A brewer's yeast engineering strain and its construction method and application

Abstract

The invention relates to the field of genetic engineering technology, and discloses a saccharomyces cerevisiae engineering strain and a construction method and application thereof. The saccharomyces cerevisiae engineering strain KY001 has a deposit number of CCTCC NO: M2024707. The saccharomyces cerevisiae engineering strain improves its reducing ability to sodium selenite by regulating the tolerance module and antioxidant module of saccharomyces cerevisiae. The tolerance module includes cysteine ​​lyase, sulfite transporter, monothiol glutaredoxin and flocculation gene of the saccharomyces cerevisiae strain. The antioxidant module includes superoxide dismutase and glutathione peroxidase. The invention not only improves the tolerance of saccharomyces cerevisiae to sodium selenite through the up-regulated expression of the tolerance module and the antioxidant module, so that the saccharomyces cerevisiae can survive and grow in high-concentration sodium selenite, but also enhances the antioxidant capacity of saccharomyces cerevisiae, which is beneficial to improving the survival rate of yeast cells, thereby improving the yield of biological nano-selenium.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to an engineering strain of brewer's yeast and a construction method and application thereof. Background Art

[0002] Selenium is an essential trace element for the human body and is vital to human and livestock health. Among the three forms of inorganic selenium (sodium selenite), organic selenium (selenoamino acids) and nanoparticle selenium, nanoparticle selenium has lower toxicity, higher bioavailability, lower excretion, and better biological activity than inorganic and organic selenium. The results of many studies have shown that selenium nanoparticles have significant positive effects on the physiological activities of plants and animals, such as promoting plant growth, improving the quality of crop products, promoting stress tolerance, and enhancing the antioxidant capacity and immunomodulatory capacity of poultry.

[0003] At present, the existing nano-selenium synthesis technology has the problems of high cost, low efficiency and product safety. Its synthesis mode has three methods: physical, chemical and biological. The chemical synthesis method requires chemical reducing agents and stabilizers, and these preparations may be toxic, and the prepared nano-selenium has a lower biological uptake rate and utilization. The biological synthesis method can achieve low-cost and harmless nano-selenium production. The biological synthesis method is mainly divided into multiple synthesis methods such as bacteria, fungi, algae, plant extracts and protein molecules. Compared with these two methods of plant extracts and protein molecules, the synthesis that relies on bacteria or fungi has higher controllability, high efficiency and relatively simple production process. However, there is a problem of safety and production efficiency in the nano-selenium synthesized by biological method, so the present invention improves it as follows:

[0004] 1) Ensure product safety: The safety of the product is related to the production strain. Although some existing technologies have developed efficient biological nano-selenium synthesis strains, the chassis microorganisms do not meet my country's safety regulations for adding microbial agents to food, feed, and fertilizer. Saccharomyces cerevisiae is a safe strain that is allowed to be used as a feed additive and fertilizer additive, and yeast selenium is also a form of feed additive that is allowed to be used. Therefore, the present invention selects Saccharomyces cerevisiae for genetic engineering modification to produce biological nano-selenium.

[0005] 2) Production efficiency: The efficiency of brewer's yeast in synthesizing bio-nanoselenium is low. The purpose of the present invention is to overcome this shortcoming, improve the production efficiency and product safety of its bio-nanoselenium by genetically engineering brewer's yeast, and realize a low-cost, high-efficiency and safe bio-nanoselenium production method to meet the needs of industrial-scale production. Summary of the invention

[0006] In view of the problems in the related art, the present invention proposes an engineered strain of Saccharomyces cerevisiae and a construction method and application thereof to overcome the above-mentioned technical problems existing in the existing related art.

[0007] To this end, the specific technical solution adopted by the present invention is as follows:

[0008] According to one aspect of the present invention, a saccharomyces cerevisiae engineering strain is provided, the saccharomyces cerevisiae engineering strain is the saccharomyces cerevisiae engineering strain KY001, and its deposit number is CCTCC NO: M2024707; the chassis microorganism used in the saccharomyces cerevisiae engineering strain is saccharomyces cerevisiae, which is a eukaryotic microorganism most commonly used for fermenting bread and brewing wine, and the saccharomyces cerevisiae engineering strain is optimized by genetic engineering methods, and its reducing ability to sodium selenite is significantly improved by regulating the tolerance module and antioxidant module of saccharomyces cerevisiae;

[0009] The tolerance module includes cysteine ​​lyase (CYS3), sulfite transporter (SSU1), monothiol glutaredoxin (GRX5) and flocculation gene (FLO1) of Saccharomyces cerevisiae strain; the antioxidant module includes superoxide dismutase (SOD1) and glutathione peroxidase (GPX2).

[0010] The improvement of tolerance module and antioxidant module is achieved by promoter engineering. The expression frames of tolerance module and antioxidant module are inserted into CYS3, X-2 and X-3 sites respectively by CRISPR / Cas9 gene editing technology.

[0011] According to another aspect of the present invention, a method for constructing an engineering strain of Saccharomyces cerevisiae is provided, wherein a constitutive promoter-driven expression frame is inserted into the genome of Saccharomyces cerevisiae using a pML104 plasmid expressing a CRISPR / Cas9 system to enhance the expression of a tolerance module and an antioxidant module, thereby obtaining an engineering strain of Saccharomyces cerevisiae that efficiently synthesizes bio-nanoselenium, and specifically comprises the following steps:

[0012] Design the sgRNA sequence targeting the target gene CYS3 and connect it with the pML104 linearized plasmid to obtain the pML104-CYS3 editing plasmid;

[0013] Build Repair Template RE CYS3 , which contains the upstream homology arm of the gene, the replacement sequence (P TEF1 -SSU1-P TDH3 -GRX5) and downstream homology arms, and pML104-CYS3 and repair template RE CYS3 transfection into Saccharomyces cerevisiae strain SC1;

[0014] PCR screening was used to replace CYS3 with P TEF1 -SSU1-P TDH3 -GRX5 positive clones, mutant strain SC2 was obtained after eliminating the editing plasmid pML104-CYS3;

[0015] Design the sgRNA sequence targeting the target site X-2 and connect it with the pML104 linearized plasmid to obtain the pML104-X-2 plasmid;

[0016] Build Repair Template RE X-2 , which contains the upstream of X-2 site, the replacement sequence (P PGK1 -FLO1) and X-2 sites, and pML104-X-2 and the repair template RE X-2 transfected into Saccharomyces cerevisiae strain SC2;

[0017] PCR technology was used to screen for P insertion at X-2 site. PGK1 -FLO1 positive clones, mutant strain SC3 was obtained after eliminating the editing plasmid pML104-X-2;

[0018] Design the sgRNA sequence targeting the target site X-3 and connect it with the pML104 linearized plasmid to obtain the pML104-X-3 plasmid;

[0019] Build Repair Template RE X-3 , which contains the X-3 site upstream, the replacement sequence (P TEF1 -SOD1-P PGK1 -GPX2) and X-3 site downstream. X-3 transfected into Saccharomyces cerevisiae strain SC3;

[0020] PCR technology was used to screen for P insertion at X-3 site. TEF1 -SOD1-P PGK1 -GPX2 positive clones, mutant strain KY001 was obtained after eliminating the editing plasmid pML104-X-3;

[0021] Among them, the gene number of gene CYS3 is YAL012W; the gene number of gene SSU1 is YPL092W; the gene number of gene GRX5 is YPL059W; the gene number of gene FLO1 is YAR050W; the gene number of gene SOD1 is YJR104C; and the gene number of gene GPX2 is YBR244W.

[0022] According to another aspect of the present invention, a method for preparing bio-nanoselenium by an engineered strain of Saccharomyces cerevisiae is provided, wherein the high cell density fermentation technology is used to achieve efficient synthesis of bio-nanoselenium, and the method specifically comprises the following steps:

[0023] The engineered strain of Saccharomyces cerevisiae KY001 was transferred into the seed fermentation broth and cultured at 30°C and 200 rpm for 24 h;

[0024] Transfer 10% of the seed solution (v:v) to a 5L fermenter and grow the strain to the end of the logarithmic phase at 30°C, an initial pH of 6.0, and 30% dissolved oxygen. Add sodium selenite at the preset flow rate and incubate;

[0025] The fermented cells and the supernatant are separated. The supernatant is centrifuged at high speed, and then collected and washed to obtain bio-nanoselenium.

[0026] The shaking culture temperature is 30°C, the shaking time is 24h, and the rotation speed is 200rpm.

[0027] The volume ratio of the seed solution to the total solution is 10:100, the volume of the fermenter is 5 L, the culture temperature in the fermenter is 30° C., the culture time is 23 h, the initial pH is 6.0, and the dissolved oxygen is 30%.

[0028] The preset flow rate of adding sodium selenite is 2 ml·h -1 , the final concentration was 4.4 g / L, and the static culture time was 120 h.

[0029] Compared with the prior art, the present invention provides an engineered strain of Saccharomyces cerevisiae and a method for constructing the same, which has the following features:

[0030] Beneficial effects:

[0031] (1) The present invention utilizes CRISPR / Cas9 gene editing technology to knock out cysteine ​​cleavage enzyme (CYS3) and uses a strong constitutive promoter P TEF1 driven by the sulfite transporter (SSU1) and the strong constitutive promoter P TEF1 The monothiol glutaredoxin (GRX5) expression cassette is driven by a strong constitutive promoter P PGK1 The expression cassette of the flocculation gene (FLO1) driven by the saccharomyces cerevisiae was used to improve the tolerance of Saccharomyces cerevisiae to sodium selenite. On this basis, a strong constitutive promoter P was inserted into the X-3 site. TEF1 Driven by superoxide dismutase (SOD1) and a strong constitutive promoter P PGK1 The expression box of glutathione peroxidase (GPX2) driven by the cytochrome P404 gene was used to enhance the ability of Saccharomyces cerevisiae to resist reactive oxygen species. Finally, high cell density culture technology was used to increase cell biomass and improve the yield of selenium nanoparticles biosynthesized by Saccharomyces cerevisiae.

[0032] (2) The present invention is the first to carry out engineering modification from two aspects: improving the tolerance of brewer's yeast to sodium selenite and improving the ability of brewer's yeast to resist oxidative stress, thereby improving the conversion efficiency of brewer's yeast in synthesizing biological nano-selenium.

[0033] (3) The present invention not only improves the yield of bio-nanoselenium, reaching a conversion level of 4.11 g / L (bioconversion rate reaches 92.9%), but also obtains high-purity, small-particle bio-nanoselenium (average particle size is 79.3 nm, 93.9% of the particle size is less than 100 nm) by optimizing the extraction and purification methods. It has important practical value in the industrial-scale production of bio-nanoselenium.

[0034] (4) The present invention not only improves the tolerance of Saccharomyces cerevisiae to sodium selenite by up-regulating the expression of the sodium selenite tolerance module and the antioxidant module, so that it can survive and grow in a high concentration of sodium selenite environment, but also enhances the antioxidant capacity of Saccharomyces cerevisiae, which is beneficial to improving the survival rate of yeast cells, thereby increasing the production of bio-nanoselenium.

[0035] Strain deposit information:

[0036] The brewer's yeast engineered strain provided by the present invention is classified as Saccharomyces cerevisiae KY001, Saccharomyces species KY001, deposited in China Center for Type Culture Collection (CCTCC) on April 18, 2024, with a deposit number of CCTCC NO: M 2024707, and a deposit address: Wuhan University, Wuhan, China, postal code: 430072; telephone: 027-68754052. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0038] Figure 1 is a growth curve of the strain in the embodiment of the present invention in YPD medium containing 1 mM sodium selenite;

[0039] Figure 2 is the conversion rate of the strain in the embodiment of the present invention to 10 mM sodium selenite in YPD medium;

[0040] Figure 3 is a scanning electron microscope picture of biological nanoselenium in an embodiment of the present invention;

[0041] Figure 4 is a particle size distribution diagram of bio-nanoselenium statistically analyzed by ImageJ in an embodiment of the present invention;

[0042] Figure 5 is a data graph of sodium selenite reduction rate of the engineered strain KY001 in two fermentation modes in an embodiment of the present invention;

[0043] Figure 6This is a data graph showing the effect of incubation time on the particle size of biological nano-selenium in an embodiment of the present invention. DETAILED DESCRIPTION

[0044] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] The strains, genes and primers used in the present invention are shown in Table 1 below:

[0046] Table 1 Strains, genes and primers

[0047]

[0048]

[0049] Example 1: Construction of an engineered strain for upregulating substrate tolerance module and antioxidant module

[0050] 1. Construction of editing plasmid targeting target gene

[0051] Prepare the required culture media and reagents: Escherichia coli is cultured in LB medium, which contains 5g / L yeast extract, 10g / L tryptone, and 10g / L sodium chloride. LB medium is sterilized by high pressure steam at 121℃ for 20min. Unless otherwise specified, Saccharomyces cerevisiae usually uses YPD medium, which contains 10g / L yeast extract, 20g / L peptone, and 20g / L glucose. SC-URA medium for screening positive clones contains 1.7g / L yeast basic nitrogen source, 20g / L glucose, 5g / L ammonium sulfate, and 0.1g / L 16 kinds of amino acid mixed solution. YPD medium and SC-URA medium are sterilized by high pressure steam at 105℃ for 30min. Yeast cell transfection buffer is prepared and stored at -20℃ for future use. Its components are 200mM lithium acetate, 100mM dithiothreitol, and 40% (w / v) polyethylene glycol. SC+FOA medium was used to eliminate the editing plasmid, and its composition was 1.45 g / L yeast basal nitrogen source, 20 g / L glucose, 5 g / L ammonium sulfate, 2 g / L total amino acid mixed solution and 1 g / L 5-fluoroorotic acid. YPD medium, SC-URA medium and SC+FOA medium were sterilized by high pressure steam at 105°C for 30 min.

[0052] Plasmid construction: Use the webpage http: / / chopchop.cbu.uib.no / system to design sgRNA oligonucleotide sequences targeting CYS3, X-2, and X-3 sites. The positive strand of sgRNA adds a GATC sticky end at the 5' end to facilitate ligation with the restriction plasmid. Annealing is performed using a temperature controller to hybridize the forward and reverse sgRNA oligonucleotide sequences to form a double-stranded nucleotide sequence. Digest with Swal enzyme at 25°C for 12h, then digest with BcII enzyme at 50°C for 4h, and then use a purification kit to obtain the pML104 linearized plasmid. Incubate the pML104 linearized plasmid, sgRNA oligonucleotide sequence, T4 ligase, and Buffer at 16°C overnight, and obtain the complete plasmids pML104-CYS3, pML104-X-2, and pML104-X-3 by enzyme ligation.

[0053] Plasmid transformation: Add the plasmid to the competent cell E.coli Turbo solution, and use the heat shock method (42°C, 90s) to chemically transfect the plasmid into E.coli. Add 500μl sterile LB solution, incubate at 37°C for 1.5h, spread the bacterial solution on the LB plate containing 100μg / ml ampicillin, and then incubate at 37°C for 20h, pick the growing single clone, and use PCR technology to verify and select the strain with successful plasmid construction. After expansion culture, use the plasmid extraction kit to extract pML104-CYS3, pML104-X-2 and pML104-X-3 plasmids for standby use.

[0054] 2. Construction of an engineered strain of Saccharomyces cerevisiae with enhanced sodium selenite tolerance

[0055] Preparation of double-stranded DNA repair template: Design a template containing 500 bp upstream homology arms and replacement sequence (P TEF1 -SSU1-P TDH3 -GRX5, SEQ ID NO: 1), homologous recombination repair template RE of downstream 500 bp homology arm CYS3 . Using PCR technology, we obtained upstream homology arms and replacement sequences (P TEF1 -SSU1-P TDH3 -GRX5) and the downstream homology arm DNA fragment. The above three DNA sequences were connected into a DNA sequence repair template using Gibson. After amplification using PCR technology, a pure double-stranded DNA repair template RE was obtained according to the method of the purification kit. CYS3 spare.

[0056] Editing the Saccharomyces cerevisiae genome: Activate the Saccharomyces cerevisiae strain SC1 and culture the strain to the late logarithmic phase. Pipette 125 μl of bacterial solution and use a centrifuge (12000 rpm, 30 s) to collect the bacterial sediment. Use 25 μl of yeast cell transfection buffer to fully resuspend the bacteria, then add salmon sperm DNA, editing plasmid pML104-CYS3 and repair template RE CYS3 After 30 min in a 42°C water bath, the cells were plated on SC-URA yeast solid medium without uracil. After 3 days of culture at 30°C, PCR was used to screen whether the gene CYS3 was successfully replaced by P TEF1 -SSU1-P TDH3 - GRX5 engineered strains.

[0057] Elimination of the editing plasmid: The positive clones were plated on SC+FOA medium solid plates. Single clones were selected and plated on YPD medium and SC-URA respectively to screen out the engineered strain SC2 that eliminated the plasmid pML104-CYS3.

[0058] Construction of engineered strain SC2: The editing plasmid pML104-X-2 has been constructed in the step of "Constructing an editing plasmid targeting the target gene". According to the aforementioned method of "Preparing double-stranded DNA repair template", PCR technology was used to amplify the upstream of the X-2 site and the replacement sequence (P PGK1 -FLO1, SEQ ID NO: 2), and a DNA fragment downstream of the X-2 site. The three fragments were connected as a repair template RE using Gibson. X-2 According to the aforementioned "Editing the Saccharomyces cerevisiae genome" method, the editing plasmid pML104-X-2 and the repair template RE X-2 Transfected into Saccharomyces cerevisiae. PCR was used to screen for insertion of P at X-2 site. PGK1 -FLO1 positive clones. According to the aforementioned "elimination of editing plasmid" method, after the bacterial solution was spread on the SC+FOA medium solid plate to eliminate the editing plasmid, the engineered strain SC2 without plasmid was obtained.

[0059] 3. Construction of an engineered strain of Saccharomyces cerevisiae with enhanced sodium selenite reduction ability

[0060] Construction of engineering strain KY001: The editing plasmid pML104-X-3 has been constructed in the step of "Constructing an editing plasmid targeting the target gene". According to the above-mentioned "Preparation of double-stranded DNA repair template" method, PCR technology was used to amplify the upstream of the X-3 site and the replacement sequence (P TEF1 -SOD1-P PGK1 -GPX2, SEQ ID NO: 3), a DNA fragment downstream of the X-3 site, and the three fragments were connected as a repair template RE using Gibson. X-3According to the above-mentioned "Editing the Saccharomyces cerevisiae genome" method, the editing plasmid pML104-X-3 and the repair template RE X-3 Transfected into Saccharomyces cerevisiae. PCR was used to screen for insertion of P at X-3 site TEF1 -SOD1-P PGK1 -GPX2 positive clones. According to the aforementioned "elimination of editing plasmid" method, after the bacterial solution was applied to the SC+FOA medium solid plate to eliminate the editing plasmid, the engineered strain KY001 without plasmid was obtained, and its deposit number is CCTCC NO: M 2024707.

[0061] Example 1 Editing Saccharomyces cerevisiae by CRISPR / Cas9 technology to achieve the construction of Saccharomyces cerevisiae engineering strain KY001. The CYS3 gene was replaced by a strong constitutive promoter P TEF1 driven by the sulfite transporter (SSU1) and the strong constitutive promoter P TEF1 A strong constitutive promoter P was inserted into the X-2 site to express the monothiol glutaredoxin (GRX5) expression cassette. PGK1 The expression cassette of the flocculation gene (FLO1) driven by the X-3 locus was inserted into the X-3 locus to enhance the tolerance and stress resistance of Saccharomyces cerevisiae. TEF1 Driven by superoxide dismutase (SOD1) and a strong constitutive promoter P PGK1 The glutathione peroxidase (GPX2) expression cassette driven by GFP can enhance the antioxidant capacity of Saccharomyces cerevisiae.

[0062] Example 2: Performance test of engineered strains of Saccharomyces cerevisiae

[0063] Sodium selenite tolerance test: Add sodium selenite to YPD medium and filter sterilize to obtain fresh sterile YPD medium containing 1mM sodium selenite. Pipette 198μl and place in a sterile 96-well plate. Pick the original strain SC1 and the engineered strain SC2 monoclonal clones and transfer them to 5ml YPD medium. After culturing at 30℃ for 24h, 1% (v:v) is transferred to a 96-well plate containing sterile YPD medium containing 1mM sodium selenite. Monitor the optical density curve of brewer's yeast over time in a microplate reader with a wavelength of 600nm. The results are as follows: Figure 1 As shown, the tolerance of the engineered strain SC2 to sodium selenite was significantly improved compared with the original strain SC1.

[0064] Detection method of sodium selenite in culture medium: Use YPD culture medium to prepare 0, 0.5, 1, 2, 3, 4mM sodium selenite solution. Dilute the above sodium selenite standard sample solution and the sample to be tested 10 times with deionized water. Add 80μl of 0.5M hydrochloric acid solution, 40μl of 0.1M EDTA-2Na solution, 20μl of 0.1M NaF solution, 20μl of 0.1M sodium oxalate solution, 250μl of 10-fold diluted standard solution or sample to be tested, and 100μl of 0.1% 2,3-diaminonaphthalene (add in dark, prepare immediately before use) to a 1.5ml EP tube. After 40 minutes in a 40℃ water bath, draw 100μl into a 96-well plate and place it in an ELISA reader to detect the absorbance at a wavelength of 377nm.

[0065] Sodium selenite reduction ability test: Single clones of the original strain SC1 and the engineered strain KY001 were selected and transferred to 5 ml YPD medium, and activated at 30°C, 200 rpm for 24 hours. 1% was transferred to 100 ml YPD medium, cultured at 30°C, 200 rpm for 12 hours, and then 10 mM sodium selenite was added. After incubation at 30°C for 24 hours, the concentration of sodium selenite in the medium was detected, and the bioconversion rate of sodium selenite was calculated. The results are as follows: Figure 2 As shown, the bioconversion rate of sodium selenite by the engineered strain KY001 (60.43%) was 11.67% higher than that of the original strain SC1 (48.76%).

[0066] Extraction and purification of bio-nanoselenium: Take 2 ml of bacterial solution in an EP tube, centrifuge and remove the supernatant to collect the bacteria. Add 200 μl of deionized water and freeze and thaw repeatedly at -80°C and 37°C for 3 times. Set the ultrasonic cell disruptor at 35% power, with a cycle of 2s on and 2s off for 10 minutes to further disrupt the cells. Finally, wash the cells twice with anhydrous ethanol at 14000g for 2min, resuspend in 200 μl of anhydrous ethanol and let stand at 37°C for 24h. Centrifuge at 15000g for 10min, wash twice with ultrapure water, and finally resuspend in 100 μl of ultrapure water.

[0067] Detect the particle size of biological nano-selenium: drop the above liquid on the silicon wafer, wait for the water to evaporate completely, and use a scanning electron microscope to photograph the surface morphology of biological nano-selenium. The results are as follows: Figure 3 The particle size of 500 nanoparticles in the electron microscope image was measured using ImageJ software, and the proportion of particles with a particle size less than 100 nm was counted. The results are shown in Figure 4 The average particle size was determined to be 73.7 nm by fitting. 81.2% of the particles were less than 100 nm in size.

[0068] Example 2 The performance of the engineered strain was demonstrated through experiments. The growth test demonstrated that the tolerance of the engineered strain SC2 to sodium selenite was significantly improved. The sodium selenite reduction test demonstrated that the reduction ability of KY001 to sodium selenite was 11.67% higher than that of SC1.

[0069] Example 3: Synthesis of biological nano-selenium by high cell density fermentation of recombinant engineered yeast

[0070] Preparation of culture medium and reagent solutions: The engineered strain uses an optimized fermentation medium, which consists of 10 g / L peptone, 5 g / L yeast extract, 50 g / L glucose, 2 g / L potassium dihydrogen phosphate, 4 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 1.5 g / L methionine, 1 g / L glutamic acid, 1 g / L glycine, and 1 g / L cysteine. The pH was adjusted to 6.0. In order to achieve high cell density fermentation of Saccharomyces cerevisiae, 50 g / L syrup solution and 50 g / L corn extract solution were prepared as feed. In order to synthesize biological nano-selenium, 200 mM sodium selenite mother liquor was prepared as substrate feed. The above culture medium and reagents were sterilized by high pressure steam at 105°C for 30 min.

[0071] High cell density fermentation of engineered yeast strains: Pick a single clone of the KY001 strain from the plate and transfer it to 200 ml of seed medium YPD medium. After culturing at 30°C for 24 hours, 10% (v:v) was transferred to a 5L fermentor. The initial working volume of the fermentor is 2L of fermentation medium. The fermentor was operated at 30°C and 500rpm, maintaining dissolved oxygen at 30%, and using 6.5mM ammonia water to control the pH to 6.0. When the concentration of glucose dropped to 5g / L, 50g / L syrup solution and 50g / L corn extract solution were added to the fermentor at flow rates of 1.5g / L / h and 0.4g / L / h, respectively.

[0072] Synthesis of biological nano-selenium: When the bacterial liquid grows to the end of the logarithmic phase (23h), the fermentation tank speed is reduced to 50rpm. 200mM sodium selenite mother solution is added to the fermentation tank at a flow rate of 2ml / h. When the final concentration of sodium selenite in the fermentation tank reaches 4.4g / L, stirring is stopped and the fermentation tank is incubated at 30℃. The concentration of sodium selenite in the fermentation broth is monitored according to the method described above. The results are as follows Figure 5 As shown, the final conversion rate of sodium selenite reached 92.9%.

[0073] Extraction of biological nano-selenium: The KY001 strain enhanced the expression of the flocculation gene FLO1. After static incubation, the cells settled in the lower layer of the tank. The supernatant contained almost no cells and was rich in biological nano-selenium. The supernatant was collected by centrifugation at a speed of 14000g, and then washed with anhydrous ethanol and deionized water to obtain a pure biological nano-selenium solution. The morphology of biological nano-selenium was observed using a scanning electron microscope. The average particle size was 79.3nm, and no cells were observed.

[0074] Particle size monitoring of bio-nanoselenium: According to the method described in "Detection of particle size of bio-nanoselenium", the particle size of bio-nanoselenium in the supernatant was detected after incubation in YPD medium for 23h, 53h, 72h, 96h and 120h. The results are as follows Figure 6 As shown, the particle size of bionanoselenium slightly increased with the extension of incubation time, the average particle size was below 79.3 nm, and 93.9% of the particles were less than 100 nm in size.

[0075] Example 3: Bio-nanoselenium was synthesized by high cell density fermentation using an engineered strain. The final conversion rate was 32.47% higher than the final conversion rate of sodium selenite by shake flask fermentation. The synthesized bio-nanoselenium had the characteristic of low particle size.

[0076] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A Saccharomyces cerevisiae engineered strain, characterized in that: The Saccharomyces cerevisiae engineered strain is the Saccharomyces cerevisiae engineered strain KY001, and its deposit number is CCTCC NO: M 2024707; The engineered strain of Saccharomyces cerevisiae improves its reducing ability to sodium selenite by regulating the tolerance module and antioxidant module of Saccharomyces cerevisiae; Wherein, the tolerance module includes cysteine ​​lyase, sulfite transporter, monothiol glutaredoxin and flocculation gene of Saccharomyces cerevisiae strain; The antioxidant modules are superoxide dismutase and glutathione peroxidase.

2. The method for preparing bio-nanoselenium by using an engineered strain of Saccharomyces cerevisiae as claimed in claim 1, characterized in that: The following steps are involved: The engineered strain of Saccharomyces cerevisiae was transferred into the seed fermentation liquid and subjected to shaking culture; Transferring the seed solution of a preset volume ratio to a fermentation tank for cultivation, adding sodium selenite at a preset flow rate, and culturing statically; The fermented cells and the supernatant are separated, the supernatant is centrifuged at high speed, and then collected and washed to obtain bio-nanoselenium.

3. The method for preparing bio-nanoselenium by using an engineered strain of Saccharomyces cerevisiae according to claim 2, characterized in that: The shaking culture was carried out at a temperature of 30°C, a shaking time of 24 h, and a rotation speed of 200 rpm.

4. The method for preparing bio-nanoselenium by using an engineered strain of Saccharomyces cerevisiae according to claim 2, characterized in that: The volume ratio of the seed solution to the total solution is 10:100, the volume of the fermenter is 5 L, the culture temperature in the fermenter is 30° C., the culture time is 24 h, the initial pH is 6.0, and the dissolved oxygen is 30%.

5. The method for preparing bio-nanoselenium by using an engineered strain of Saccharomyces cerevisiae according to claim 2, characterized in that: After adding 4.4 g / L sodium selenite, the mixture was left to stand to synthesize bio-nanoselenium.

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