High-yield ryegrass antifreeze protein strain as well as construction method and application thereof

Abstract

The invention provides a high-yield ryegrass antifreeze protein strain and a construction method and application thereof.The strain is a saccharomyces cerevisiae mutant strain and is classified and named as saccharomyces cerevisiae, and the preservation number of the strain is CGMCC No.31079. The strain is subjected to rearrangement, high-throughput screening and secondary screening, and finally the yield of target protein is increased; compared with an original strain with the ryegrass antifreeze protein production capacity, the strain shows a higher level in the ryegrass antifreeze protein production aspect, the yield of the ryegrass antifreeze protein mutant strain can be effectively improved, the screening flux of the ryegrass antifreeze protein strain can also be improved, the cost is lower, the efficiency is higher, and no pollution is caused; the strain is used as a production host of the antifreeze protein of ryegrass, the shake flask yield of the obtained antifreeze protein is close to 400mg / L, the yield of a 5L fermentation tank is close to 2g / L, and the obtained antifreeze protein can be used as a probiotic cryopreservation protective agent.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a high-yield ryegrass antifreeze protein strain and a construction method and application thereof. Background Art

[0002] Antifreeze proteins (AFPs) are functional proteins evolved by organisms to survive prolonged low temperatures, helping them resist the damage caused by freezing. AFPs were first discovered in deepwater fish in the 1960s and 1970s. With increasing research, they have been found in plants, insects, and various microorganisms. AFPs exert their activity primarily in three ways: First, they modify and alter ice crystal morphology during ice formation, a process known as dynamic recrystallization. Second, large ice crystals form at the expense of smaller ones, a process AFPs can inhibit, a property known as ice recrystallization inhibition (IRI). Third, in materials containing AFPs, particularly those derived from insects, a nonequilibrium freezing point depression has been observed, while the melting point remains unchanged. This phenomenon is known as the Kelvin effect, and the difference between the melting and freezing points is defined as thermal hysteresis (TH). Due to their excellent biocompatibility and exceptional antifreeze properties, AFPs hold great potential for application in food preservation, agriculture, industry, and cryopreservation of biological tissues.

[0003] Lolium antifreeze protein (LpAFP) has the best ice recrystallization inhibition (IRI) ability among natural antifreeze proteins. The protein consists of 118 amino acids, and the gene contains 357 nucleotides, consisting of 25% aspartic acid, 16% valine, 15% serine, and 10% threonine. It contains virtually no aromatic or other hydrophobic groups. It also contains a seven-amino acid repeating sequence (XXNXVXG) and six potential glycosylation sites. Fourier transform infrared spectroscopy reveals a large number of exposed β-sheets, with each loop presumably composed of two repeating sequences. Furthermore, Lolium antifreeze protein remains stable at 100°C. Its thermal stability offers significant advantages for the food industry involving thermal processing. Furthermore, its potent ability to inhibit ice recrystallization can reduce ice crystal damage to cellular structures.

[0004] However, natural extraction of plant-derived antifreeze proteins presents a series of challenges, including long production cycles, difficult isolation and purification, high costs, and unstable activity. This necessitates the use of synthetic biology techniques for efficient heterologous expression. Currently, heterologous expression of ryegrass antifreeze proteins yields only milligram levels, necessitating the development of efficient heterologous expression strains to meet market demands. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a high-yield ryegrass antifreeze protein strain.

[0006] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned high-yield ryegrass antifreeze protein strain.

[0007] Another technical problem to be solved by the present invention is to provide an application of the above-mentioned high-yield ryegrass antifreeze protein strain.

[0008] In order to solve the above technical problems, the technical solution of the present invention is:

[0009] The invention discloses a high-yield ryegrass antifreeze protein strain, which is a saccharomyces cerevisiae mutant strain ZYFW035, classified as saccharomyces cerevisiae, and has a deposit number of CGMCC No. 31079.

[0010] The method for constructing the above-mentioned high-yield ryegrass antifreeze protein strain comprises the following specific steps:

[0011] S1: The initial ryegrass antifreeze protein-producing strain was inoculated into 3 mL of YPD liquid medium and cultured at 30°C for 8-12 h, with the OD maintained at 0.6-0.8, to obtain a bacterial solution in the logarithmic growth phase;

[0012] S2: Wash 1 mL of bacterial solution obtained in S1 twice with sterile water, then resuspend the cells with 1 mL of 0.1 M lithium acetate and gently place on ice for 5-10 minutes; then add 620 μL of polyethylene glycol (PEG) 3350, 40 μL of salmon sperm DNA (boiled in advance for 12 minutes and placed on ice for later use), 90 μL of 1 M LiOAc and 100 ng of target DNA in a new sterile tube as the transformation system and place it on ice; soak the pretreated cells (100 μL) in the transformation buffer system, gently pipette to mix, and incubate at 30°C for 30 minutes; add 90 μL of DMSO, invert several times to mix, heat shock at 42°C for 18 minutes, collect the cells and discard the supernatant, add 400 μL of 5 mM CaCl2 solution to resuspend the cells, and let it stand for 5-10 minutes; collect the cells and discard the supernatant, resuspend with sterile water and evenly spread on solid culture medium (SC-HIS), and culture at 30°C for 3 days;

[0013] S3: Pick the transformed strain obtained in step S2 that has good growth, clear colony edges, and large colonies and culture it in 3 mL of YPD liquid medium at 30°C and 220 rpm overnight. Collect the cells by centrifugation to extract the genome and perform transformation verification.

[0014] S4: Pick a single colony of the yeast transformant from step S3 and streak it on the SC-His plate for purification. Pick a single colony and culture it in yeast liquid medium until the OD 6000.4-0.6, take 1 mL and transfer to 5 mL of galactose-His medium containing 1 mM estradiol, culture at 30°C, 220 rpm shaker for 8-10 h for SCRaMbLE, dilute 10 -5 ~10 -8 Single colonies were obtained by spreading on SC-His plates and cultured at 30°C for 2-3 days to obtain a high-yielding ryegrass antifreeze protein strain library;

[0015] S5: The strains before and after SCRaMbLE were inoculated into YPD liquid medium and cultured until the stable phase. 1 mL of bacteria (about 10 7 The cells were washed twice with PBS and resuspended. The cells were sorted using the flow cytometer with an excitation wavelength of 488 nm, an absorption wavelength of 610 nm, and a sorting rate of 6000 cells / sec. A gate was set and cells with the top 0.1% fluorescence intensity were placed in a 96-well plate (with yeast culture medium and chloramphenicol placed in advance). The plates were incubated at 30°C and 220 rpm until turbidity occurred.

[0016] S6: Culture the cells obtained by flow cytometry sorting, calculate the lethality rate, and rescreen using a microplate reader and shake flasks;

[0017] S7: The screened high-yield strain was fermented, the cells were broken and purified by nickel column to obtain purified ryegrass antifreeze protein.

[0018] Preferably, the method for constructing the above-mentioned high-yield ryegrass antifreeze protein strain is used to test the IRI performance of the ryegrass antifreeze protein purified by S7, and the specific steps are as follows: a 10 μL droplet is dropped from a height of 1-1.5 m onto a crucible placed on liquid nitrogen to instantly form a thin layer of ice; the crucible is quickly transferred to a liquid nitrogen pre-cooled platform (-60°C), equilibrated for 3 minutes, heated to -8°C at -20°C / min and incubated for 30 minutes to allow recrystallization, the ice crystal morphology is recorded every 10 minutes, and the ice crystal image is observed and saved; the image is processed using the provided NIS-Elements BR software, and the mean grain area (MGA) of the ice crystals is obtained by calculating the number of ice crystals in the same area, and compared with the PBS positive control; the smaller the MGA, the stronger the IR1 activity; the selected area is 1 / 3 of the field of view; all experiments are performed in parallel at least 3 times.

[0019] Preferably, in the method for constructing the high-yield ryegrass antifreeze protein strain, step S2 is:

[0020] S2-1: The open reading frame (ORF) of URA3 was amplified by PCR, and the DNA fragment was recovered from agarose gel;

[0021] S2-2: Overlap PCR was used to add homology arms to both ends of URA3 and sequenced;

[0022] S2-3: The fragment obtained in S2 was transferred into Saccharomyces cerevisiae and coated with SC-Ura complete synthetic medium;

[0023] S2-4: Retrieve the ryegrass antifreeze protein sequence (3ULT) from the PDB protein database, obtain the DNA sequence using the codon table and combined with codon bias, and perform gene synthesis;

[0024] S2-5: The codon-optimized ryegrass antifreeze protein gene was transferred into a yeast strain containing a URA3 tag, and the resultant was spread on a 5-fluoroorotic acid plate after dilution. The nucleotide sequence of the ryegrass antifreeze protein gene is shown in SEQ ID NO. 1 in the sequence listing.

[0025] S2-6: Single colonies were selected in liquid YPD, and the genome sequence was extracted for PCR verification;

[0026] S2-7: Inoculate the S5 strain into a shake flask, culture for 48-72 hours, and observe under a fluorescence microscope;

[0027] S2-8: Generation of a large mutant library by estradiol- and galactose-induced genome rearrangement in Saccharomyces cerevisiae;

[0028] S2-9: High-throughput screening was performed by flow cytometry, rescreening was performed by microplate reader, and the yield of high-yield strains was verified by shake flask fermentation.

[0029] Preferably, in the method for constructing the high-yield ryegrass antifreeze protein strain, in steps S2-1 and S2-2, homology arms are added to both ends of the URA3 gene by Overlap PCR, and the target fragment is constructed by fragment combination.

[0030] Preferably, in the method for constructing the high-yield ryegrass antifreeze protein strain, the rearrangement conditions in step S2-8 are: estradiol concentration of 1 mM, galactose concentration of 20 g / L, and rearrangement time of 8 h.

[0031] Preferably, in the method for constructing the above-mentioned high-yield ryegrass antifreeze protein strain, the flow cytometer sorting conditions in step S2-9 are: setting the excitation wavelength to 488 nm, the absorption wavelength to 610 nm, the sorting speed to 3000-8000 cells / sec, and setting a gate to select cells with fluorescence intensity in the top 0.01%-0.1% to be placed in a 96-well plate (yeast culture medium and chloramphenicol are placed in advance).

[0032] The application of the high-yield ryegrass antifreeze protein strain in the production of ryegrass antifreeze protein.

[0033] Preferably, the above-mentioned ryegrass antifreeze protein is used to carry out large-scale fermentation of a high-yielding strain, by adding high-abundance amino acids of ryegrass antifreeze protein to the fermentation broth, adjusting and optimizing the fermentation temperature (28-32°C), pH (5-7) value and fermentation time (60-120h), and feeding a mixture of a carbon source (glucose 6mL / h) and a nitrogen source (ammonia water) to achieve high-level expression of ryegrass antifreeze protein.

[0034] Preferably, the application of the ryegrass antifreeze protein can yield the highest protein yield by continuously adding a mixture of glucose and nitrogen source at a rate of 6-10 mL / h from 12 h to 96 h of fermentation according to the optimized conditions.

[0035] The above-mentioned ryegrass antifreeze protein is used as a cryoprotectant for probiotics.

[0036] Preferably, the application of the ryegrass antifreeze protein comprises the following steps: adding the ryegrass antifreeze protein solution after continuous subculture of the probiotics, pre-freezing at -80°C for 24 hours, and freeze-drying.

[0037] After pre-freezing at -80℃ for 24 hours and freeze-drying, the freezing survival rate and morphological characteristics before and after freezing were measured. The active enzymes such as lactate dehydrogenase and β-galactosidase in the probiotics before and after freezing and thawing were detected to judge the effect of the new antifreeze protein on the activity of probiotics after cold storage, and to determine whether the new antifreeze protein is more helpful in maintaining the advantages of the life characteristics of probiotics compared with natural antifreeze proteins and commercial refrigerants.

[0038] Preferably, in the application of the above-mentioned ryegrass antifreeze protein, the probiotics are Lactobacillus plantarum, Streptococcus thermophilus, Pediococcus pentosaceus and / or Lactobacillus bulgaricus.

[0039] Different concentrations of ryegrass antifreeze protein solutions were used to conduct a probiotic freezing experiment. The specific probiotics included Lactobacillus plantarum, Streptococcus thermophilus, Pediococcus pentosaceus and Lactobacillus bulgaricus. The freezing steps were as follows: the probiotics were continuously subcultured and then added with ryegrass antifreeze protein solution. After pre-freezing at -80℃ for 24 hours and freeze-drying, their freezing survival rate and morphological characteristics before and after freezing were measured. The active enzymes such as lactate dehydrogenase and β-galactosidase in the probiotics before and after freezing and thawing were detected to determine the effect of the new antifreeze protein on the activity of probiotics after cold storage and to determine whether the new antifreeze protein is more helpful in maintaining the life characteristics of Streptococcus thermophilus compared with natural antifreeze protein and commercial refrigerants.

[0040] Beneficial effects:

[0041] The high-yield ryegrass antifreeze protein strain is a synthetic cerevisiae yeast. It is used as the production host of ryegrass antifreeze protein. The mutant genome library was constructed by SCRaMbLE and combined with the high-throughput screening strategy of flow cytometry. After four rounds of iterative genome rearrangement screening, the mutant genome library was obtained from 2×10 7 2,400 high-yield mutants were initially screened out from a mutant library, and further rescreening was performed using 96-well plates and shake flasks to obtain 4 high-yield mutant strains, and finally the strain CGMCC No.31079 with the most significant yield-enhancing effect was screened; in its construction method, the cerevisiae strain was rearranged, high-throughput screened and rescreened to ultimately achieve an increase in the yield of the target protein. Compared with the original strain with the ability to produce ryegrass antifreeze protein, the strain showed a higher level of ryegrass antifreeze protein production, which not only effectively increased the yield of ryegrass antifreeze protein in the mutant strain, but also increased the screening throughput of ryegrass antifreeze protein strains, with low cost, high efficiency and no pollution; the shake flask yield of the strain was close to 400 mg / L, which was 3.33 times higher than that of the initial strain, and the yield in a 5L fermentation tank was close to 2 g / L. The obtained antifreeze protein can be used as a cryoprotectant for probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Screening strategies for high-yielding ryegrass antifreeze protein strains;

[0043] Figure 2 This is the IRI result of ryegrass antifreeze protein;

[0044] Figure 3 The figure is a comparison chart of enzyme survival rate;

[0045] Figure 4 Construction process for strains;

[0046] Figure 5 Fermentation results in a 5-L fermentor, including (a, b) growth and fermentation of the strain, glucose consumption, and accumulation of ethanol and acetic acid under batch fermentation conditions; (c, d) growth and fermentation of the strain, and accumulation of by-products when a mixture of glucose and a nitrogen source was continuously added at a rate of 6 mL / h.

[0047] Preservation Instructions

[0048] The high-yield ryegrass antifreeze protein strain obtained by screening in the present invention has been deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC) on June 25, 2024, with the deposit number CGMCC No.31079 and the classification name Saccharomyces cerevisiae. DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0050] Example 1

[0051] Construction of a haploid Saccharomyces cerevisiae strain producing ryegrass antifreeze protein

[0052] The CAN site (YEL063C) of the artificial synthetic chromosome V was selected as the insertion site, the functional URA3 gene was selected as the reverse screening marker, and a synthetic cerevisiae yeast containing the URA3 gene at the target site was constructed as an intermediate strain. The fusion ryegrass antifreeze protein gene containing a green fluorescent protein tag was transformed into the intermediate strain containing the URA3 gene. The strain that could grow normally on the 5-fluoroorotic acid plate was selected and transferred to the YPD liquid medium. The strain was cultured overnight, the genome was extracted, and preliminary verification was performed by PCR. Then, the strain was observed under a fluorescence microscope, and attention was paid to avoiding light. If a clear fluorescent signal can be seen, it means that the ryegrass antifreeze protein gene is successfully expressed. Figure 4 As shown, the specific construction process is as follows:

[0053] S1: The open reading frame (ORF) of URA3 was amplified by PCR technology, and the DNA fragment was recovered from agarose gel. The nucleotide sequence of URA3 is shown in SEQ ID NO.2 in the sequence listing.

[0054] S2: Overlap PCR was used to add homology arms to both ends of URA3 and sequenced.

[0055] S3: Prepare SC-Ura medium with the following formula: 20 g / L glucose, 5-8 g / L yeast nitrogen-free medium (YNB), 1-3 g / L amino acid drop-out mix, 10-20 mL / L His, 10-20 mL / L Ura, 10-20 mL / L Leu, and 10-20 mL / L Trp. Autoclave at 121°C for 30 min. Transform the fragment obtained in S2 into Saccharomyces cerevisiae yXZX236 (see "Design and Construction of Saccharomyces cerevisiae Chromosome V," a 2017 doctoral dissertation from Tianjin University). Apply SC-Ura complete synthetic medium to obtain the optimized Saccharomyces cerevisiae (designated zWYF005).

[0056] S4: The ryegrass antifreeze protein sequence (3ULT) was retrieved from the PDB protein database. The optimized ryegrass antifreeze protein gene was obtained by using the codon table and combining the codon preference. The nucleotide sequence is shown in the sequence listing SEQ ID NO.1.

[0057] S5: Transform the optimized ryegrass antifreeze protein gene into a yeast strain containing the URA3 tag:

[0058] (1) Saccharomyces cerevisiae (zWYF005) was inoculated into 3 mL of YPD liquid medium and cultured at 30°C for 8-12 h with the OD maintained at 0.6-0.8 to obtain a bacterial solution in the logarithmic growth phase;

[0059] (2) Wash the obtained 1 mL bacterial solution twice with sterile water, then resuspend the cells with 1 mL of 0.1 M lithium acetate and gently place on ice for 5-10 minutes; then add 620 μL of polyethylene glycol (PEG) 3350, 40 μL of salmon sperm DNA (boiled in advance for 12 minutes and placed on ice for later use), 90 μL of 1 M LiOAc and 100 ng of target DNA in a new sterile tube as the transformation system and place it on ice; soak the pretreated cells (100 μL) in the transformation buffer system, gently pipette to mix, and incubate at 30°C for 30 minutes; add 90 μL of DMSO, invert several times to mix, heat shock at 42°C for 18 minutes, collect the cells and discard the supernatant, add 400 μL of 5 mM CaCl2 solution to resuspend the cells, and let it stand for 5-10 minutes; collect the cells and discard the supernatant, resuspend with sterile water and evenly spread on solid culture medium (SC-HIS), and culture at 30°C for 3 days.

[0060] After dilution, spread it on a 5-fluoroorotic acid plate. The 5-fluoroorotic acid plate formula is as follows: Solution A: sterilize with 750 mL double-distilled water + 20 g agar; Solution B: 5-10 g ammonium sulfate, 1-3 g YNB, 10-20 g glucose, 1-2 g 5-FOA, 0.1-0.2 g His (histidine), 0.1-0.2 g Leu (leucine), 0.05-0.1 g Met (methionine), 0.05-0.1 g Ura (uracil), 250 mL water, ultrasonically shake for 3-5 hours until the 5-fluoroorotic acid is completely dissolved, then filter with a 0.22 μm mixed fiber (MCE) microporous membrane, add Solution B to Solution A which is not hot to the touch (55-65°C), and pour onto the plate.

[0061] S6: Prepare YPD medium: 20 g / L glucose, 20 g / L tryptone, 10 g / L yeast extract. Add 15 g / L agar powder to the solid medium. Autoclave at 121°C for 25 min, separating the glucose from other nitrogen sources to minimize Maillard reactions. Single colonies were isolated in liquid YPD, and the genome sequence was extracted and verified by PCR to obtain the yeast strain (designated zWYF006).

[0062] S7: Inoculate the S6 strain into a shake flask, culture for 48-72 hours, and observe under a fluorescence microscope. Protect from light during observation.

[0063] Example 2

[0064] Optimization of Saccharomyces cerevisiae genome shuffling conditions

[0065] Saccharomyces cerevisiae (zYFW006) undergoes genome rearrangements under the induction of estradiol and galactose, designing chromosomal structure insertions, deletions, duplications, and translocations. The presence of a large number of essential gene deletions and lethal gene mutations can lead to yeast lethality. Therefore, exploring and optimizing rearrangement conditions is very important. This example focuses on the rearrangement time and estradiol concentration. The specific implementation process is as follows:

[0066] S1: The plasmid pCRE4 carrying the Cre enzyme gene and the control plasmid pRS413 were respectively transformed into the initial strain (zYFW005).

[0067] S2: Cultured under the following four conditions: (1) SC-His glucose medium without estradiol; (2) SC-His glucose medium with 1 μM estradiol; (3) SGal-His galactose medium without estradiol; (4) SGal-His galactose medium with 1 μM estradiol. Lethality was observed by spot-plate serial dilution.

[0068] S3: The strain containing the pCRE4 plasmid in S1 was subjected to SCRaMbLE, and the rearrangement time was set to 4h, 6h, 8h, 10h and 12h. The dilution plates were observed respectively, and the optimal time was determined to be 8h.

[0069] S4: The strain containing the pCRE4 plasmid in S1 was subjected to SCRaMbLE, and the estradiol concentrations were set to 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1.0mM, 1.2mM and 1.4mM. The rearranged strains were diluted and spot-plated for observation to determine the optimal estradiol concentration of 1.0mM.

[0070] Example 3

[0071] Construction of a library of high-yielding ryegrass antifreeze protein strains

[0072] Artificially synthesized chromosomes can produce a wide range of genetically diverse rearrangements when induced by Cre recombinase. A green fluorescent-tagged ryegrass antifreeze protein gene was integrated into the synthetic Saccharomyces cerevisiae strain, and the diversity of ryegrass antifreeze protein production was defined by changes in fluorescence intensity. During induction, essential gene deletions or synthetic lethal gene interactions caused by chromosomal rearrangement events can lead to high cell mortality. The varying fluorescence intensities of surviving strains after chromosomal rearrangement reflect fluctuations in ryegrass antifreeze protein production. The specific construction process is as follows:

[0073] S1: Pick a single colony carrying pCRE4 from the SC-His plate, inoculate it into 3 mL of SC-His medium and culture it at 30°C, 220 rpm for 24 hours.

[0074] S2: Centrifuge at 5000 rpm for 2 min at room temperature, remove the supernatant, collect the cells and wash them three times with sterile water.

[0075] S3: The cells in S2 were transferred to 3 mL of synthetic complete (SC) medium without histidine and glucose and containing 2% glycerol as a carbon source, and cultured with shaking at 30°C overnight.

[0076] S4: Centrifuge at 5000 rpm for 2 min at room temperature, remove the supernatant, collect the cells and wash them three times with sterile water.

[0077] S5: Resuspend the cells in 3 mL of complete synthetic (SC) medium without histidine and glucose and containing 2% galactose as a carbon source, with an initial OD600 of 0.6-1.0, add estradiol to a concentration of 1 mM, and culture with shaking at 30°C for 8 hours. Perform SCRaMbLE to complete library construction.

[0078] Example 4

[0079] Screening of high-yielding ryegrass antifreeze protein strains

[0080] like Figure 1 As shown, SCRaMbLE generates a large mutant library, and high-yielding strains are isolated by flow cytometry using fluorescence intensity as a screening marker. The mutant library contains lethal strains, which can be identified through plate rescreening and shake flask screening. Multiple rounds of iteration can further increase strain yield. The specific screening process is as follows:

[0081] S1: Collect the mutant strain library obtained by SCRaMbLE and resuspend it in PBS.

[0082] S2: The peak single-cell fluorescence intensity of the initial strain was used as the screening threshold. Yeast strains with fluorescence above the threshold were selected and cultured in 96-well plates. Fluorescence screening conditions were: excitation wavelength 488nm, absorption wavelength 520nm, sorting speed 6000 cells / sec, and gated on cells with fluorescence intensity in the top 0.1% to be added to a 96-well plate (yeast culture medium and chloramphenicol were placed in advance). Four rounds of screening were performed, with 3748576, 4012201, 6996092, and 7381528 strains screened in each round, respectively, for an average of 600 strains screened per round.

[0083] S3: Perform secondary sorting using an enzyme-labeled instrument to screen out strains with high fluorescence intensity and good growth conditions.

[0084] S4: Through further optimization through shake flask fermentation, the yield of each round continued to increase compared with the previous round, with each round increasing by 0.82-fold, 0.06-fold, 0.64-fold, and 0.38-fold, respectively. Finally, a high-yield ryegrass antifreeze protein strain was obtained (deposit number CGMCC No. 31079). After fermentation process optimization in a 5-L fermenter, its yield was close to 2 g / L.

[0085] Example 5

[0086] The antifreeze protein was produced by fermentation of ryegrass antifreeze protein strain CGMCC No.31079 in a 5L fermenter. The fermentation broth temperature was adjusted to 30°C and the pH value was 6-6.5. 2g / L of lysine, valine and histidine, and 2.5g / L of glycine and aspartic acid were added to the fermentation broth. The test was carried out in the fermenter. From 12h, a mixture of glucose (carbon source) and ammonia (nitrogen source) was continuously added at a rate of 6mL / h for fermentation until 96h. Figure 5 As shown in c, the biomass reached 65 g / L at 84 h, an increase of 35%. The highest protein yield could be harvested after 96 h of fermentation, reaching 1.9 g / L, which was 3.47 times the yield of shake flask fermentation of the same strain and the highest yield of ryegrass antifreeze protein reported so far.

[0087] In addition, if Figure 5 As shown in Figure 2, after 72 h of fermentation, the antifreeze protein production of strain CGMCC No.31079 reached 1.5 g / L, which was 2.6 times higher than that of shake flask fermentation ( Figure 5 a). Byproducts such as ethanol and acetic acid mainly accumulate in the early stage of fermentation, reaching a peak at 48 hours, but are further metabolized in the later stage ( Figure 5 b). The final titers of metabolic byproducts ethanol and acetic acid were 3.2 and 1.6 g / L ( Figure 5 d).

[0088] Example 6

[0089] IRI activity assay

[0090] The ability to inhibit ice crystal recrystallization is an important property of antifreeze proteins. This example uses a "splat-cooling" assay to measure the ability of recombinant ryegrass antifreeze protein to inhibit ice crystal recrystallization. The specific steps are as follows:

[0091] S1: Drop a 10 μL droplet from a height of 1-1.5 m onto a crucible placed on liquid nitrogen, and a thin layer of ice forms instantly.

[0092] S2: The crucible was quickly transferred to a liquid nitrogen pre-cooled platform (-60°C), equilibrated for 3 minutes, heated at -20°C / min to -8°C and incubated for 30 minutes to allow recrystallization. The ice crystal morphology was recorded every 10 minutes, and the ice crystal image was observed and saved.

[0093] S3: The images were processed using the provided NIS-Elements BR software. The mean grain area (MGA) of the ice crystals was calculated by counting the number of ice crystals in the same area and compared with the PBS positive control. The smaller the MGA, the stronger the IRI activity. The selected area was 1 / 3 of the field of view. All experiments were performed at least three times in parallel. Figure 2 As shown in the figure, at a concentration of 0.15 mg / mL, the ice crystals of the ryegrass antifreeze protein solution still showed fine ice crystals after 30 minutes of growth compared with the PBS solution, which was able to completely inhibit the growth of ice crystals.

[0094] Example 7

[0095] Application of antifreeze proteins in cell cryopreservation

[0096] Antifreeze proteins derived from ryegrass remain stable at 100°C and exhibit the strongest IRI performance among natural antifreeze proteins. Their powerful ability to inhibit ice recrystallization can reduce cell damage. In this example, they are applied to the cryopreservation of probiotics such as lactic acid bacteria and Streptococcus thermophilus. The specific steps are as follows:

[0097] S1: After continuous subculture of probiotics, antifreeze proteins and other cryoprotectants such as dimethyl sulfoxide (DMSO) are added.

[0098] S2: After pre-freezing at -80℃ for 24 hours and freeze-drying, the freezing survival rate and morphological characteristics before and after freezing were measured to observe whether the cells were damaged. The results showed that the survival rate of probiotics containing ryegrass antifreeze protein after freezing was above 80%.

[0099] S3: Detect the active enzymes such as lactate dehydrogenase and β-galactosidase in probiotics before and after freezing and thawing, such as Figure 3 As shown, the results showed that the enzyme activity of the group containing antifreeze protein was more than 3 times that of the control group.

[0100] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. Improvements and modifications such as strain transformation carried out by technicians in this technical field based on the method of the present invention or on the basis of the method are considered to be within the scope of protection of the present invention.

Claims

1. A high-yield ryegrass antifreeze protein strain, characterized by: Accession number: CGMCC No.31079.

2. The method for constructing the high-yield ryegrass antifreeze protein strain according to claim 1, characterized in that: The specific steps are as follows: S1: The ryegrass antifreeze protein-producing strain was inoculated into YPD liquid medium and cultured, with the OD maintained at 0.6-0.8, to obtain a bacterial solution in the logarithmic growth phase; S2: Wash the bacterial solution obtained in S1 with water, resuspend the cells with lithium acetate, and gently place on ice; add polyethylene glycol 3350, salmon sperm DNA, LiOAc, and target DNA in a new sterile tube in sequence as the transformation system, and place on ice; soak the pretreated cells in the transformation buffer system, gently pipette and mix, and incubate; add DMSO, mix, heat shock, collect the cells, discard the supernatant, add CaCl2 solution to resuspend the cells, and let it stand; Collect the cells and discard the supernatant, resuspend them with sterile water, and evenly spread them on solid culture medium, culture at 30°C for 3 days; S3: The transformed strain from step S2 was placed in 3 mL of YPD liquid medium, cultured overnight, and the cells were collected by centrifugation to extract the genome and perform transformation verification; S4: Pick a single colony of the yeast transformant from step S3 and streak it on the SC-His plate for purification. Pick a single colony and culture it in yeast liquid medium until OD 600 0.4-0.6, transferred to galactose-His medium containing estradiol, cultured on a shaking platform for SCRaMbLE, diluted 10 -5 ~10 -8 Single colonies were obtained by plating on SC-His plates and cultured for 2-3 days to obtain a high-yielding ryegrass antifreeze protein strain library; S5: The strains before and after SCRaMbLE were inoculated into YPD liquid medium and cultured until the stationary phase. The cells were washed with PBS and resuspended. The cells were sorted using the cell sorting function of a flow cytometer with an excitation wavelength of 488 nm, an absorption wavelength of 610 nm, and a sorting rate of 6000 cells / sec. The cells with the top 0.1% fluorescence intensity were gated and transferred to a 96-well plate and cultured until turbidity occurred. S6: Culture the cells obtained by flow cytometry sorting, calculate the lethality rate, and rescreen using a microplate reader and shake flasks; S7: The screened high-yield strain was fermented, the cells were broken and purified by nickel column to obtain purified ryegrass antifreeze protein.

3. The method for constructing a high-yield ryegrass antifreeze protein strain according to claim 2, characterized in that: The specific method of step S2 is: S2-1: The open reading frame of URA3 was amplified by PCR, and the DNA fragment was recovered by agarose gel; S2-2: Overlap PCR was used to add homology arms to both ends of URA3 and sequenced; S2-3: The fragment obtained in S2 was transferred into Saccharomyces cerevisiae and coated with SC-Ura complete synthetic medium; S2-4: Retrieve the ryegrass antifreeze protein sequence from the PDB protein database, obtain the DNA sequence using the codon table and combined with codon bias, and perform gene synthesis; S2-5: The codon-optimized ryegrass antifreeze protein gene was transferred into a yeast strain containing a URA3 tag, and the resultant was spread on a 5-fluoroorotic acid plate after dilution. The nucleotide sequence of the ryegrass antifreeze protein gene is shown in SEQ ID NO. 1 in the sequence listing. S2-6: Single colonies were selected in liquid YPD, and the genome sequence was extracted for PCR verification; S2-7: Inoculate the S5 strain into a shake flask, culture for 48-72 hours, and observe under a fluorescence microscope; S2-8: Generation of a large mutant library by estradiol- and galactose-induced genome rearrangement in Saccharomyces cerevisiae; S2-9: High-throughput screening was performed by flow cytometry, rescreening was performed by microplate reader, and the yield of high-yield strains was verified by shake flask fermentation.

4. The method for constructing a high-yield ryegrass antifreeze protein strain according to claim 2, characterized in that: The IRI performance of the purified ryegrass antifreeze protein from step S7 was tested as follows: a 10 μL droplet was dropped from a height of 1-1.5 m onto a crucible placed on liquid nitrogen, instantly forming a thin layer of ice; the crucible was quickly transferred to a liquid nitrogen pre-cooled platform, equilibrated for 3 minutes, heated at -20°C / min to -8°C, and incubated for 30 minutes to allow recrystallization; the ice crystal morphology was recorded every 10 minutes, and ice crystal images were observed and saved; the images were processed using the provided NIS-Elements BR software, and the average crystal area of ​​the ice crystals was calculated by counting the number of ice crystals in the same area, and compared with the PBS positive control; the smaller the MGA, the stronger the IRI activity; The selected area was 1 / 3 of the visual field; all experiments were performed at least three times in parallel.

5. Use of the high-yield ryegrass antifreeze protein strain according to claim 1 in producing ryegrass antifreeze protein.

6. The use according to claim 5, characterized in that: The high-yield strain was fermented on a large scale. By adding high-abundance amino acids of ryegrass antifreeze protein to the fermentation broth, adjusting and optimizing the fermentation temperature to 28-32°C, pH value to 5-7 and fermentation time to 60-120h, and feeding a mixture of carbon source glucose and nitrogen source ammonia water, high-level expression of ryegrass antifreeze protein was achieved.

7. The use according to claim 6, characterized in that: From 12 h onwards, glucose and nitrogen source mixture were continuously added at a rate of 6-10 mL / h and fermented until 96 h.

8. Use of the ryegrass antifreeze protein according to claim 5 as a cryoprotectant for probiotics.

9. The use according to claim 8, characterized in that: The specific steps are as follows: after continuous subculture of the probiotics, add ryegrass antifreeze protein solution, pre-freeze at -80℃ for 24 hours and freeze-dry.

10. The use according to claim 8, characterized in that: The probiotics are Lactobacillus plantarum, Streptococcus thermophilus, Pediococcus pentosaceus and / or Lactobacillus bulgaricus.

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