Saccharomyces cerevisiae for producing squalene as well as screening method and application of saccharomyces cerevisiae

Abstract

The invention provides Saccharomyces cerevisiae for producing squalene as well as a screening method and application of the Saccharomyces cerevisiae. The method comprises the following steps: culturing a Saccharomyces cerevisiae engineering strain ySC782 for synthesizing squalene to obtain a to-be-mutagenized bacterial solution, carrying out ARTP mutagenesis on the to-be-mutagenized bacterial solution, adding the to-be-mutagenized bacterial solution into a YPD liquid culture medium, culturing to obtain a resuscitation bacterial solution, adding the resuscitation bacterial solution into a sucrose solution with gradient concentration distribution, screening, and repeating the ARTP mutagenesis-sucrose solution screening step to obtain the squalene mutant strain. And finally, the saccharomyces cerevisiae strain ySC782-M5 is obtained through screening, and the saccharomyces cerevisiae strain ySC782-M5 is obtained through screening. The yield of squalene produced by the saccharomyces cerevisiae ySC782-M5 reaches 49.83 g / L, so that a solution is provided for breaking through the yield bottleneck of squalene synthesized by yeast.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to a squalene-producing Saccharomyces cerevisiae strain and a screening method and application thereof. BACKGROUND

[0002] Squalene (also known as squalane, molecular formula C 30 H 50 ) is a high-value terpenoid compound, which is widely used in the fields of medicine, cosmetics and functional food due to its strong biological activity. However, the traditional natural extraction method faces the limitations of ecological protection pressure and high cost; therefore, the microbial fermentation method gradually becomes a more promising alternative solution due to its sustainability advantage. Yeast, as an important microbial chassis, naturally has the mevalonate pathway (MVA pathway), a clear genetic background, mature molecular operation tools and fermentation process, which makes it an ideal host for squalene engineering and production.

[0003] However, the existing squalene engineering yeast strain modification faces significant bottlenecks: (1) the strategy based on multiple rounds of gene editing has problems of long cycle, complex operation and high cost, and is easy to cause genomic instability and metabolic imbalance, thereby inhibiting cell growth and accumulating byproducts, which requires a large amount of resources for subsequent optimization and verification; (2) the traditional physical / chemical mutagenesis method is relatively simple in operation, but has high randomness of mutation, extremely low positive mutation rate, and lacks efficient and suitable technical means for screening squalene high-yield strains from ultra-large-scale mutation library.

[0004] ARTP mutagenesis (atmospheric room temperature plasma) technology has shown good application prospects in microbial breeding due to its high mutation rate, convenient operation and safety. However, when it is applied to the improvement of squalene yeast engineering strains, there are still problems, such as the limited number of positive mutants generated by single-ARTP mutagenesis and the lack of high-throughput methods for efficiently and quickly screening squalene high-yield yeast mutant strains. Therefore, it is urgent to develop an efficient integrated mutagenesis-screening strategy to break through the above bottlenecks and accelerate the breeding process of squalene high-yield yeast engineering strains. SUMMARY In view of the defects of the prior art, the present application provides a squalene-producing Saccharomyces cerevisiae strain and a screening method and application thereof. The present application performs continuous ARTP mutagenesis and density gradient centrifugation enrichment on a Saccharomyces cerevisiae engineering strain ySC782, effectively screens an engineering strain for improving squalene yield, and finally screens a Saccharomyces cerevisiae (S. cerevisiae) strain ySC782-M5, which has a squalene yield of 49.83 g / L, thereby providing a solution to break through the squalene yield bottleneck of yeast synthesis. Saccharomyces cerevisiae

[0005] ​In order to achieve the above object, the present application adopts the following technical solutions: In a first aspect, the present application provides a squalene-producing Saccharomyces cerevisiae strain, which is Saccharomyces cerevisiae (S. cerevisiae) strain ySC782-M5, deposited on January 12, 2022 in the China Center for Type Culture Collection (CCTCC) and assigned accession number CCTCC M 2026087. Saccharomyces cerevisiae In a first aspect, the present application provides a squalene-producing Saccharomyces cerevisiae strain, which is Saccharomyces cerevisiae (S. cerevisiae) strain ySC782-M5, deposited on January 12, 2022 in the China Center for Type Culture Collection (CCTCC) and assigned accession number CCTCC M 2026087.

[0006] In a second aspect, the present application further provides a screening method for the squalene-producing Saccharomyces cerevisiae strain, comprising the following steps: S1, inoculating the Saccharomyces cerevisiae engineering strain ySC782 on a YPD solid plate by streaking and culturing to single colonies; S2, inoculating the single colonies into YPD liquid medium, culturing to obtain a seed liquid; S3, inoculating the seed liquid into YPD liquid medium, culturing to obtain a bacterial suspension; S4, centrifuging the bacterial suspension, discarding the supernatant to obtain bacterial cells; washing the bacterial cells, and resuspending them in a glycerol solution to obtain a mutagenesis bacterial liquid; S5, performing ARTP mutagenesis on the mutagenesis bacterial liquid; S6, culturing the mutagenized bacterial liquid in YPD liquid medium to obtain a recovered bacterial liquid; S7, adding sucrose solutions with concentrations in a gradient distribution from high to low into centrifuge tubes in sequence, then adding the recovered bacterial liquid into the centrifuge tubes, centrifuging, and taking the top enrichment liquid to obtain a screening liquid; S8, adding the screening liquid into YPD liquid medium, culturing to obtain a new bacterial suspension; repeating steps S4-S7; S9, spreading the screening liquid of the last round on a YPD solid plate to separate single colonies, and screening to obtain the Saccharomyces cerevisiae (S. cerevisiae) strain ySC782-M5. Saccharomyces cerevisiae S9, spreading the screening liquid of the last round on a YPD solid plate to separate single colonies, and screening to obtain the Saccharomyces cerevisiae (S. cerevisiae) strain ySC782-M5.

[0007] Preferably, in the step of performing ARTP mutagenesis on the mutagenesis bacterial liquid, the mutagenesis parameters are as follows: radio frequency power is 120-130 W, helium flow rate is 10-15 SLM, and treatment time is 25-30 s.

[0008] Preferably, the sucrose solutions with concentrations of 10% w / v, 8% w / v, 6% w / v, 4% w / v and 2% w / v are added into centrifuge tubes in sequence, then the recovered bacterial liquid is added into the centrifuge tubes, centrifuged at 4000-4500 rpm for 30-35 min at 4-5℃, and the top enrichment liquid is taken to obtain the screening liquid.

[0009] Preferably, the engineered Saccharomyces cerevisiae strain ySC782 is streaked on a YPD solid plate and incubated at 28~32℃ for 44~52 h until the single colony diameter is 1~2 mm. The single colony is inoculated into YPD liquid medium and incubated at 28~32℃ with 180~220 rpm shaking for 14~18 h to obtain a seed liquid with OD 600 >10.0.

[0010] Preferably, the seed liquid is inoculated into YPD medium to obtain a cell concentration of 0.15~0.25 OD 600 / mL, and incubated at 28~32℃ with 180~220 rpm shaking to obtain a cell suspension with OD 600 0.6~1.0. The cell suspension is centrifuged, and the supernatant is discarded to obtain the cell body; the cell body is washed with 4~6% w / v glycerol solution and resuspended in 4~6% w / v glycerol solution to obtain the mutagenesis bacteria liquid.

[0011] Preferably, the mutagenized bacteria liquid is incubated in YPD liquid medium at 28~32℃ with 180~220 rpm shaking until the OD 600 is 4.5~5.5 to obtain a recovery bacteria liquid. The screening liquid is added to YPD liquid medium and incubated at 28~32℃ with 240~260 rpm shaking until the OD 600 is 0.6~1.0 to obtain a new cell suspension.

[0012] Preferably, the YPD liquid medium comprises the following components: peptone 18~22 g / L, yeast powder 9~11 g / L, glucose 18~22 g / L, and the rest is water. The YPD solid plate comprises the following components: peptone 18~22 g / L, yeast powder 9~11 g / L, glucose 18~22 g / L, agar 18~22 g / L, and the rest is water.

[0013] In a third aspect, the present application further provides the Saccharomyces cerevisiae (S. cerevisiae) Saccharomyces cerevisiae ) strain ySC782-M5 for producing squalene.

[0014] Preferably, the application inoculates the Saccharomyces cerevisiae strain into fermentation medium for culture, collects the fermentation product to obtain squalene.

[0015] The squalene-producing Saccharomyces cerevisiae strain, the screening method and the application thereof have the following beneficial effects compared with the prior art: The application uses a squalene-synthesizing Saccharomyces cerevisiae engineering strain ySC782, and the strain is cultured to obtain a to-be-mutagenized bacterial liquid, the to-be-mutagenized bacterial liquid is subjected to ARTP mutagenesis, and then is added into YPD liquid culture medium to culture to obtain a recovery bacterial liquid, and then is added into a sucrose solution with a gradient distribution of concentration to perform screening, and the ARTP mutagenization-sucrose solution screening step is repeated, and finally, a Saccharomyces cerevisiae (S. cerevisiae) strain ySC782-M5 is screened. Saccharomyces cerevisiae The squalene yield of the S. cerevisiae strain ySC782-M5 reaches 49.83 g / L, and the application provides a solution to break through the bottleneck of yeast squalene synthesis yield. BRIEF DESCRIPTION OF DRAWINGS

[0016] The above and other objects, features and advantages of the present disclosure exemplary embodiments will be more apparent from the following detailed description read in conjunction with the accompanying drawings. In the drawings, several embodiments of the present disclosure are illustrated by way of example and not limitation in which like references indicate similar, or corresponding, elements in which: Figure 1 A schematic diagram of the principle of centrifugation of the screening liquid using the gradient-distributed sucrose solution according to the application; Figure 2 The squalene yields of five strains ySC782-M1, ySC782-M2, ySC782-M3, ySC782-M4 and ySC782-M5 after fermentation. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present disclosure will be described clearly and completely below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are part of, rather than all of, the embodiments of the present disclosure. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present disclosure.

[0018] The specific embodiments of the present disclosure will be described in detail below in conjunction with the drawings.

[0019] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of embodiments. Furthermore, in the description of this application, the term "comprising" means "including but not limited to". Various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single digits within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any referenced number (fraction or integer) within the indicated range.

[0020] While various embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many modifications, alterations, and alternatives will occur to those skilled in the art without departing from the spirit and essence of the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in the practice of the invention. The appended claims are intended to define the scope of protection of the invention and therefore cover modular compositions, equivalents, or alternatives within the scope of these claims.

[0021] This invention provides a squalene-producing Saccharomyces cerevisiae strain, wherein the Saccharomyces cerevisiae is Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae The strain ySC782-M5 is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC M 2026087.

[0022] Based on the same inventive concept, the present invention also provides a method for screening the above-mentioned brewing yeast, comprising the following steps: S1. The engineered strain of Saccharomyces cerevisiae ySC782 was streaked onto YPD solid plates and cultured until a single colony was formed; S2. Inoculate a single colony into YPD liquid medium and culture to obtain a seed culture. S3. Inoculate the seed culture into YPD liquid medium and culture to obtain a bacterial suspension; S4. After centrifuging the bacterial suspension, discard the supernatant to obtain bacterial cells; wash the bacterial cells and resuspend them in glycerol solution to obtain the bacterial suspension to be mutated; S5. Perform ARTP mutagenesis on the bacterial culture to be mutagenized; S6. The mutagenized bacterial culture was placed in YPD liquid medium and cultured to obtain the revived bacterial culture. S7. Add sucrose solutions with a gradient distribution from high to low concentration to centrifuge tubes, then add the revived bacterial solution to the centrifuge tubes, centrifuge, and take the top enrichment to obtain the screening solution. S8. Add the screening solution to YPD liquid culture medium and culture to obtain a new bacterial suspension; repeat steps S4 to S7, repeating 2 to 6 times. S9. The final screening solution was spread onto YPD solid plates to isolate single clones, and the *Saccharomyces cerevisiae* was obtained through screening. Saccharomyces cerevisiae ) strain ySC782-M5.

[0023] This invention uses a squalene-synthesizing Saccharomyces cerevisiae engineered strain ySC782 (its construction method is described in patent CN116635527B - Exogenous promoters of Saccharomyces cerevisiae and their application in synthetic biology), to obtain a culture for mutagenesis. The culture is then subjected to ARTP mutagenesis, followed by culturing in YPD liquid medium to obtain a revived culture. This revived culture is then added to a sucrose solution with a gradient concentration for screening. The ARTP mutagenesis-sucrose solution screening steps are repeated until the Saccharomyces cerevisiae is finally obtained. Saccharomyces cerevisiae The yeast strain ySC782-M5 produced 49.83 g / L of squalene, providing a solution to overcome the bottleneck in yeast squalene synthesis.

[0024] In some embodiments, the bacterial culture to be mutated is coated onto a sterile slide and placed on the sample stage of an ARTP mutagen for ARTP mutagenesis. The mutagenesis parameters are: radio frequency power of 120~130 W, helium flow rate of 10~15 SLM, and processing time of 25~30 s.

[0025] In some embodiments, 10% w / v sucrose solution, 8% w / v sucrose solution, 6% w / v sucrose solution, 4% w / v sucrose solution, and 2% w / v sucrose solution are sequentially added to centrifuge tubes. Then, the revived bacterial culture is added to the centrifuge tubes, and the mixture is centrifuged at 4000-4500 rpm for 30-35 min at 4-5°C. The top enrichment is collected to obtain the screening solution. The gradient design is based on the fact that the increase of intracellular squalene lipid droplets in high-yielding strains leads to a decrease in overall cell density.

[0026] Further reference Figure 1As shown, 10% w / v (mass-volume concentration) sucrose solution, 8% w / v sucrose solution, 6% w / v sucrose solution, 4% w / v sucrose solution, 2% w / v sucrose solution (located at the top layer) were sequentially added to the centrifuge tube, and then the resuscitated bacterial solution was added to the centrifuge tube, at this time the resuscitated bacterial solution was located above the 2% w / v sucrose solution, after centrifugation, the top enrichment liquid was collected; the cells in the resuscitated bacterial solution have density differences: white is low-density cells (high squalene content, squalene density ≈ 0.854~0.862), black is high-density cells (low squalene content). During centrifugation, cells will "match" to the sucrose solution layer with similar density according to their own density: low-density cells (density closest to 2% w / v sucrose solution) will stay in the uppermost 2% w / v sucrose solution; high-density cells (higher density) will settle in the lower layer of higher concentration sucrose solution (4%, 6%, 8%, 10% layer). The final effect: cells of different densities are distributed in layers in the gradient, the 2% w / v top layer of bacterial solution will be enriched with more low-density (high squalene) cells, and direct collection of this layer can obtain the screening solution; the centrifugation effect without sucrose density gradient (only 2% w / v sucrose solution): there is only uniform density 2% w / v sucrose solution in the centrifuge tube, without concentration / density stratification. During centrifugation, cells only rely on centrifugal force to settle, but due to the uniform density of the solution, low-density and high-density cells cannot be stratified in different density layers, but can only settle to the bottom of the tube with the centrifugal force (only the settling speed is different). The final effect: cells of different densities will be mixed and aggregated at the bottom or middle of the tube, and precise enrichment and separation of low-density (high squalene) cells cannot be achieved. In some embodiments, the Saccharomyces cerevisiae engineering strain ySC782 is streaked on a YPD solid plate and cultured at 28~32℃ for 44~52 h until the single colony diameter is 1~2 mm; The single colony is inoculated into YPD liquid medium and cultured at 28~32℃, 180~220 rpm for 14~18 h to obtain an OD 600 >10.0 seed solution.

[0027] In some embodiments, the seed solution is inoculated into YPD liquid medium to obtain a bacterial concentration of 0.15~0.25 OD 600 / mL, and cultured at 28~32℃, 180~220 rpm to obtain an OD 600 of 0.6~1.0 bacterial suspension; After centrifugation of the bacterial suspension, the supernatant is discarded to obtain bacterial cells; the bacterial cells are washed with 4~6% w / v glycerol solution and resuspended in 4~6% w / v glycerol solution to obtain the mutagenized bacterial solution.

[0028] In some embodiments, the mutagenized bacterial solution is placed in YPD liquid medium and cultured at 28~32℃, 180~220 rpm until an OD 600is 4.5~5.5, and a recovered bacterial solution is obtained; The screening solution is added to YPD liquid medium and cultured at 28~32℃ and 240~260 rpm until OD 600 is 0.6~1.0, and a new bacterial suspension is obtained.

[0029] In some embodiments, the YPD liquid medium comprises the following components: 18~22 g / L of proteose peptone, 9~11 g / L of yeast powder, 18~22 g / L of glucose, and the rest is water (water as a solvent). The YPD solid plate (i.e., solid medium) comprises the following components: 18~22 g / L of proteose peptone, 9~11 g / L of yeast powder, 18~22 g / L of glucose, 18~22 g / L of agar, and the rest is water.

[0030] In some embodiments, the last round of screening solution is coated on a YPD plate to isolate a single clone, and a Saccharomyces cerevisiae strain ySC782-M5 is screened, specifically comprising: The last round of screening solution is coated on a YPD solid plate to isolate a single clone, and a single clone is picked up on a 96-well microplate containing 500 μL of YPD liquid medium using Q-PIX (Q-PIX microbial cloning and screening system) and cultured at 30℃ and 1,000 rpm for 24 h to obtain a seed solution, and then the seed solution is transferred to a new 96-well microplate containing 500 μL of YPD liquid medium at 0.2 OD 600 / mL by an automatic pipetting workstation (specifically, a Tecan pipetting workstation Fluent 780), and fed with glucose at a final concentration of 50 g / L every 24 h, and fermented for 72 h, and five strains are screened and named as ySC782-M1, ySC782-M2, ySC782-M3, ySC782-M4, and ySC782-M5 (i.e., the Saccharomyces cerevisiae strain ySC782-M5 of the present application).

[0031] Based on the same inventive concept, the present application also provides an application of the above-mentioned Saccharomyces cerevisiae strain ySC782-M5 in the production of squalene.

[0032] In some embodiments, the above-mentioned Saccharomyces cerevisiae strain ySC782-M5 is inoculated in a fermentation medium for culture, and a fermentation product is collected to obtain squalene.

[0033] Specifically, 1 mL of glycerol bacteria containing Saccharomyces cerevisiae bacteria ySC782-M5 (i.e., Saccharomyces cerevisiae bacteria ySC782-M5 is added to glycerol to obtain glycerol bacteria containing Saccharomyces cerevisiae bacteria ySC782-M5, and the content of the bacteria in the glycerol bacteria is 10 7 ~10 9 CFU / mL) is inoculated in a 250 ml conical flask containing 30 ml of YPD liquid medium, and is cultured overnight at 30°C and 200 rpm to obtain a primary seed; then the primary seed is transferred to a 1 L conical flask containing 500 ml of YPD culture solution, the initial OD 600 is 0.2, and is cultured at 30°C and 200 rpm for 16 h to obtain a secondary seed. The secondary seed is inoculated in a 10 L fermenter containing 5 L of YPD liquid medium at an inoculation amount of 10%, and is fermented, in which the temperature is controlled at 30°C, the pH is controlled at 5.0 by using 25% ammonia water, the DO > 30%, and the vvm (vvm is the volume of air per unit volume of fermentation broth per minute, unit is L / (L min), which is abbreviated as vvm) is 1. During the fermentation, the DO (dissolved oxygen) first decreases and then increases, and when the DO rises, 50% galactose is added, and the addition rate of galactose is adjusted according to the ethanol and residual sugar content, so as to control the ethanol content in the fermentation broth <10 g / L and the residual sugar content <2 g / L. After 120 h of fermentation, the squalene content is detected.

[0034] The squalene-producing Saccharomyces cerevisiae bacteria, the screening method and the application thereof of the present application are further described below. This part further describes the content of the present application in combination with specific examples, but should not be understood as a limitation of the present application. Unless otherwise specified, the technical means used in the examples is the conventional means known to those skilled in the art. Unless otherwise specified, the reagents, methods and devices used in the present application are the conventional reagents, methods and devices in the art.

[0035] In the following examples, the YPD liquid medium includes the following components: peptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L, and the rest is water; The YPD solid plate includes the following components: peptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L, agar 20 g / L, and the rest is water; The 5% w / v glycerol solution is a 50 g / L glycerol aqueous solution; 10% w / v sucrose solution, 8% w / v sucrose solution, 6% w / v sucrose solution, 4% w / v sucrose solution, 2% w / v sucrose solution are sucrose aqueous solutions with concentrations of 100 g / L, 80 g / L, 60 g / L, 40 g / L, 20 g / L respectively.

[0036] Example 1 The present embodiment provides a screening method of Saccharomyces cerevisiae, comprising the following steps: S1, inoculating Saccharomyces cerevisiae engineering strain ySC782 on a YPD solid plate and culturing at 30°C for 48 h until the single colony diameter is 1-2 mm; S2, inoculating the single colony into YPD liquid medium and culturing at 30°C, 200 rpm for 16 h to obtain seed liquid with OD 600 >10.0; S3, inoculating the seed liquid into YPD liquid medium to make the cell concentration 0.2 OD 600 / mL, culturing at 30°C, 200 rpm to obtain cell suspension with OD 600 =0.8; S4, centrifuging the cell suspension at 4000 rpm for 3 min and discarding the supernatant; Washing the cell suspension with 10 mL 5% glycerol solution, then centrifuging at 4000 rpm for 3 min and discarding the supernatant; finally, resuspending the cell suspension with 10 mL 5% glycerol solution to obtain the mutagenesis bacteria liquid; S5, uniformly coating 10 μL of the mutagenesis bacteria liquid on a sterile slide and placing it on the sample table of the ARTP mutagenesis instrument for mutagenesis, setting the parameters: radio frequency power 120 W, helium flow rate 10 SLM, processing time 25 s; repeating 10-20 identical samples in parallel (about 7×10 4 mutants are obtained from a single sample); S6, placing the mutagenized bacteria liquid in 2 mL YPD liquid medium and culturing at 30°C, 200 rpm to recover to OD 600 =5.0 to obtain the recovered bacteria liquid; S7, sequentially adding 2 mL of 10% w / v sucrose solution, 8% w / v sucrose solution, 6% w / v sucrose solution, 4% w / v sucrose solution, 2% w / v sucrose solution into a 15 mL centrifuge tube, finally adding 200 μL of the recovered bacteria liquid on top, and centrifuging at 4°C, 4000 rpm for 30 min after standing for 10 min, taking 200 μL of the top enrichment liquid (i.e. corresponding to 2% w / v sucrose solution) to a sterile shake flask to obtain the screening liquid; S8, adding the screening liquid into YPD liquid medium to a total volume of 10 mL and culturing at 30°C, 260 rpm until OD600 =0.8, obtain a new bacterial suspension; repeat steps S4-S7; S9, the last round of screening liquid is coated on YPD plate to separate single clone, and the single clone is picked by Q-PIX (Q-PIX microbial clone screening system) to a 96-well microplate containing 500 μL YPD liquid medium, and is cultured at 30°C and 1,000 rpm for 24 hours to obtain a seed liquid, and then the seed liquid is transferred to a new 96-well microplate containing 500 μL YPD liquid medium at 0.2 OD 600 / mL by an automatic pipetting workstation (specifically, a Tecan pipetting workstation Fluent 780), and is fed every 24 hours to a final concentration of 50 g / L of glucose, and is fermented for 72 hours.

[0037] S10, the strain screened by the 96-well microplate fermentation is taken, 1 mL of a glycerol bacteria (Saccharomyces cerevisiae bacteria ySC782-M5 are added to glycerol to obtain a glycerol bacteria containing Saccharomyces cerevisiae bacteria ySC782-M5, and the content of the bacteria in the glycerol bacteria is 10 9 CFU / mL) is inoculated in a 250 ml conical flask containing 30 ml of YPD liquid medium, and is cultured at 30°C and 200 rpm on a shaker for 16 hours to obtain a primary seed. Then the primary seed is transferred to a 1 L conical flask containing 500 ml of YPD culture solution, so that the initial OD is 0.2, and is cultured at 30°C and 200 rpm on a shaker for 16 hours to obtain a secondary seed. The secondary seed is inoculated in a 10 L fermenter containing 5 L of YPD liquid medium at a 10% inoculation amount, and is fermented, in which the temperature is controlled at 30°C, the pH is controlled at 5.0 by using 25% ammonia water, the DO is greater than 30%, and the vvm is 1. During the fermentation, the DO decreases first and then increases, and when the DO increases, 50% galactose is added, and the addition rate of the galactose is adjusted according to the ethanol and residual sugar amounts, so that the ethanol content in the fermentation liquid is less than 10 g / L, and the residual sugar amount is less than 2 g / L. After 120 hours of fermentation, the squalene content is detected.

[0038] S11, product squalene detection 200 μL of the fermentation liquid is collected, glass beads with a diameter of 400 μm are added, and the glass beads are ground and broken on a low-temperature grinding instrument at 65 Hz and for 60 seconds. Then 300 μL of methanol and 300 μL of n-heptane are added, and the mixture is shaken for 30 minutes. The mixture is centrifuged at 13,000 rpm for 1 minute, 150 μL of the supernatant is taken and placed in a new 1.5 ml EP tube for detection, and the sample is filtered through a 0.45m organic filter before being injected.

[0039] Quantitative detection was performed using an Agilent 8860 gas chromatograph, HP-5(MS) (30.0 m x 250 μm x 0.25 μm, i.e. film thickness 0.25 µm, inner diameter 0.25 mm, length 30.0 m) gas chromatography column, with an FID detector. The injection port temperature was 250°C, the FID detector temperature was 300°C, the split ratio was 20:1, the carrier gas flow rate was 2 mL / min, the air was 400 mL / min, the hydrogen was 40 mL / min, the tail blow was 30 mL / min, the column temperature was 160°C for 1 min, increased to 220°C at 15°C / min, then increased to 280°C at 5°C / min, maintained for 10 min, then ran at 300°C for 2 min, the injection amount was 1 μL, and the quantification was performed according to the peak area with a standard curve of squalene standard.

[0040] Positive mutation rate statistics: in the fermentation of 96-well plates, the squalene content detected after fermentation was significantly higher than that of the control strain without mutagenesis, and the strain with significantly higher squalene content was defined as a positive mutant. The positive mutation rate (%) = the number of positive mutants / the number of mutagenized strain fermentation strains.

[0041] Example 2 According to the method in Example 1, 10 μL of the mutagenized bacterial solution was uniformly coated on a sterile slide and placed on the sample table of the ARTP mutagenesis instrument for mutagenesis. The parameters were set as follows: radio frequency power 120 W, helium flow rate 10 SLM, and treatment time 25 s. The mutagenized bacterial solution was placed in 2 mL of YPD liquid medium and cultured at 30°C with 200 rpm shaking until the OD 600 =5.0, to obtain a recovered bacterial solution; 200 μL of the recovered bacterial solution was added to 2 mL of 10% w / v sucrose solution, 8% w / v sucrose solution, 6% w / v sucrose solution, 4% w / v sucrose solution, and 2% w / v sucrose solution, respectively. After standing for 10 min, the mixture was centrifuged at 4°C and 4000 rpm for 30 min. 200 μL of the top enriched liquid was taken and added to a sterile flask to obtain a screening bacterial population 1. The screening bacterial solution was added to YPD liquid medium to a total volume of 10 mL, and cultured at 30°C with 260 rpm shaking until the OD 600 =0.8, to obtain a new bacterial suspension. According to the method of Example 1, steps S4-S7 were repeated twice to obtain a screening bacterial population 2 and a screening bacterial population 3. Screening bacterial flora 1, screening bacterial flora 2, screening bacterial flora 3 were coated on YPD solid plates, and 950 strains of single colonies were picked by Q-Pix to 96-well plates (one unmutated ySC782 was set as a control for each plate); the automatic pipetting station performed fed-batch fermentation, and 200 μL of fermentation broth was collected after fermentation for product detection, and the positive mutation rate is shown in Table 1 as follows: Table 1-Positive mutation rate data

[0042] The five strains with the highest yield obtained by screening were named ySC782-M1, ySC782-M2, ySC782-M3, ySC782-M4, and ySC782-M5, respectively, and were fermented in a 10 L tank for verification, and the fermentation process was as follows: 1 mL of glycerol bacteria (yeast saccharomyces cerevisiae ySC782-M1, ySC782-M2, ySC782-M3, ySC782-M4, and ySC782-M5 were added to glycerol to obtain glycerol bacteria containing yeast saccharomyces cerevisiae, and the strain content in the glycerol bacteria was 10 9 CFU / mL) in a 250 ml conical flask containing 30 ml of YPD liquid medium, 30°C 200 rpm shaking bed overnight culture 16h to obtain the first seed. Then the first seed was transferred to a 1 L conical flask containing 500 ml of YPD liquid medium, the starting OD 600 was 0.2, and was placed in a 30°C 200 rpm shaking bed for 16h to obtain the second seed. The second seed was inoculated into a 10L fermentation tank containing 5L YPD liquid medium at a 10% inoculation amount for fermentation, and during the fermentation process, the temperature was controlled at 30°C, the pH was controlled at 5.0 with 25% ammonia water, DO>30%, and vvm was 1. With the progress of fermentation, DO first decreased and then increased, and when DO rose, 50% galactose was added, and the galactose feeding rate was adjusted according to the ethanol and residual sugar content to control the ethanol content in the fermentation broth <10g / L, and the residual sugar content <2g / L. After 120h of fermentation, the squalene yield was detected, and the results are shown in Figure 1 Table 2, the original strain ySC782 reached 38.27 g / L after 72h of fermentation, and the squalene yields of the mutagenized strains ySC782-M1, ySC782-M2, ySC782-M3, ySC782-M4, and ySC782-M5 reached 46.97 g / L, 44.27 g / L, 48.97 g / L, 44.67 g / L, and 49.83 g / L, respectively; among them, the squalene content of ySC782-M5 increased by 29.43% compared with the original strain.

[0043] It can be understood that any combination of the technical features in the above-described embodiments can be made, and for the sake of brevity, not all possible combinations of the technical features in the above-described embodiments are described, however, as long as the combination of the technical features does not exist contradictory, it should be considered within the scope of the present disclosure.

[0044] The above are only the preferred embodiments of the present application, and only the technical principles of the present application are specifically described, and these descriptions are only for the purpose of explaining the principles of the present application, and cannot be interpreted in any way as a limitation on the scope of protection of the present application. Based on the explanation here, any modification, equivalent replacement and improvement made within the spirit and principle of the present application, and other specific embodiments of the present application which can be easily thought by those skilled in the art without creative labor, should be included in the protection scope of the present application.

Claims

1. A squalene-producing Saccharin yeast, characterized in that, The brewer's yeast is brewer's yeast (Saccharomyces cerevisiae). Saccharomyces cerevisiae The strain ySC782-M5 is deposited at the China Center for Type Culture Collection, with accession number CCTCC M 2026087.

2. A method for screening *Saccharomyces cerevisiae* as described in claim 1, characterized in that, Includes the following steps: S1. The engineered strain of Saccharomyces cerevisiae ySC782 was streaked onto YPD solid plates and cultured until a single colony was formed. S2. Inoculate a single colony into YPD liquid medium and culture to obtain a seed culture. S3. Inoculate the seed culture into YPD liquid medium and culture to obtain a bacterial suspension; S4. After centrifuging the bacterial suspension, discard the supernatant to obtain the bacterial cells; wash the bacterial cells and resuspend them in glycerol solution to obtain the bacterial suspension to be mutated. S5. Perform ARTP mutagenesis on the bacterial culture to be mutagenized; S6. Place the mutagenized bacterial culture in YPD liquid medium and culture to obtain the revived bacterial culture; S7. Add sucrose solutions with a gradient distribution from high to low concentration to centrifuge tubes, then add the revived bacterial solution to the centrifuge tubes, centrifuge, and take the top enrichment to obtain the screening solution. S8. Add the screening solution to YPD liquid culture medium and culture to obtain a new bacterial suspension; repeat steps S4 to S7. S9. The final screening solution was spread onto YPD solid plates to isolate single clones, and the *Saccharomyces cerevisiae* was obtained through screening. Saccharomycescerevisiae ) strain ySC782-M5.

3. The method for screening Saccharomyces cerevisiae as described in claim 2, characterized in that, In the ARTP mutagenesis procedure for the bacterial culture to be induced, the mutagenesis parameters are: radio frequency power of 120~130 W, helium flow rate of 10~15 SLM, and processing time of 25~30 s.

4. The method for screening Saccharomyces cerevisiae as described in claim 2, characterized in that, Add 10% w / v sucrose solution, 8% w / v sucrose solution, 6% w / v sucrose solution, 4% w / v sucrose solution and 2% w / v sucrose solution to centrifuge tubes in sequence, then add the revived bacterial culture to the centrifuge tubes, centrifuge at 4000-4500 rpm for 30-35 min at 4-5℃, and take the top enrichment to obtain the screening solution.

5. The method for screening Saccharomyces cerevisiae as described in claim 2, characterized in that, The engineered strain of Saccharomyces cerevisiae ySC782 was streaked onto YPD solid plates and cultured at 28-32℃ for 44-52 h until the diameter of a single colony was 1-2 mm. Single colonies were inoculated into YPD liquid medium and cultured at 28–32°C with shaking at 180–220 rpm for 14–18 h to obtain OD. 600 Seed solution >10.

0.

6. The method for screening Saccharomyces cerevisiae as described in claim 2, characterized in that, The seed culture was inoculated into YPD medium to achieve a cell concentration of 0.15–0.25 OD. 600 / mL, incubated at 28~32℃ with shaking at 180~220 rpm to obtain OD 600 A bacterial suspension with a concentration of 0.6–1.0; After centrifuging the bacterial suspension, discard the supernatant to obtain bacterial cells; wash the bacterial cells with 4-6% w / v glycerol solution, and then resuspend them in 4-6% w / v glycerol solution to obtain the bacterial suspension to be mutated.

7. The method for screening Saccharomyces cerevisiae as described in claim 2, characterized in that, The mutagenized bacterial culture was placed in YPD liquid medium and cultured at 28-32℃ with shaking at 180-220 rpm until OD reached. 600 The concentration was set at 4.5~5.5 to obtain the resuscitation bacterial solution; Add the screening solution to YPD liquid culture medium and incubate at 28-32℃ and 240-260 rpm with shaking until OD reaches the target value. 600 The concentration was 0.6~1.0, and a new bacterial suspension was obtained.

8. The method for screening Saccharomyces cerevisiae as described in claim 2, characterized in that, The YPD liquid culture medium comprises the following components: peptone 18-22 g / L, yeast extract 9-11 g / L, glucose 18-22 g / L, and the remainder is water; YPD solid plates consist of the following components: peptone 18-22 g / L, yeast extract 9-11 g / L, glucose 18-22 g / L, agar 18-22 g / L, and the remainder is water.

9. A brewing yeast as described in claim 1 ( Saccharomyces cerevisiae Application of ySC782-M5 bacteria in the production of squalene.

10. The application as described in claim 9, characterized in that, The Saccharomyces cerevisiae was inoculated into a fermentation medium and cultured. The fermentation products were then collected to obtain squalene.

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