Kluyveromyces marxianus capable of synthesizing beta-casein of dairy cow and application of Kluyveromyces marxianus in feed
Patent Information
- Application Number
- CN202511020829.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-24
- Publication Date
- 2026-01-13
Smart Images
Figure CN121320122A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biotechnology, in particular to a method for synthesizing cow beta-casein by using yeast and its application in feed. BACKGROUND
[0002] Casein is the most abundant protein in dairy products, and is the most abundant protein in the milk secreted by various mammals. It is also the most consumed protein. For example, the protein in cow's milk is mainly composed of casein, whey protein and lactoglobulin, among which casein accounts for the highest proportion, about 80%, whey protein accounts for about 11.5%, and lactoglobulin accounts for about 3.3%. These proteins have many benefits to the human body, such as providing energy, promoting growth and development, maintaining tissue repair, etc. The content of casein determines the content of protein in dairy products, so casein plays an important role in the quality control of dairy products. Casein is composed of four proteins, such as the four different caseins in cow's milk, namely alpha-S-1 (alpha-S-1, abbreviated as αs1-casein, CSN1S1), alpha-S-2 (alpha-S-1, αs2-casein, CSN1S2), beta-casein (Beta-casein, CSN2), and kappa-casein (kappa-casein, abbreviated as κ-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 of caseins (alpha-S-1, alpha-S-2, beta and 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 increasing, and the content of protein in the feed is one of the key factors to evaluate the quality of the feed. At present, soybean meal is still the main source of feed protein, and a large amount of soybeans are imported from abroad every year to produce soybean meal. SUMMARY
[0010] The purpose of the present application is to provide a method for synthesizing cow beta-casein by using yeast and its application in feed, to solve the problems of labor-intensive, high cost, large amount of animal pollution, and easy to cause serious environmental pollution by improper treatment when obtaining casein by feeding cows in farms; and the problem of importing a large amount of soybeans from abroad every year to produce soybean meal as feed.
[0011] To achieve the above purpose, the present application provides the following technical solutions: A Kluyveromyces marxianus capable of synthesizing cow beta-casein is obtained by the following steps: (1) The promoter adopts km.PDC1, the terminator adopts aep gene promoter, the screening marker adopts hygromycin B resistance gene sequence-hph, and the homologous arm adopts 26S rDNA gene sequence; the synthesized target gene is cloned into the vector by using restriction enzyme KpnI to cut the vector, and a new vector pSWV-km.PDC-CSN2 is formed; (2) The ligation product is transformed into Escherichia coli, and a large amount of plasmid vector is extracted, and the vector is cut by using restriction enzyme EcoRI, and the DNA fragment larger than 5 kb is recovered for transformation of Kluyveromyces marxianus; (3) After transformation, the transformation liquid containing yeast cells is completely coated on YPD solid screening plate containing 500 micrograms / milliliter of hygromycin B; (4) Cultured in a 30-degree incubator for 3-5 days, and observe whether single colonies grow; after growing, the grown single colonies are transferred one by one to fresh screening solid culture medium, cultured in a 30-degree incubator for 3-5 days, and transferred again on the screening medium to obtain pure culture transformants; (5) Extract the genomic DNA of the pure culture transformants, and perform PCR amplification by using a pair of primers of the cow beta-casein gene, and the sequence of the pair of primers is: 5'-ACCATGGTGAACAAACACTTCTTGTC-3', 5'-CCAATGAGGCCACCAGTGGTACCAGTTG-3', to obtain a DNA band with a size of 700 bp, and Kluyveromyces marxianus without CSN2 gene as a comparative strain cannot expand the 700 bp DNA, and the 700 bp amplified is sequenced, and the result shows that it is the sequence of alpha s1-casein gene, proving that the pure culture transformants are the transformants containing the target CSN2 gene; The solution of hygromycin B needs to be sterilized; The yeast contains a gene encoding cow beta-casein, the gene of CSN2, and can secrete the synthesized target protein cow beta-casein to the extracellular space and combine with the foam; The engineered Kluyveromyces marxianus is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC35181.
[0012] Further, the engineered Kluyveromyces marxianus CGMCC35181 contains the DNA base of sequence SEQ ID No. 1.
[0013] Further, the sequence of the DNA base of SEQ ID No. 1 comprises a secretion signal peptide sequence encoding 31 amino acids, a cow beta-casein gene sequence, and a hydrophobic peptide sequence encoding 11 hydrophobic polypeptides.
[0014] The application also provides the following scheme: A method for synthesizing cow beta-casein, characterized in that the Kluyveromyces marxianus engineering strain is fermented to obtain cow beta-casein, and the specific steps are as follows: (1) inoculate the engineered Kluyveromyces marxianus CGMCC35181 in a shake flask or a fermenter containing a rich culture medium, and ferment at 30-45°C, 100-800 rpm, and under aeration for 24-100 hours; (2) collect a large amount of fermentation foam generated during the fermentation process under sterile conditions, and let it stand so that the yeast cells on the foam precipitate, and then return the yeast cells to the shake flask or the fermenter; the supernatant after the separation of the precipitated foam contains cow beta-casein, which is concentrated, frozen, and freeze-dried to obtain a freeze-dried powder containing cow beta-casein; (3) after the fermentation is completed, separate the yeast cells in the shake flask or the fermenter, and spray dry to obtain yeast cell powder.
[0015] Further, the culture medium contains a carbon source and a nitrogen source.
[0016] The application also provides the following scheme: The application of the synthesized cow beta-casein in feed, and the freeze-dried powder of the cow beta-casein and the yeast cell powder as a source of animal protein in feed.
[0017] The application has the advantages that the Kluyveromyces marxianus of the application can produce more foam and separate and purify the target protein from the foam; the microbial fermentation synthesis is convenient to operate, has low culture cost, and causes little pollution; a large amount of yeast cell obtained is also one of good sources of animal protein feed; and the dependence on foreign imports is reduced.
[0018] Therefore, it is of great value to synthesize these different kinds of casein in microbial cells. Because microbial fermentation synthesis is convenient, the culture cost is low, and it does not occupy too much land area. After fermentation, the yeast cells obtained can also be used as high-protein single-cell animal feed, achieving two goals at once.
[0019] The present application adopts the food safety microorganism Kluyveromyces marxianus as a protein synthesis host, transfers the gene of beta-casein into the yeast, and obtains engineered Kluyveromyces marxianus containing the gene of beta-casein. A large amount of beta-casein is obtained by fermentation in a fermenter, and a large amount of yeast cells are also obtained, which contain up to 40% of single-cell protein and are also one of the good animal protein feed sources.
[0020] The dependence on foreign imports is reduced. Kluyveromyces marxianus is a safe microorganism approved for use, has the advantages of high temperature resistance, strong stress resistance, strong protein expression and secretion capacity, rapid growth, wide raw material utilization range, and is widely used as a protein expression host. The present application uses Kluyveromyces marxianus as an expression host of beta-casein (CSN2) of a cow, and links the gene encoding the protein with a hydrophobic polypeptide coding gene to improve the hydrophobicity of CSN2, so that the target protein secreted by Kluyveromyces marxianus is enriched on the foam, which is beneficial to the separation and purification of the target protein from the foam.
[0021] The above description is only a summary of the technical solutions of the present application. In order to more clearly understand the technical means of the present application, and to implement the content of the description, the following will be described in detail with reference to the preferred embodiments of the present application and in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 The above description is only a summary of the technical solutions of the present application. In order to more clearly understand the technical means of the present application, and to implement the content of the description, the following will be described in detail with reference to the preferred embodiments of the present application and in conjunction with the accompanying drawings.
[0023] Figure 2 The above description is only a summary of the technical solutions of the present application. In order to more clearly understand the technical means of the present application, and to implement the content of the description, the following will be described in detail with reference to the preferred embodiments of the present application and in conjunction with the accompanying drawings.
[0024] Figure 3 The above description is only a summary of the technical solutions of the present application. In order to more clearly understand the technical means of the present application, and to implement the content of the description, the following will be described in detail with reference to the preferred embodiments of the present application and in conjunction with the accompanying drawings. Figure 2 The above description is only a summary of the technical solutions of the present application. In order to more clearly understand the technical means of the present application, and to implement the content of the description, the following will be described in detail with reference to the preferred embodiments of the present application and in conjunction with the accompanying drawings.
[0025] Figure 4 The above description is only a summary of the technical solutions of the present application. In order to more clearly understand the technical means of the present application, and to implement the content of the description, the following will be described in detail with reference to the preferred embodiments of the present application and in conjunction with the accompanying drawings.
[0026] Figure 5Schematic diagram of fermentation of engineered K. marxianus containing hydrophobic peptide.
[0027] Figure 6 Schematic diagram of fermentation of engineered K. marxianus not containing hydrophobic peptide. DETAILED DESCRIPTION
[0028] The technical solutions of the present application will be described below in conjunction with the drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.
[0029] In the description of the present application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings, and are only for the purpose of facilitating the description of the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application. In addition, the terms "first", "second", "third" are only for descriptive purposes and cannot be understood as indicating or implying relative importance.
[0030] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connection" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.
[0031] In addition, the technical features involved in different embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.
[0032] Please refer to Figure 1 A K. marxianus capable of synthesizing bovine β-casein is constructed by the following steps: (1) The promoter uses km.PDC1, the terminator uses the promoter of aep gene, the selection marker uses the hygromycin B resistance gene sequence-hph, and the homologous arm uses the 26S rDNA gene sequence. The synthesized target gene is cloned into the vector by using restriction enzyme Kpnl to cut the vector, and a new vector pSWV-km.PDC-CSN2 is constructed. (2) Transform the ligation product into E. coli, extract the plasmid vector in large quantities, and use the vector to recover the DNA fragment larger than 5 kb by EcoRI restriction enzyme for transforming K. marxianus; (3) After transformation, the transformation liquid containing the yeast cells is entirely coated on the YPD solid screening plate containing 500 micrograms / milliliter of hygromycin B; (4) Please refer to Figures 2-3 Cultivate in a 30-degree incubator for 3-5 days, and observe whether single colonies grow; after growth, the grown single colonies are individually transferred to fresh screening solid culture medium, cultivated in a 30-degree incubator for 3-5 days, and repeatedly transferred on the screening culture medium once again to obtain pure culture transformants; (5) Please refer to Figure 4 Extract the genomic DNA of the pure culture transformants, and use a pair of primers of the bovine beta-casein gene for PCR amplification, and the pair of primer sequences are: 5'-ACCATGGTGAACAAACACTTCTTGTC-3', 5'-CCAATGAGGCCACCAGTGGTACCAGTTG-3', to obtain a DNA band with a size of 700 bp, while the K. marxianus without the CSN2 gene as a comparative strain cannot expand the 700 bp DNA, and the 700 bp is sequenced, and the result shows the sequence of the alpha s1-casein gene, proving that the pure culture transformants are the transformants containing the target CSN2 gene, and the comparative strain without the CSN2 gene cannot be expanded as indicated by the arrow; The solution of hygromycin B needs to be sterilized; Please refer to Figures 5-6 The yeast contains the gene encoding the bovine beta-casein, and the bovine beta-casein is beta-casein, CSN2, and can secrete the synthesized target protein bovine beta-casein to the extracellular space, combine with the foam, and produce more foam; the K. marxianus without the hydrophobic peptide as a comparative strain has very little foam; The engineered K. marxianus is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC35181.
[0033] The engineered K. marxianus CGMCC35181 contains the DNA base of sequence SEQ ID No. 1.
[0034] The sequence of the DNA base of SEQ ID No. 1 comprises a secretion signal peptide sequence, a bovine beta-casein gene sequence, and a hydrophobic peptide sequence, the secretion signal peptide sequence encodes 31 amino acids, and the bovine beta-casein gene sequence and the hydrophobic peptide sequence encode 11 hydrophobic polypeptides.
[0035] The application also provides the following scheme: The application discloses a method for synthesizing bovine beta-casein, and belongs to the field of microbial fermentation. (1) inoculate the engineered Kluyveromyces marxianus strain CGMCC35181 into a shake flask or a fermenter containing a rich medium, and then perform fermentation culture under the conditions of 30-45°C, 100-800 rpm and aeration for 24-100 hours; (2) collect a large amount of fermentation foam generated in the fermentation process under a sterile condition, and then perform standing treatment, so that the yeast cells on the foam are precipitated, the yeast cells are returned to the shake flask or the fermenter, the supernatant after separation of the precipitated foam contains bovine beta-casein, the supernatant is concentrated, the concentrated solution is frozen and freeze-dried, and finally, a freeze-dried powder containing bovine beta-casein is obtained; (3) after the fermentation is completed, the yeast cells in the shake flask or the fermenter are separated, and then spray drying is performed to obtain a yeast cell powder.
[0036] The medium contains a carbon source and a nitrogen source, the carbon source is glucose, fructose, glycerol, xylose or biomass hydrolysate, and the nitrogen source is an inorganic nitrogen source containing ammonium or an organic nitrogen source.
[0037] The application further provides the following solutions. The application further provides the following solutions.
[0038] Example 1: Kluyveromyces marxianus CGMCC35181 is used to ferment and synthesize beta-casein by taking glucose as a carbon source A liquid medium 1.5 liter was prepared in a 3 liter fermentor as follows: glucose monohydrate 150 g / l, yeast extract 8 g / l, peptone 2 g / l, diammonium phosphate 2 g / l, ammonium citrate 2 g / l, the carbon source and nitrogen source were sterilized separately, and mixed after sterilization. After cooling, 100 ml of yeast seed liquid was added. The fermentation was carried out at a temperature of 30°C, with a stirring speed of 200-800 rpm, and an aeration rate of 3 liters / 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 35, and foam began to be produced. No antifoam agent was added throughout the 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 centrifuged supernatant was freeze-dried to obtain white protein powder. After the end of the single batch fermentation (complete glucose consumption), the fermentation broth was centrifuged to obtain yeast cells, which were freeze-dried to obtain yeast cell powder. The fermentation broth was passed through a 10 KDa filter membrane to separate the target protein. Analysis showed that the content of the target protein in the separated foam was 24.6 μg / ml, and the content of the target protein in the supernatant after separation of the fermentation broth was 8.5 μg / ml. The determination method of the 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. It was shown that the C-terminal of β-casein combined with a hydrophobic peptide, which increased its hydrophobicity and improved its ability to combine with the foam, which was beneficial to the separation and purification of the target protein. The total protein content in the obtained yeast powder was determined to be 45 ± 1.5% of the dry weight. Figures 5-6 The foam production state in the 5 liter fermentor was very rich, while the amount of foam of the comparative strain was very small.
[0039] Example 2: Fermentation of Kluyveromyces marxianus CGMCC 35181 to synthesize β-casein using fructose as the carbon source A liquid medium 1.5 liter was prepared in a 3 liter fermentor as follows: crystalline fructose 150 g / liter, and the rest of the nutrient components were the same as in Example 1. The fermentation conditions and the analytical tests were the same as in Example 1. After the fermentation was completed, it was analyzed that the content of the target protein in the separated foam was 26.4 μg / ml, and the content of the target protein in the supernatant after the separation of the fermentation broth was 6.8 μg / ml. It can be seen that the results using fructose as the carbon source were basically the same as 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 combined with the foam in the form of hydrophobic protein. It was indicated that the C-terminal of β-casein combined with a hydrophobic peptide could increase its hydrophobicity and improve its ability to combine with the foam, which was beneficial to the separation and purification of the target protein. The total protein content in the obtained yeast powder was determined to be 43±1.6% of the dry weight.
[0040] Example 3: Fermentation of Kluyveromyces marxianus CGMCC 35181 to synthesize β-casein using glycerol as the carbon source A liquid medium 1.5 liter was prepared in a 3 liter fermentor as follows: glycerol 150 g / liter, and the rest of the nutrient components were the same as in Example 1. The fermentation conditions and the analytical tests were the same as in Example 1. After the fermentation was completed, it was analyzed that the content of the target protein in the separated foam was 23.8 μg / ml, and the content of the target protein in the supernatant after the separation of the fermentation broth was 4.9 μg / ml. It can be seen that the protein content using glycerol as the carbon source was slightly lower than that using glucose as the carbon source. However, the content of the target protein in the foam was still 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. It was indicated that the C-terminal of β-casein combined with a hydrophobic peptide could increase its hydrophobicity and improve its ability to combine with the foam, which was beneficial to the separation and purification of the target protein. The total protein content in the obtained yeast powder was determined to be 42±2.5% of the dry weight.
[0041] Example 4: Fermentation of Kluyveromyces marxianus CGMCC 35181 to synthesize β-casein using xylose as the carbon source A liquid medium 1.5 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 22.6 μg / mL, and the content of the target protein in the supernatant after separation of the broth was 4.8 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 the target protein is mostly combined with the foam in the form of hydrophobic protein. This indicates that the C-terminal of β-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 determined to be 44 ± 1.8% of the dry weight. K. marxianus can utilize xylose, but it is not as effective as using glucose or fructose as the carbon source. Glucose is preferred as the carbon source.
[0042] Example 5: K. marxianus CGMCC 35181 Fermentation to Synthesize β-Casein Using Biomass Hydrolysate as the Carbon Source A liquid medium 1.5 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 14 μg / mL, and the content of the target protein in the supernatant after separation of the broth was 3.6 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 the target protein is mostly combined with the foam in the form of hydrophobic protein. This indicates that the C-terminal of β-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 determined to be 32.2% of the dry weight. K. marxianus can utilize biomass hydrolysate, but it is not as effective as using glucose or fructose as the carbon source. Glucose is preferred as the carbon source.
[0043] Example 6: K. marxianus CGMCC 35181 Fermentation to Synthesize β-Casein Using Glucose as the Carbon Source at 40°C A liquid medium of 1.5 liters was prepared in a 3-liter fermentor as follows: glucose 150 g / L, and other nutrient components were the same as in Example 1. The fermentation temperature was 40°C, and other conditions and analytical tests were the same as in Example 1. After the fermentation was completed, it was analyzed that the content of the target protein in the separated foam was 33 μg / mL, and the content of the target protein in the supernatant after the fermentation broth was separated was 7.2 μg / mL. It can be seen that the protein content is slightly higher when the fermentation is carried out at 40°C than at 30°C. The content of the target protein in the foam is significantly higher than that in the fermentation broth, indicating that most of the target protein is combined with the foam in the form of hydrophobic protein. It is shown that the C-terminal of β-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 determined to be 42.5±1.5% of the dry weight. The Kluyveromyces marxianus is a heat-resistant yeast strain, which can grow well at 45°C.
[0044] Example 7: Kluyveromyces marxianus CGMCC35181 was used to ferment and synthesize β-casein with glucose as carbon source at 45°C A liquid medium of 1.5 liters was prepared in a 3-liter fermentor as follows: glucose 150 g / L, and other nutrient components were the same as in Example 1. The fermentation temperature was 45°C, and other conditions and analytical tests were the same as in Example 1. After the fermentation was completed, it was analyzed that the content of the target protein in the separated foam was 26.4 μg / mL, and the content of the target protein in the supernatant after the fermentation broth was separated was 5.2 μg / mL. It can be seen that the protein content is lower when the fermentation is carried out at 45°C than at 40°C. The content of the target protein in the foam is significantly higher than that in the fermentation broth, indicating that most of the target protein is combined with the foam in the form of hydrophobic protein. It is shown that the C-terminal of β-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 determined to be 41.5% of the dry weight. The Kluyveromyces marxianus is a heat-resistant yeast strain, which can grow well at 45°C, but it is not the optimal growth temperature, resulting in a decrease in the protein synthesis ability, so it is preferred to carry out the fermentation at 40°C.
[0045] Example 8: Mice were fed with Kluyveromyces marxianus CGMCC35181 cell powder The yeast cell powder obtained from Example 1 is mixed with common mouse feed at a ratio of 5% and fed to mice. The mice are 4-week-old weaned mice, each weighing 20±2 grams. A total of 10 mice are continuously fed for 30 days, and the body weight is measured and compared with a control group (fed with common feed, also 10 mice). After 30 days of continuous feeding, it is found that the average body weight of the mice fed with the yeast cell powder (10 mice) is 31±1.5 grams, and the average body weight of the mice fed with the common feed (control group) is 26±1.5 grams. This shows that the mice fed with the yeast cell powder have a greater increase in body weight than the control group. This result indicates that if other animals (such as poultry and pigs) are fed with feed containing the yeast cell powder, they are likely to have a greater increase in body weight. This is beneficial to the growth of animals.
[0046] In summary, the present application provides a method for synthesizing bovine β-casein using yeast and its application in feed. The Maxxkruse yeast in the application can produce more foam, and the target protein can be separated and purified from the foam. The microbial fermentation synthesis is easy to operate, has low culture cost and small pollution. A large amount of yeast cells obtained is also a good source of animal protein feed. The dependence on imported products from abroad is reduced.
[0047] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not contradict each other, they should be considered within the scope of the present application.
[0048] The above-described embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.
Claims
1. A Kluyveromyces macrocephala strain capable of synthesizing bovine β-casein, 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 selection marker used was the hygromycin B resistance gene sequence-hph, and the homologous 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 form a new vector pSWV-km.PDC-CSN2. (2) The ligation product was transformed into Escherichia coli, a large amount of plasmid vector was extracted, the vector was digested with restriction endonuclease EcoRI, and DNA fragments larger than 5kb were recovered for transformation of Kluyveromyces martensii. (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) Incubate in a 30-degree incubator for 3-5 days and observe whether single colonies grow. After they grow, transfer each single colony to a fresh screening solid medium and incubate in a 30-degree incubator for 3-5 days. Then repeat the transfer on the screening medium to obtain pure culture transformants. (5) Genomic DNA was extracted from the pure culture transformants and amplified by PCR using a pair of primers for the bovine β-casein gene. The primer sequences were: 5'-ACCATGGTGAACAAACACTTCTTGTC-3' and 5'-CCAATGAGGCCACCAGTGGTACCAGTTG-3', resulting in a DNA band of 700 bp. As a control strain, Kluyveromyces masculinii, which does not contain the CSN2 gene, could not amplify this 700 bp DNA. The amplified 700 bp was sequenced, and the result showed that it was the αs1-casein gene sequence, proving that the pure culture transformants were transformants that truly contained the target CSN2 gene. The solution of hygromycin B needs to be sterilized; This yeast contains a gene encoding bovine β-casein, specifically β-casein, CSN2, and can secrete the synthesized target protein, bovine β-casein, extracellularly, where it binds to foam. The engineered Kluyveromyces macrocephala is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC35181.
2. The *Kluyveromyces martensii* strain capable of synthesizing bovine β-casein as described in claim 1, characterized in that, The engineered Kluyveromyces macrocarpa CGMCC35181 contains DNA bases with the sequence SEQ ID No.
1.
3. The *Kluyveromyces martensii* strain capable of synthesizing bovine β-casein as described in claim 2, characterized in that, The DNA base sequence of SEQ ID No. 1 includes a secretion signal peptide sequence, a bovine β-casein gene sequence, and a hydrophobic peptide sequence. The secretion signal peptide sequence encodes 31 amino acids, and the bovine β-casein gene sequence and the hydrophobic peptide sequence encode 11 hydrophobic polypeptides.
4. A method for synthesizing bovine β-casein as described in any one of claims 1-2, characterized in that, The aforementioned Kluyveromyces macrocarpa engineered strain, through fermentation culture, yields bovine β-casein. The specific steps are as follows: (1) Inoculate engineered Kluyveromyces macrocarpa CGMCC35181 into a shake flask or fermenter containing rich culture medium and ferment for 24 to 100 hours at 30-45°C, 100-800 rpm, and with aeration. (2) Collect a large amount of fermentation foam generated during the fermentation process under sterile conditions, let it stand, so that the yeast cells on the foam precipitate, and then return the yeast cells to the shake flask or fermentation tank. The supernatant after the separated foam precipitate contains bovine β-casein. Concentrate the supernatant and freeze-dry it to obtain freeze-dried powder containing bovine β-casein. (3) After fermentation, the yeast cells in the shake flask or fermentation tank are separated and spray-dried to obtain yeast cell powder.
5. The method for synthesizing bovine β-casein as described in claim 4, characterized in that, The culture medium contains a carbon source and a nitrogen source.
6. The application of the synthetic bovine β-casein as described in claim 4 in feed, characterized in that, The freeze-dried bovine β-casein powder and the yeast cell powder serve as sources of animal protein in feed.