity of variant nucleases

[0076] The amount of enzyme used to change the ΔA260 absorbance by 1.0 in 2.625 mL of reaction system at 37°C and pH 8.0 within 30 minutes is defined as one activity unit U. It is reported that nuclease activity is higher at a final concentration of 1 to 2 mmol / L Mg 2+ The enzyme activity is highest in the presence of 5% paraformaldehyde and pH 8.0-9.2. With salmon sperm DNA as substrate, the reaction system is: 2.5 mL 0.1 mg / mL salmon sperm DNA, 1 μL test sample, 124 μL enzyme digestion buffer (50 mM Tris HCl, pH 8.0, 0.1 mg / mL BSA, 1 mM MgCl2). Mix well and incubate at 37°C for 20 minutes. After the reaction is completed, add 35 μL 20% trifluoroacetic acid (final concentration 0.27%) and mix well to terminate the reaction. Centrifuge at 13000g for 5 minutes, dilute the supernatant 5 times with sterile ddH2O, and measure the light absorption value at 260 nm.

[0077] The specific activity of the variant nuclease was found to be 2.9×10 6 U / mg. Compared with the commercially available Benzonase nuclease (average of about 1×106 U / mg), the enzyme activity of the variant nuclease of the present invention is increased by 190%.

[0078] 5. Analysis of nuclease glycosylation by PNGase F

[0079] The target protein was deglycosylated under denaturing conditions. 5% SDS and 1M DTT were added to the target protein, and 0.5M sodium phosphate buffer (pH 7.5), 10% Triton X-100, and PNGase F were added after boiling at 100°C for denaturation. The mixture was incubated at 37°C for 1 h, and then the sample was analyzed by SDS-PAGE ( Figure 4 ), the results show that the variant nuclease of the present invention has a single N-glycosylation site.

[0080] The results showed that the secretory expression of variant nucleases by the yeast engineered bacteria provided by the present invention solved the toxicity problem of nuclease expression to host cells, and the produced variant nucleases had the advantages of single glycosylation modification, high specific enzyme activity, and easy purification, and had broad application prospects.

Claims

1. A variant nuclease derived from Bacillus nuclease, having an amino acid sequence as shown in SEQ ID NO:

2.

2. A polynucleotide encoding the variant nuclease according to claim 1.

3. A yeast expression vector comprising the polynucleotide according to claim 2. The yeast expression vector according to claim 3 , which contains an α-factor signal peptide coding sequence.

5. A host cell selected from: (a) a cell having the polynucleotide according to claim 2 integrated into its chromosome; and (b) A cell containing the yeast expression vector according to claim 3. An engineered yeast comprising the polynucleotide according to claim 2.

7. The yeast engineering bacteria according to claim 6, wherein The engineered yeast expresses and secretes the variant nuclease according to claim 1.

8. A method for constructing a yeast engineering bacterium, comprising: The polynucleotide according to claim 2 is introduced into yeast.

9. The method of claim 8, wherein: The yeast is selected from the group consisting of: Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, and combinations thereof.

10. A method for preparing the variant nuclease according to claim 1, comprising: (i) culturing the yeast engineered bacteria according to claim 6 under conditions suitable for expression, so that it expresses the variant nuclease; and, (ii) purifying the variant nuclease from the culture of the engineered yeast.

11. The method of claim 10, wherein: The purification comprises performing one or more operations of filtration, ultrafiltration, diafiltration, and ion exchange on the supernatant collected in step (ii).

12. The method of claim 11, wherein: Step (ii) does not include one or more operations of collecting bacteria, washing bacteria, cell lysis, and clarifying.

13. Use of the variant nuclease according to claim 1 for controlling nucleic acid contamination.

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