Kluyveromyces marxianus capable of synthesizing dairy cow casein alpha-s-1 and application of Kluyveromyces marxianus in feed

Abstract

The invention discloses a method for synthesizing dairy cow casein alpha-S-1 by using saccharomycetes and application of the dairy cow casein alpha-S-1 in feed, and relates to the technical field of biology. According to the method for synthesizing the dairy cow casein alpha-S-1 by using the saccharomycetes and the application of the saccharomycetes in the feed, the saccharomycetes contain genes for coding the dairy cow casein alpha-S-1 and can secrete the synthesized target dairy cow casein alpha-S-1 to the outside of cells, and freeze-dried powder containing the dairy cow casein alpha-S-1 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 alpha-S-1 by using 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 about 40% of total casein. It consists of 207 amino acids and is divided into three peptide chains: αs1-CN-1, αs1-CN-I, and αs1-CN-I. αs1-CN-l 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 connected to each other through disulfide bonds, forming a large polypeptide chain complex.

[0004] αs2-casein is the second most abundant casein in bovine milk, accounting for about 30% of total casein. It consists of 207 amino acids and is divided into three peptide chains: αs2-CN-1, αs2-CN-II, and αs2-CN-I. Both αs2-CN-l and αs2-CN-II contain 99 amino acids, while αs2-CN-I contains 9 amino acids. These three peptide chains are connected to each other through disulfide bonds, forming a large polypeptide chain complex.

[0005] β-casein accounts for about 10% of total casein. It consists of 209 amino acids and is divided into two peptide chains: β-CN-I and β-CN-II. β-CN-I contains 50 amino acids, while β-CN-II contains 159 amino acids. These two peptide chains are connected to each other through non-covalent bonds, forming a large polypeptide chain complex.

[0006] κ-casein is the smallest casein in bovine milk, with the least content, about 0.1%. It consists of 169 amino acids. κ-casein has a unique structure, consisting of an α-helix and a β-sheet region. The α-helix region contains 64 amino acids, while the β-sheet region contains 48 amino acids. These two regions are connected to each other through non-covalent bonds, forming a small polypeptide chain.

[0007] Overall, the caseins in bovine milk are composed of four different caseins, including αs1-casein, αs2-casein, β-casein, and κ-casein. Each of them contains a different number of amino acids and is connected to each other through non-covalent bonds or disulfide bonds to form a polypeptide chain complex. This complex primary structure endows caseins with special functions and properties, which has important influence on the quality and nutritional value of bovine milk.

[0008] The main source of protein in the diet of Chinese residents is various animal meat and eggs. In order to obtain enough meat and eggs, it is necessary to develop the breeding industry. However, raising cows on farms to obtain casein is labor-intensive, has high cost, and produces a large amount of animal waste, which can cause serious environmental pollution if not properly treated.

[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 alpha-S-1 using yeast and its application in feed, so as to solve the problems of obtaining casein by raising dairy cows on farms, which is labor-intensive, costly, and has a large amount of animal waste discharge, which can easily cause significant environmental pollution if improperly handled; and the problem of importing a large amount of soybeans 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 for synthesizing bovine casein alpha-S-1 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-CSN1S1; (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 αs1-casein gene. The primer sequences were: 5'-ACCATGGTGAACAAACACTTCTTGTC-3', 5'-GGTGGTGGTGGTGGTGCCACAGTGG-3'. A DNA band of 700 bp was obtained. However, the Kluyveromyces marxianus strain that did not contain the CSN1S1 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 αs1-casein gene sequence, proving that the pure culture transformant was the transformant that truly contained the target CSN1S1 gene. The hygromycin B solution needs to be sterilized; The yeast contains a gene encoding cow casein alpha-S-1, which is casein alpha S1, CSN1S1 gene, and can secrete the synthesized target protein cow casein alpha-S-1 outside the cell and combine with foam; The engineered Kluyveromyces marxianus is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC35183.

[0012] Furthermore, the engineered Kluyveromyces marxianus CGMCC35183 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 cow casein alpha-S-1 gene sequence and a hydrophobic peptide sequence, wherein the secretory signal peptide sequence encodes 31 amino acids, and the cow casein alpha-S-1 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 alpha-S-1, wherein the engineered Kluyveromyces marxianus strain is fermented to obtain cow casein alpha-S-1, and the specific steps are as follows: (1) Inoculate the engineered Kluyveromyces marxianus CGMCC35183 in a shake flask or fermenter containing rich culture medium, and ferment at 30-45°C, 100-800 rpm, and aerate for 24-100 hours; (2) Collecting a large amount of fermentation foam produced during the fermentation process under sterile conditions, allowing the yeast cells on the foam to settle, and returning the yeast cells to the shake flask or fermentation tank. The supernatant after the foam is separated contains cow casein alpha-S-1, which is concentrated and the concentrate is freeze-dried to obtain a freeze-dried powder containing cow casein alpha-S-1; (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 alpha-S-1 in feed. The freeze-dried powder of the cow casein alpha-S-1 and the yeast cell powder are used as sources of animal protein in the 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 cow casein alpha S1 (CSN1S1), and the gene encoding this protein was linked to a gene encoding a hydrophobic peptide to increase the hydrophobicity of CSN1S1. This allowed the target protein secreted by Kluyveromyces marxianus to accumulate in the foam, facilitating its separation and purification 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 invention uses the food-safe microorganism Kluyveromyces marxianus as a host for protein synthesis. The gene for αs1-casein is transferred into the yeast, resulting in an engineered Kluyveromyces marxianus containing the αs1-casein gene. Fermentation in a fermenter yields large quantities of αs1-casein and yeast cells. These yeast cells contain up to 40% single-cell protein, making them an excellent 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-S-1 (CSN1S1). The gene encoding this protein is linked to a gene encoding a hydrophobic polypeptide to increase the hydrophobicity of CSN1S1. 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-CSN1S1 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-CSN1S1 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 Kluyveromyces marxianus yeast for synthesizing bovine casein alpha-S-1 as described in the present application 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-CSN1S1; (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 αs1-casein gene. The primer sequences were: 5'-ACCATGGTGAACAAACACTTCTTGTC-3', 5'-GGTGGTGGTGGTGGTGCCACAGTGG-3'. A DNA band of 700 bp was obtained. However, a control strain of Kluyveromyces marxianus that did not contain the CSN1S1 gene could not amplify this 700 bp DNA. Sequencing of the amplified 700 bp revealed the αs1-casein gene sequence, demonstrating that the pure culture transformant truly contained the target CSN1S1 gene. The control strain indicated by the arrow did not contain the CSN1S1 gene and could not be amplified. The solution of hygromycin B needs to be sterilized; See Figure 5-6 The yeast contains a gene encoding cow casein alpha-S-1, which is casein alpha S1, CSN1S1 gene, and can secrete the synthesized target protein cow casein alpha-S-1 outside the cell and combine with foam; The engineered Kluyveromyces marxianus yeast was deposited with the China General Microbiological Culture Collection under the accession number CGMCC35183. The Latin name is Kluyveromyces marxianus. The deposit date is July 10, 2025.

[0034] The engineered Kluyveromyces marxianus CGMCC35183 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 cow casein alpha-S-1 gene sequence, and a hydrophobic peptide sequence. The secretory signal peptide sequence encodes 31 amino acids, and the cow casein alpha-S-1 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 alpha-S-1, wherein an engineered strain of Kluyveromyces marxianus is fermented and cultured to obtain cow casein alpha-S-1, the specific steps being: (1) Inoculate the engineered Kluyveromyces marxianus CGMCC35183 in a shake flask or fermenter containing rich culture medium, and ferment at 30-45°C, 100-800 rpm, and aerate for 24-100 hours; (2) Collecting a large amount of fermentation foam produced during the fermentation process under sterile conditions, allowing the yeast cells on the foam to settle, and returning the yeast cells to the shake flask or fermentation tank. The supernatant after the foam is separated contains cow casein alpha-S-1, which is concentrated and the concentrate is freeze-dried to obtain a freeze-dried powder containing cow casein alpha-S-1; (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 alpha-S-1 in feed, freeze-dried powder of cow casein alpha-S-1 and yeast cell powder as sources of animal protein in feed.

[0039] Example 1: Fermentation of αs1-casein by Kluyveromyces marxianus CGMCC35183 using glucose as a carbon source In a 5-liter fermentor, prepare 3 liters of the following liquid medium: 150 g / L glucose monohydrate, 8 g / L yeast extract powder, 2 g / L peptone, 2 g / L diammonium phosphate, and 2 g / L ammonium citrate. Sterilize the carbon and nitrogen sources separately and mix them after sterilization. After cooling, inoculate 100 ml of yeast seed liquid. Fermentation is carried out at 30°C and agitation at 200-800 rpm with aeration at a rate of 3 liters / minute. Initially, the agitation speed is 200 rpm. When the dissolved oxygen (DO) drops to 50%, the speed is increased to 400 rpm, then to 600 rpm when the DO drops to 30%, and then to 800 rpm when the DO drops below 10%, where it is maintained. After 24 hours of fermentation, the cell OD600 value increases from an initial value of 0.8 to 32, and foaming begins. No defoaming agent is added during the entire fermentation process. As fermentation progresses, the cell mass gradually increases, along with the amount of foam, which is discharged from the gas outlet. The foam is collected and centrifuged, and the resulting cells are returned to the fermentor for further fermentation. The supernatant is lyophilized to produce a white protein powder. After the single batch fermentation is completed (glucose is completely consumed), the fermentation broth is centrifuged, and the yeast cells are separated and lyophilized to produce yeast cell powder. The fermentation broth is filtered through a 10 kDa filter to separate the target protein. Analysis revealed that the target protein content in the separated foam was 25 μg / mL, and in the supernatant after separation of the fermentation broth, the target protein content was 8 μg / mL. Protein content was determined according to the instructions of the Bradford protein assay kit, a commercially available kit. The target protein content in the foam was significantly higher than that in the fermentation broth, indicating 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 αs1-casein increases its hydrophobicity, enhancing its ability to bind to the foam and facilitating its isolation and purification. Protein content in the resulting yeast powder was determined to be 45% total protein by dry weight. Figure 4 This is the state of foaming in a 5-liter fermentation tank. The foam is very rich, while the control bacteria has very little foam.

[0040] Example 2: Fermentation of αs1-casein by Kluyveromyces marxianus CGMCC35183 using fructose as a carbon source In a 5-liter fermentation tank, 3 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. The fermentation conditions and analytical tests were the same as in Example 1. After the fermentation was completed, analysis showed that the content of the target protein in the separated foam was 28 μg / mL, and after the fermentation broth was separated, the content of the target protein in the supernatant was 7.2 μg / mL. It can be seen that using fructose as the carbon source yielded results that were basically consistent with those using glucose as the carbon source. 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 bound to the foam in the form of hydrophobic protein. This indicates that the C-terminus of αs1-casein, after binding to a hydrophobic peptide, can increase its hydrophobicity and improve its ability to bind to the foam, which is beneficial to the separation and purification of the target protein. The protein in the obtained yeast powder was measured to have a total protein content of 45% of the dry weight.

[0041] Example 3: Fermentation of αs1-casein by Kluyveromyces marxianus CGMCC35183 using glycerol as a carbon source In a 5-liter fermentor, 3 liters of the following liquid culture medium were 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 μg / mL, and after separation of the fermentation broth, the target protein content in the supernatant was 5.4 μg / mL. This indicates that using glycerol as the carbon source resulted in a 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 hydrophobic proteins. This suggests that the binding of the C-terminus of αs1-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 42% of the dry weight.

[0042] Example 4: Fermentation of αs1-casein by Kluyveromyces marxianus CGMCC35183 using xylose as a carbon source In a 5-liter fermentor, 3 liters of the following liquid culture medium were prepared: 150 g / L xylose, with the remaining nutrients 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 22 μg / mL, and that in the supernatant after separation of the fermentation broth was 4.2 μg / mL. This indicates that using xylose as the carbon source resulted in 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 hydrophobic proteins. This suggests that the binding of the hydrophobic peptide to the C-terminus of αs1-casein increases its hydrophobicity, enhancing its ability to bind to the foam and facilitating the isolation and purification of the target protein. The protein content of the resulting yeast powder was determined to be 42.6% of the dry weight. Kluyveromyces marxianus can utilize xylose, but not as effectively as using glucose or fructose as carbon sources. Glucose is preferred as the carbon source.

[0043] Example 5: Fermentation of αs1-casein by Kluyveromyces marxianus CGMCC35183 using biomass hydrolysate as a carbon source In a 5-liter fermentor, 3 liters of the following liquid culture medium were prepared: 300 g / L corncob hydrolyzate (50% soluble solids, i.e., 150 g / L soluble solids). 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 revealed that the target protein content in the isolated foam was 16 μg / mL, and in the supernatant after separation of the fermentation broth, the target protein content was 3.2 μg / mL. This indicates that using corncob hydrolyzate as a carbon source significantly reduces the protein content compared to using glucose as a 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 primarily bound to the foam via hydrophobic proteins. This suggests that the binding of the hydrophobic peptide to the C-terminus of αs1-casein increases its hydrophobicity, enhancing its ability to bind to the foam and facilitating the isolation and purification of the target protein. Protein content in the resulting yeast powder was determined to be 32.6% of the total protein by dry weight. Kluyveromyces marxianus can utilize biomass hydrolyzate, but not as effectively as glucose or fructose as a carbon source. Glucose is preferred as a carbon source.

[0044] Example 6: Synthesis of αs1-casein by fermentation of Kluyveromyces marxianus CGMCC35183 using glucose as a carbon source at 40°C In a 5-liter fermentor, 3 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 31 μg / mL, and in the supernatant after separation of the fermentation broth, the target protein content was 8.2 μg / mL. This indicates that fermentation using glucose as the carbon source at 40°C resulted in slightly higher protein content than that observed at 30°C. The significantly higher target protein content in the foam compared to the fermentation broth indicates that the target protein is largely bound to the foam via a hydrophobic peptide. This suggests that the binding of the C-terminus of αs1-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 44.5% of the total protein by dry weight. Kluyveromyces marxianus is a thermotolerant yeast that grows well at 45°C.

[0045] Example 7: Synthesis of αs1-casein by fermentation of Kluyveromyces marxianus CGMCC35183 using glucose as a carbon source at 45°C In a 5-liter fermentor, 3 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 27 μg / mL, and in the supernatant after separation of the fermentation broth, the target protein content was 6.2 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 largely bound to the foam via a hydrophobic peptide. This suggests that the binding of the C-terminus of αs1-casein to a hydrophobic peptide 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 42.5% 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 CGMCC35183 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 30±1.5 grams, while the average weight of the control group (fed ordinary feed) was 27±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] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

[0048] In summary, the present invention provides a method for synthesizing cow casein alpha-S-1 using yeast and its application in feed. The Kluyveromyces marxianus of the present invention can produce more foam and separate and purify the target protein from the foam; the microbial fermentation synthesis is easy to operate, has low culture cost and low pollution; the large amount of yeast cells obtained is also a good source of animal protein feed, reducing dependence on foreign imports.

[0049] 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.

[0050] 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 for synthesizing cow casein alpha-S-1, 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-CSN1S1; (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 once on 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 αs1-casein gene. The primer sequences were: 5'-ACCATGGTGAACAAACACTTCTTGTC-3', 5'-GGTGGTGGTGGTGGTGCCACAGTGG-3'. A DNA band of 700 bp was obtained. However, the Kluyveromyces marxianus strain that did not contain the CSN1S1 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 αs1-casein gene sequence, proving that the pure culture transformant was the transformant that truly contained the target CSN1S1 gene. The hygromycin B solution needs to be sterilized; The yeast contains a gene encoding cow casein alpha-S-1, which is caseinalpha S1, CSN1S1 gene, and can secrete the synthesized target protein cow casein alpha-S-1 outside the cell and combine with foam; The engineered Kluyveromyces marxianus is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC35183.

2. The Kluyveromyces marxianus for synthesizing cow casein alpha-S-1 according to claim 1, characterized in that: The engineered Kluyveromyces marxianus CGMCC35183 contains the DNA base sequence of SEQ ID No.

1.

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

4. A method for synthesizing cow casein alpha-S-1 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 alpha-S-1, specifically in the following steps: (1) Inoculate the engineered Kluyveromyces marxianus CGMCC35183 in a shake flask or fermenter containing rich culture medium, and ferment at 30-45°C, 100-800 rpm, and aerate for 24-100 hours; (2) Collecting a large amount of fermentation foam produced during the fermentation process under sterile conditions, allowing the yeast cells on the foam to settle, and returning the yeast cells to the shake flask or fermentation tank. The supernatant after the foam is separated contains cow casein alpha-S-1, which is concentrated and the concentrate is freeze-dried to obtain a freeze-dried powder containing cow casein alpha-S-1; (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 casein alpha-S-1 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 alpha-S-1 in feed according to claim 4, characterized in that: The freeze-dried powder of cow casein alpha-S-1 and the yeast cell powder are used as sources of animal protein in feed.

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