Kluyveromyces marxianus capable of synthesizing casein alpha s2 of dairy cow and application of Kluyveromyces marxianus in feed

Abstract

The invention discloses a method for synthesizing dairy cow casein alpha s2 by using saccharomycetes and application of the dairy cow casein alpha s2 in feed, and relates to the technical field of biology. According to the method for synthesizing the dairy cow casein alpha s2 by using the saccharomycetes and the application of the saccharomycetes in the feed, the saccharomycetes contain genes for coding the dairy cow casein alpha s2 and can secrete the synthesized target dairy cow casein alpha s2 to the outside of cells, and freeze-dried powder containing the dairy cow casein alpha s2 and saccharomycetes cells are used as the feed. The kluyveromyces marxianus can generate more foam, and target protein is separated and purified from the foam; the microbial fermentation synthesis operation is convenient, the culture cost is low, and the pollution is small; the obtained plenty of saccharomycetes cells are also one of good animal protein feed sources; and the dependence on foreign import is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for synthesizing cow casein αs2 by utilizing yeast and an application thereof in feed. Background Art

[0002] Casein is the most abundant protein in dairy products. It is the most abundant protein in the milk secreted by various mammals and is also the most consumed protein. For example, the proteins in cow's milk are primarily composed of casein, whey protein, and lactoglobulin. Casein accounts for the largest proportion, approximately 80%, while whey protein accounts for approximately 11.5%, and lactoglobulin accounts for approximately 3.3%. These proteins have many benefits for the human body, such as providing energy, promoting growth and development, and maintaining tissue repair. The level of casein determines the protein content of dairy products, making it crucial for controlling dairy product quality. Casein is composed of four different types of proteins. For example, the main components of casein in cow's milk are αs1-casein (alpha-S-1, abbreviated as αs1-casein, CSN1S1), αs2-casein (alpha-S-1, αs2-casein, CSN1S2), β-casein (beta-casein, CSN2), and κ-casein (kappa-casein, CSN3). Each casein is composed of multiple peptide chains, which are connected to each other by bonds to form a polypeptide chain complex (Reference: Hassanin AA, Osman A, Atallah OO, El-Saadony MT, Abdelnour SA, Taha HSA,Awad MF, Elkashef H, Ahmed AE, Abd El-Rahim I, Mohamed A and Eldomiaty AS(2022) Phylogenetic comparative analysis: Chemical and biological features ofcaseins (alpha-S-1, alpha-S-2, betaand kappa-) in domestic dairy animals. Front. Vet. Sci. 9:952319. doi: 10.3389 / fvets.2022.952319).

[0003] αs1-casein is the most abundant casein in bovine milk, accounting for approximately 40% of the total casein content. It is composed of 207 amino acids and is divided into three peptide chains: αs1-CN-1, αs1-CN-I, and αs1-CN-I. αs1-CN-I is the largest peptide chain, containing 92 amino acids; αs1-CN-I contains 90 amino acids; and αs1-CN-I contains 25 amino acids. These three peptide chains are interconnected by disulfide bonds, forming a large polypeptide chain complex.

[0004] αs2-casein is the second most abundant casein in bovine milk, accounting for approximately 30% of the total casein content. It is composed of 207 amino acids, divided into three peptide chains: αs2-CN-1, αs2-CN-II, and αs2-CN-I. αs2-CN-1 and αs2-CN-II each contain 99 amino acids, while αs2-CN-I consists of 9 amino acids. These three peptide chains are interconnected by sulfide bonds, forming a large polypeptide chain complex.

[0005] β-casein accounts for approximately 10% of total casein. It is composed of 209 amino acids, divided into two peptide chains: β-CN-I and β-CN-II. β-NI contains 50 amino acids, while β-CN-II contains 159 amino acids. These two peptide chains are linked by non-covalent bonds to form a large polypeptide chain complex.

[0006] κ-casein is the smallest casein in bovine milk and is also the least abundant, occurring in approximately 0.1% of milk. It is composed of 169 amino acids. κ-casein has a unique structure, consisting of an α-helical region and a β-pleated region. The α-helical region contains 64 amino acids, while the β-pleated region contains 48 amino acids. These two regions are linked by non-covalent bonds to form a small polypeptide chain.

[0007] Generally speaking, casein in cow's milk is composed of four different caseins: αs1-casein, αs2-casein, β-casein, and κ-casein. Each of these contains a different number of amino acids, interconnected by non-covalent or disulfide bonds to form a complex polypeptide chain. This complex primary structure confers unique functions and properties to caseins, significantly impacting the quality and nutritional value of cow's milk.

[0008] The main sources of protein in the diet of Chinese residents are various animal meats and eggs. In order to obtain enough meat and eggs, it is necessary to vigorously develop the breeding industry. However, obtaining casein by raising dairy cows on farms is labor-intensive, costly, and has a large amount of animal waste discharge. Improper handling can easily cause serious pollution to the environment.

[0009] At the same time, the demand for feed is growing, and the protein content in feed is one of the key factors in evaluating feed quality. Currently, soybean meal is still the main source of feed protein, and large quantities of soybeans are imported from abroad each year to produce soybean meal. Summary of the Invention

[0010] The purpose of the present invention is to provide a method for synthesizing cow casein AS2 using yeast and its application in feed, so as to solve the problems that obtaining casein by raising dairy cows on farms is labor-intensive, costly, has a large amount of animal waste discharge, and can easily cause significant environmental pollution if improperly handled; and that a large amount of soybeans must be imported from abroad each year to produce soybean meal as feed.

[0011] In order to achieve the above object, the present invention provides the following technical solutions: A Kluyveromyces marxianus yeast capable of synthesizing cow casein αs2 is constructed by the following steps: (1) The promoter is km.PDC1, the terminator is the aep gene promoter, the selection marker is the hygromycin B resistance gene sequence (hph), and the homology arm is the 26S rDNA gene sequence. The vector is digested with the restriction endonuclease KpnI and the synthesized target gene is cloned into the vector to construct the new vector pSWV-km.PDC-CSN1S2; (2) The ligation product was transformed into Escherichia coli, and the plasmid vector was extracted in large quantities. The vector was digested with the restriction endonuclease EcoRI, and DNA fragments larger than 5 kb were recovered and used to transform Kluyveromyces marxianus; (3) After transformation, the entire transformation solution containing yeast cells was spread on a YPD solid screening plate containing 500 μg / ml hygromycin B; (4) Culture in a 30-degree incubator for 3-5 days and observe whether single colonies have grown. After growth, transfer the single colonies one by one to fresh screening solid culture medium, culture in a 30-degree incubator for 3-5 days, and repeat the transfer to the screening medium to obtain pure culture transformants; (5) The genomic DNA of the pure culture transformant was extracted and PCR amplified using a pair of primers for the αs2-casein gene. The primer sequences were: 5'-ACCATGGTGAACAAACACTTCTTGTC-3', 5'-CCAATGAGGCCACCAGTGGTACCAGTTG-3'. A DNA band of 700 bp was obtained. However, the Kluyveromyces marxianus strain that did not contain the CSN1S2 gene was used as a control strain and could not amplify this 700 bp DNA. The amplified 700 bp was sequenced and the results showed that it was the αs2-casein gene sequence, proving that the pure culture transformant was the transformant that truly contained the target CSN1S2 gene. The hygromycin B solution needs to be sterilized; The yeast contains a gene encoding cow casein alphaS2, which is alpha-S-2, casein alphaS2, CSN1S2 gene, and can secrete the synthesized target protein casein alpha-S-2 outside the cell and combine with foam; The engineered Kluyveromyces marxianus is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC35182.

[0012] Furthermore, the engineered Kluyveromyces marxianus CGMCC35182 contains the DNA base sequence of SEQ ID No.1.

[0013] Furthermore, the DNA base sequence of SEQ ID No. 1 contains a secretory signal peptide sequence, a casein αs2 gene sequence and a hydrophobic peptide sequence, wherein the secretory signal peptide sequence encodes 31 amino acids, and the cow β-casein gene sequence and the hydrophobic peptide sequence encode 11 hydrophobic polypeptides.

[0014] The present invention also provides the following solution: A method for synthesizing cow casein αs2, wherein the engineered Kluyveromyces marxianus strain is fermented and cultured to obtain cow casein αs2, and the specific steps are: (1) Inoculate the engineered Kluyveromyces marxianus CGMCC35182 in a shake flask or fermenter containing rich culture medium, and ferment at 30-45°C, 200-800 rpm, and aerate for 24-100 hours; (2) Collecting a large amount of fermentation foam produced during the fermentation process under sterile conditions, allowing it to stand to allow the yeast cells on the foam to precipitate, and then returning the yeast cells to a shake flask or fermentation tank. The supernatant after the foam precipitation contains cow casein αs2, which is concentrated and freeze-dried to obtain a freeze-dried powder containing cow casein αs2; (3) After fermentation, separate the yeast cells in the shake flask or fermentation tank and spray dry them to obtain yeast cell powder.

[0015] Furthermore, the culture medium contains a carbon source and a nitrogen source.

[0016] The present invention also provides the following solution: The invention discloses an application of synthetic cow casein αs2 in feed, wherein the freeze-dried powder of the cow casein αs2 and the yeast cell powder are used as sources of animal protein in feed.

[0017] The beneficial effects of the present invention are: the Kluyveromyces marxianus of the present application can produce more foam and separate and purify the target protein from the foam; the microbial fermentation synthesis operation is convenient, the culture cost is low, and the pollution is small; a large number of yeast cells obtained are also one of the good sources of animal protein feed; and the dependence on foreign imports is reduced.

[0018] Kluyveromyces marxianus was used as the expression host for bovine casein alpha S2 (alpha-S-2, casein alpha S2, CSN1S2), and the gene encoding this protein was linked to a gene encoding a hydrophobic peptide to increase the hydrophobicity of CSN1S2, thereby allowing the target protein secreted by Kluyveromyces marxianus to be enriched in the foam, which is beneficial for the separation and purification of the target protein from the foam.

[0019] Therefore, synthesizing these different casein types in microbial cells is of great value. Microbial fermentation is easy to operate, inexpensive to cultivate, and requires minimal land. The resulting yeast cells can also be used as high-protein feed for single-celled animals, killing two birds with one stone.

[0020] This patented method uses the food-safe microorganism Kluyveromyces marxianus as a host for protein synthesis. The gene for αs2-casein is transferred into the yeast, resulting in an engineered Kluyveromyces marxianus containing the αs2-casein gene. Fermentation in a fermenter yields large quantities of αs2-casein and yeast cells, which contain up to 40% single-cell protein and are a valuable source of animal protein feed.

[0021] Reducing dependence on foreign imports, Kluyveromyces marxianus, as a safe microorganism approved for use, is widely used as a protein expression host due to its advantages, including high temperature resistance, strong stress tolerance, robust protein expression and secretion capabilities, rapid growth, and wide raw material utilization. This invention utilizes Kluyveromyces marxianus as the expression host for bovine casein alpha S2 (alpha-S-2, casein alpha S2, CSN1S2). The gene encoding this protein is linked to a gene encoding a hydrophobic polypeptide to increase the hydrophobicity of CSN1S2. This allows the target protein secreted by Kluyveromyces marxianus to accumulate in the foam, facilitating its separation and purification from the foam.

[0022] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Schematic diagram of the Kluyveromyces marxianus expression vector pSWV-km.PDC-CSN1S2 of the present invention.

[0024] Figure 2 This is a diagram showing the cultivation status of transformants obtained by transforming Kluyveromyces marxianus with pSWV-km.PDC-CSN1S2 on a plate containing hygromycin.

[0025] Figure 3 For Figure 2 Diagram of the cultivation status of the transformants grown in the medium for transfer to pure culture.

[0026] Figure 4 Schematic diagram of PCR identification of pure culture transformants.

[0027] Figure 5 Schematic diagram of fermentation of engineered Kluyveromyces marxianus containing hydrophobic peptides.

[0028] Figure 6 Schematic diagram of fermentation of engineered Kluyveromyces marxianus without hydrophobic peptides. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0030] In the description of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0031] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on the specific circumstances.

[0032] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0033] See Figure 1 The present application discloses a Kluyveromyces marxianus yeast capable of synthesizing bovine casein αs2, which is constructed by the following steps: (1) The promoter is km.PDC1, the terminator is the aep gene promoter, the selection marker is the hygromycin B resistance gene sequence (hph), and the homology arm is the 26S rDNA gene sequence. The vector is digested with the restriction endonuclease KpnI and the synthesized target gene is cloned into the vector to construct the new vector pSWV-km.PDC-CSN1S2; (2) The ligation product was transformed into Escherichia coli, and the plasmid vector was extracted in large quantities. The vector was digested with the restriction endonuclease EcoRI, and DNA fragments larger than 5 kb were recovered and used to transform Kluyveromyces marxianus; (3) After transformation, the entire transformation solution containing yeast cells was spread on a YPD solid screening plate containing 500 μg / ml hygromycin B; (4) See Figure 2-3 Incubate the cells in a 30°C incubator for 3-5 days and observe whether individual colonies have grown. Once grown, transfer the colonies one by one to fresh screening solid medium and incubate them in a 30°C incubator for 3-5 days. Repeat the transfer to the screening medium to obtain pure culture transformants. (5) See Figure 4 Genomic DNA of the pure culture transformant was extracted and PCR amplified using a pair of primers for the αs2-casein gene. The primer sequences were: 5'-ACCATGGTGAACAAACACTTCTTGTC-3', 5'-CCAATGAGGCCACCAGTGGTACCAGTTG-3'. A 700 bp DNA band was obtained. However, a control strain of Kluyveromyces marxianus that did not contain the CSN1S2 gene could not amplify this 700 bp DNA. Sequencing of the amplified 700 bp revealed the αs2-casein gene sequence, demonstrating that the pure culture transformant truly contained the target CSN1S2 gene. The control strain indicated by the arrow did not contain the CSN1S2 gene and could not be amplified. The solution of hygromycin B needs to be sterilized. Its ratio per liter is: 30 g glucose, 10 g yeast extract powder, 5 g peptone, and 20 g agar powder. See Figure 5-6The yeast contains a gene encoding cow casein alphas2, which is alpha-S-2, casein alpha S2, CSN1S2 gene, and can secrete the synthesized target protein casein alpha-S-2 to the extracellular space and combine with foam; The engineered Kluyveromyces marxianus strain was deposited with the China General Microbiological Culture Collection under the accession number CGMCC35182. The taxonomic name is Kluyveromyces marxianus. The deposit date is July 10, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0034] The engineered Kluyveromyces marxianus CGMCC35182 contains the DNA base sequence of SEQ ID No.1.

[0035] The DNA base sequence of SEQ ID No. 1 includes a secretory signal peptide sequence, a casein αs2 gene sequence and a hydrophobic peptide sequence. The secretory signal peptide sequence encodes 31 amino acids, and the cow β-casein gene sequence and the hydrophobic peptide sequence encode 11 hydrophobic polypeptides.

[0036] The present invention also provides the following solution: A method for synthesizing cow casein αs2, wherein an engineered strain of Kluyveromyces marxianus is fermented and cultured to obtain cow casein αs2, the specific steps being: (1) Inoculate the engineered Kluyveromyces marxianus CGMCC35182 in a shake flask or fermenter containing rich culture medium, and ferment at 30-45°C, 200-800 rpm, and aerate for 24-100 hours; (2) Collecting a large amount of fermentation foam produced during the fermentation process under sterile conditions, allowing it to stand to allow the yeast cells on the foam to precipitate, and then returning the yeast cells to a shake flask or fermentation tank. The supernatant after the foam precipitation contains cow casein αs2, which is concentrated and freeze-dried to obtain a freeze-dried powder containing cow casein αs2; (3) After fermentation, separate the yeast cells in the shake flask or fermentation tank and spray dry them to obtain yeast cell powder.

[0037] The culture medium contains carbon source and nitrogen source. The carbon source is glucose, fructose, glycerol, xylose and biomass hydrolyzate. The nitrogen source is inorganic nitrogen source containing ammonium and organic nitrogen source. The organic nitrogen source includes yeast extract powder, peptone and corn steep liquor powder.

[0038] The present invention also provides the following solution: The application of synthetic cow casein αs2 in feed, freeze-dried powder of cow casein αs2 and yeast cell powder as sources of animal protein in feed.

[0039] Example 1: Maxicell Kluyveromyces marxianus CGMCC 35182 fermenting to produce αs2-casein with glucose as carbon source The following liquid medium 1.6 liters was prepared in a 3 liter fermentor: glucose monohydrate 150 g / L, yeast extract 8 g / L, peptone 2 g / L, diammonium phosphate 2 g / L, ammonium citrate 2 g / L, carbon source and nitrogen source were sterilized separately, and mixed after sterilization. After cooling, 100 ml of yeast seed liquid was inoculated. The fermentation was carried out at a temperature of 30°C, with stirring at a speed of 200-800 rpm, and aeration at a rate of 3 L / min. The stirring speed was 200 rpm at the beginning of the fermentation, and was increased to 400 rpm when the dissolved oxygen decreased to 50%, to 600 rpm when the dissolved oxygen decreased to 30%, to 800 rpm when the dissolved oxygen decreased to 10% or less, and was maintained at 800 rpm. At 24 hours of fermentation, the OD600 value of the cells increased from the initial 0.8 to 30, and foam began to be produced. No antifoam agent was added during the entire process. As the fermentation proceeded, the amount of cells increased gradually, and the foam also gradually increased and was discharged from the gas outlet. The foam was collected and centrifuged, and the obtained cells were returned to the fermentor for continued fermentation. The supernatant from the centrifugation was lyophilized to obtain white protein powder. After the completion of a single batch of fermentation (complete consumption of glucose), the fermentation broth was centrifuged to obtain yeast cells, which were lyophilized to obtain yeast cell powder. The fermentation broth was separated by a 10 KDa filter membrane to obtain the target protein. Analysis showed that the content of the target protein in the separated foam was 23.5 μg / ml, and the content of the target protein in the supernatant after separation of the fermentation broth was 6.7 μg / ml. The determination method of protein content was according to the instructions of the commercially available Bradford protein determination kit. It can be seen that the content of the target protein in the foam was significantly higher than that in the fermentation broth, indicating that most of the target protein was combined with the foam in the form of hydrophobic protein. This indicates that the C-terminal of αs2-casein combined with a hydrophobic peptide can increase its hydrophobicity and improve its ability to combine with foam, which is beneficial to the separation and purification of the target protein. The total protein content in the obtained yeast powder was determined to be 43.2% of the dry weight. Figure 4 The state of foam production in a 5 liter fermentor was very rich, while the amount of foam of the comparative strain was very small.

[0040] Example 2: Maxicell Kluyveromyces marxianus CGMCC 35182 fermenting to produce αs2-casein with fructose as carbon source In a 3-liter fermenter, 1.6 liters of the following liquid culture medium was prepared: 150 g / L of crystalline fructose, and the remaining nutrients were the same as in Example 1. Fermentation conditions and analytical testing were the same as in Example 1. After fermentation, analysis showed that the target protein content in the separated foam was 26.6 μg / mL, and after separation of the fermentation broth, the target protein content in the supernatant was 7.4 μg / mL. This shows that using fructose as the carbon source yielded results that were essentially consistent with those using glucose as the carbon source. The target protein content in the foam was significantly higher than that in the fermentation broth, indicating that most of the target protein was bound to the foam in the form of hydrophobic proteins. This suggests that the C-terminus of αs2-casein, bound to a hydrophobic peptide, increases its hydrophobicity and improves its ability to bind to the foam, facilitating the separation and purification of the target protein. The protein content in the resulting yeast powder was determined to be 43.3% of the dry weight.

[0041] Example 3: Fermentation of αs2-casein by Kluyveromyces marxianus CGMCC35182 using glycerol as a carbon source In a 3-liter fermentor, 1.6 liters of the following liquid culture medium was prepared: 150 g / L glycerol, with the remaining nutrients being the same as in Example 1. Fermentation conditions and analytical testing were the same as in Example 1. After fermentation, analysis revealed that the target protein content in the isolated foam was 24.6 μg / mL, and after separation of the fermentation broth, the target protein content in the supernatant was 5.8 μg / mL. This indicates that using glycerol as the carbon source resulted in slightly lower protein content than using glucose as the carbon source. However, the target protein content in the foam was still significantly higher than that in the fermentation broth, indicating that the target protein was largely bound to the foam via a hydrophobic peptide. This suggests that the binding of the C-terminus of αs2-casein to a hydrophobic peptide increases its hydrophobicity, improving its ability to bind to the foam and facilitating the separation and purification of the target protein. The protein content in the resulting yeast powder was determined to be 43.2% of the total protein by dry weight.

[0042] Example 4: Fermentation of αs2-casein by Kluyveromyces marxianus CGMCC35182 using xylose as a carbon source A liquid medium 1.6 liter was prepared in a 3 liter fermentor as follows: 150 g / L xylose, and the rest of the nutrient components were the same as in Example 1. The fermentation conditions and analysis were the same as in Example 1. After the fermentation, the content of the target protein in the separated foam was 24.2 μg / mL, and the content of the target protein in the supernatant after separation of the broth was 5.2 μg / mL. It can be seen that the protein content is lower when using xylose as the carbon source than when using glucose as the carbon source. However, the content of the target protein in the foam is still significantly higher than the content of the target protein in the broth, indicating that most of the target protein is combined with the foam in the form of hydrophobic protein. This indicates that the C-terminal of αs2-casein combined with a hydrophobic peptide can increase its hydrophobicity and improve its ability to combine with the foam, which is beneficial to the separation and purification of the target protein. The total protein content in the obtained yeast powder was 43.6% of the dry weight. K. marxianus can use xylose, but it is not as effective as using glucose or fructose as the carbon source. Glucose is preferred as the carbon source.

[0043] Example 5: K. marxianus CGMCC 35182 Fermentation to Synthesize αs2-Casein Using Biomass Hydrolysate as the Carbon Source A liquid medium 1.6 liter was prepared in a 3 liter fermentor as follows: 300 g / L corn cob hydrolysate (soluble solid content 50%, i.e. containing 150 g / L soluble solids), and the rest of the nutrient components were the same as in Example 1. The fermentation conditions and analysis were the same as in Example 1. After the fermentation, the content of the target protein in the separated foam was 17.4 μg / mL, and the content of the target protein in the supernatant after separation of the broth was 4.2 μg / mL. It can be seen that the protein content is much lower when using corn cob hydrolysate as the carbon source than when using glucose as the carbon source. However, the content of the target protein in the foam is still significantly higher than the content of the target protein in the broth, indicating that most of the target protein is combined with the foam in the form of hydrophobic protein. This indicates that the C-terminal of αs2-casein combined with a hydrophobic peptide can increase its hydrophobicity and improve its ability to combine with the foam, which is beneficial to the separation and purification of the target protein. The total protein content in the obtained yeast powder was 38.6% of the dry weight. K. marxianus can use biomass hydrolysate, but it is not as effective as using glucose or fructose as the carbon source. Glucose is preferred as the carbon source.

[0044] Example 6: K. marxianus CGMCC 35182 Fermentation to Synthesize αs2-Casein Using Glucose as the Carbon Source at 40°C In a 3-liter fermentor, 1.6 liters of the following liquid culture medium were prepared: 150 g / L glucose, with the remaining nutrients as in Example 1. The fermentation temperature was 40°C, and all other conditions and analytical testing were the same as in Example 1. After fermentation, analysis revealed that the target protein content in the isolated foam was 33.7 μg / mL, and in the supernatant after separation of the fermentation broth, it was 7.6 μg / mL. This indicates that fermentation using glucose as the carbon source at 40°C resulted in slightly higher protein content than that achieved at 30°C. The significantly higher target protein content in the foam compared to the fermentation broth indicates that the target protein is primarily bound to the foam via a hydrophobic peptide. This suggests that the binding of the C-terminus of αs2-casein to a hydrophobic peptide increases its hydrophobicity, enhancing its ability to bind to the foam and facilitating the isolation and purification of the target protein. The resulting yeast powder was measured to contain 43.5% of the total protein by dry weight. Kluyveromyces marxianus is a thermotolerant yeast that grows well at 45°C.

[0045] Example 7: Synthesis of αs2-casein by fermentation of Kluyveromyces marxianus CGMCC35182 using glucose as a carbon source at 45°C In a 3-liter fermentor, 1.6 liters of the following liquid culture medium were prepared: 150 g / L glucose, with the remaining nutrients as in Example 1. The fermentation temperature was 45°C, and all other conditions and analytical testing were the same as in Example 1. After fermentation, analysis revealed that the target protein content in the isolated foam was 29.4 μg / mL, and in the supernatant after separation of the fermentation broth, the target protein content was 7.7 μg / mL. This indicates that the protein content in the foam, using glucose as the carbon source, was lower than that in the fermentation broth at 40°C. The significantly higher target protein content in the foam than in the fermentation broth indicates that the target protein was primarily bound to the foam via a hydrophobic peptide. This suggests that the binding of the hydrophobic peptide to the C-terminus of αs2-casein increases its hydrophobicity, improving its ability to bind to the foam and facilitating the isolation and purification of the target protein. The resulting yeast powder was assayed to contain 44.5% of the total protein by dry weight. Kluyveromyces marxianus is a heat-resistant yeast. Although it can grow at 45 degrees, it is not the optimal growth temperature, which leads to a decrease in protein synthesis ability. Therefore, it is preferably fermented at 40 degrees.

[0046] Example 8: Experiment on feeding mice with Kluyveromyces marxianus CGMCC35182 cell powder The yeast cell powder obtained in Example 1 was mixed with ordinary mouse feed at a ratio of 5% and then fed to mice. The mice were weaned at 4 weeks of age, weighing 20±2 grams each. Ten mice were fed for 30 consecutive days and weighed. The weights were compared with a control group (also 10 mice fed only ordinary feed). After 30 consecutive days of feeding, the average weight of the 10 mice fed with K. marxianus yeast powder was 33±1.5 grams, while the average weight of the control group (fed ordinary feed) was 26±1.5 grams. This indicates that mice fed with yeast powder gained more weight than the control group. This result suggests that feeding other animals (such as poultry and pigs) with feed containing K. marxianus yeast powder may result in even greater weight gain, potentially benefiting animal growth.

[0047] In summary, the present invention provides a method for synthesizing cow casein AS2 using yeast and its application in feed. The Kluyveromyces marxianus of the present application can produce more foam and separate and purify the target protein from the foam; the microbial fermentation synthesis is easy to operate, with low culture cost and low pollution; the large amount of yeast cells obtained is also one of the good sources of animal protein feed, reducing dependence on foreign imports.

[0048] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0049] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A Kluyveromyces marxianus yeast capable of synthesizing cow casein αs2, characterized in that: It is constructed by the following steps: (1) The promoter used was km.PDC1, the terminator used was the aep gene promoter, the screening marker used was the hygromycin B resistance gene sequence (hph), and the homology arm used was the 26S rDNA gene sequence. The vector was digested with restriction endonuclease KpnI, and the synthesized target gene was cloned into the vector to construct a new vector pSWV-km.PDC-CSN1S2; (2) The ligation product was transformed into Escherichia coli, and the plasmid vector was extracted in large quantities. The vector was digested with the restriction endonuclease EcoRI, and DNA fragments larger than 5 kb were recovered and used to transform Kluyveromyces marxianus; (3) After transformation, the entire transformation solution containing yeast cells was spread on a YPD solid screening plate containing 500 μg / ml hygromycin B; (4) Culture in a 30-degree incubator for 3-5 days and observe whether single colonies have grown. After growth, transfer the single colonies one by one to fresh screening solid culture medium, culture in a 30-degree incubator for 3-5 days, and repeat the transfer to the screening medium to obtain pure culture transformants; (5) The genomic DNA of the pure culture transformant was extracted and PCR amplified using a pair of primers for the αs2-casein gene. The primer sequences were: 5'-ACCATGGTGAACAAACACTTCTTGTC-3', 5'-CCAATGAGGC CACCAGTGGTACCAGTTG-3'. A DNA band of 700 bp was obtained. However, the Kluyveromyces marxianus strain that did not contain the CSN1S2 gene was used as a control strain and could not amplify this 700 bp DNA. The amplified 700 bp was sequenced and the results showed that it was the αs2-casein gene sequence, proving that the pure culture transformant was the transformant that truly contained the target CSN1S2 gene. The hygromycin B solution needs to be sterilized; The yeast contains a gene encoding cow casein αs2, which is alpha-S-2, casein alpha S2, CSN1S2 gene, and can secrete the synthesized target protein casein alpha-S-2 outside the cell and combine with foam. The engineered Kluyveromyces marxianus is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC35182.

2. The Kluyveromyces marxianus capable of synthesizing cow casein αs2 according to claim 1, characterized in that: The engineered Kluyveromyces marxianus CGMCC35182 contains the DNA base sequence of SEQ ID No.

1.

3. The Kluyveromyces marxianus capable of synthesizing cow casein αs2 according to claim 2, characterized in that: The DNA base sequence of SEQ ID No. 1 comprises a secretory signal peptide sequence, a casein αs2 gene sequence and a hydrophobic peptide sequence, wherein the secretory signal peptide sequence encodes 31 amino acids, and the cow β-casein gene sequence and the hydrophobic peptide sequence encode 11 hydrophobic polypeptides.

4. A method for synthesizing cow casein αs2 according to any one of claims 1 to 2, characterized in that: The engineered Kluyveromyces marxianus strain is fermented and cultured to obtain cow casein αs2, specifically in the following steps: (1) Inoculate the engineered Kluyveromyces marxianus CGMCC35182 in a shake flask or fermenter containing rich culture medium, and ferment at 30-45°C, 200-800 rpm, and aerate for 24-100 hours; (2) Collecting a large amount of fermentation foam produced during the fermentation process under sterile conditions, allowing it to stand to allow the yeast cells on the foam to precipitate, and then returning the yeast cells to a shake flask or fermentation tank. The supernatant after the foam precipitation contains cow casein αs2, which is concentrated and freeze-dried to obtain a freeze-dried powder containing cow casein αs2; (3) After fermentation, separate the yeast cells in the shake flask or fermentation tank and spray dry them to obtain yeast cell powder.

5. The method for synthesizing cow β-casein according to claim 4, characterized in that: The culture medium contains a carbon source and a nitrogen source.

6. The use of the synthetic cow casein αs2 in feed according to claim 4, characterized in that: The freeze-dried powder of cow casein αs2 and the yeast cell powder are used as sources of animal protein in feed.

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