DESC:The present invention broadly relates to the field of biotechnology. More particularly, the present invention relates to expression vector systems for co-expression of variant or mutant casein kinases for the increased expression of target animal-free milk proteins and a process for the increasing the production animal free milk proteins in optimized nutrient conditions. Further, the present invention provides vectors, and expression cassettes for the expression of animal-free milk proteins in host cells.
Background Ar
Globally, more than 7.5 billion people around the world consume milk and milk products. Demand for cow milk and dairy products is expected to keep increasing due to increased reliance on these products in developing countries as well as growth in the human population, which is expected to exceed 9 billion people by 2050.
Relying on animal agriculture to meet the growing demand for food is not a sustainable solution. According to the Food & Agriculture Organization of the United Nations, animal agriculture is responsible for 18% of all greenhouse gases, more than the entire transportation sector combined. Dairy cows alone account for 3% of this total. Various studies have identified that the dairy industry is one of the most common contributors to the greenhouse gas emissions. More particularly, it is known that dairy production is the contributor for emission of greenhouse gases such as methane, nitrous oxide and carbon dioxide. As a result, by avoiding meat and dairy products in our day-to-day diet, it can substantially reduce the impact on the planet. Further people with lactose intolerance are unable to fully digest the sugar (lactose) in natural cow milk. Lactose intolerance is nothing but the inability to break down the lactose and the reason for the same is due to humans losing ability to produce enough lactase enzyme as they age to digest and break down the lactose. The natural cow milk also contains antibiotics, hormone, cholesterol and saturated fat which may not be always good for humans.
In addition to impacting the environment, animal agriculture poses a serious risk to human health. A startling 80% of antibiotics used in the United States go towards treating animals, resulting in the development of antibiotic resistant microorganisms also known as superbugs. For years, food companies and farmers have administered antibiotics not only to sick animals, but also to healthy animals, to prevent illness. In September 2016, the United Nations announced the use of antibiotics in the food system as a crisis on par with Ebola and HIV.
It is estimated that cow milk accounts for 83% of global milk production. Accordingly, there is an urgent need for to provide bovine milk and/or essential high-quality proteins from bovine milk in a more sustainable and humane manner, instead of solely relying on animal farming.
In recent times, cellular agriculture has gained tremendous interest and has a variety of applications to address public health, environmental and animal welfare, and food security. The cellular agriculture is defined as the production of agricultural products from cell cultures rather than from whole plants or animals. The cellular agriculture is a promising technology in the food science, environmental science, nutrition, and dietetics research areas.

In the article published by Harvard Library, 90 Reasons to Consider Cellular Agriculture, disclosed that due to contamination-free production methodologies of cellular agriculture, foods can stay safer for a greater duration, ultimately granting a longer shelf life. The cellular agriculture can supply the growing demand for animal products, feeding the increasing world population of almost 10 billion by 2050. Moreover, the cellular agriculture dairy production processes may require 97% less land and 99.6% less water, may produce 65% less greenhouse gas emissions. There would be less waste of dairy products in a cellular agriculture system, since there is greater product control.

Recombinant proteins have gained considerable attention in various fields, including biotechnology and pharmaceuticals, due to their diverse applications. Bovine caseins refer to a group of proteins found in cow's milk, primarily comprising alpha-casein, beta-casein, and kappa-casein. Bovine caseins make up about 80% of the total protein content in cow's milk. The major types are alpha-casein (about 35-40%), beta-casein (30-35%), and kappa-casein (10-15%). Caseins are phosphoproteins, meaning they contain phosphate groups. They have a unique structure characterized by hydrophilic and hydrophobic regions, which allows them to form micelles in milk, contributing to its colloidal stability. In milk, caseins play a crucial role in providing essential amino acids and minerals to support the growth and development of young mammals. They also contribute to the viscosity and texture of milk products. Bovine caseins are used extensively in the food industry for their emulsifying, gelling, and thickening properties. They are key ingredients in the production of dairy products such as cheese, yogurt, and processed foods. Caseins, a group of phosphoproteins found in mammalian milk, hold particular significance in the food industry as emulsifiers and foaming agents. Recombinant Caseins produced in microbial expression systems offer several advantages over traditional methods of extraction from milk sources. Bacterial and Yeast systems are well-established expression systems for producing recombinant proteins due to its high expression levels, post-translational modifications, and scalability. Recombinant casein proteins expressed in microbial host cell have significant industrial and dairy applications. However, achieving high yields of these proteins is challenging due to limitations in expression levels and protein stability. Traditional methods often result in low yields and inefficient protein production and is not able to meet industrial demands. Previous methods have encountered limitations in optimizing expression levels, thus necessitating the development of novel strategies to enhance production efficiency.

Casein proteins undergo extensive post-translational modifications, including phosphorylation. Phosphorylation of caseins can occur on specific amino acid residues, such as serine and threonine. This post-translational modification can affect the stability, solubility, and interactions of casein proteins. Phosphorylation can alter the conformation and structure of casein proteins. This structural change may impact their biological functions, such as their ability to form micelles or interact with other molecules in milk. Phosphorylation can also affect the properties of milk, such as its texture, viscosity, and nutritional composition. Changes in casein phosphorylation patterns may alter milk's functionality in processes like cheese-making or infant nutrition. Overall, phosphorylation is a critical regulatory mechanism that influences the expression, structure, and function of casein proteins, thereby impacting various aspects of milk biology and dairy product quality.
Casein Kinase Fam20C (Fam20C) is a serine/threonine kinase with unique structural features. It contains a catalytic domain typical of serine/threonine kinases, crucial for phosphorylating substrates with Ser-x-Glu/pSer motifs. The catalytic domain of Fam20C exhibits conformational flexibility, allowing it to undergo structural changes during the catalytic cycle, including substrate binding, phosphoryl transfer, and product release. These structural features collectively define the functionality of Fam20C's catalytic domain as a serine/threonine kinase, enabling it to phosphorylate substrates with Ser-x-Glu/pSer motifs and provide desired characteristics to recombinant casein proteins expressed from Bacterial and Yeast systems.

Accordingly, there is an urgent need to provide bovine milk and/or essential high-quality proteins from bovine milk in a more sustainable and humane manner, instead of solely
relying on animal farming. There is a need for the development of a synthetic process for the preparation of milk proteins which substantially reduces the greenhouse emissions that is observed in the conventional methods.
Summary of the Invention
Accordingly, the inventors of the present disclosure have by using genetic engineering developed expression vectors and methods for increasing yields of recombinant caseins expressed in host cells by utilizing various variants and mutants of the enzyme 'Casein kinase' to modulate the phosphorylation status of recombinant Caseins, thereby enhancing their stability and expression levels within the host organism.
Thus, by incorporating these novel methods, significantly higher yields of recombinant Caseins can be obtained compared to conventional approaches. The dairy products containing such animal-free fusion proteins will be safe, delicious and identical to the naturally available bovine milk and milk proteins.
The present disclosure relates to expression vector systems for co-expression of variants or mutants’ casein kinases for the increased expression of target animal-free milk proteins and a process for the increasing the production animal free milk proteins in optimized nutrient conditions. Further, the present disclosure provides vectors, and expression cassettes for the expression of animal-free milk proteins in host cells.
Furthermore, the present disclosure relates to expression vector systems for co-expression of variants or mutants’ casein kinases in combinations thereof, to modulate the phosphorylation status and folding efficiency of recombinant milk proteins, thereby enhancing their stability and expression levels within the host organism and increased expression of target animal-free milk proteins and a process for the increasing the production animal free milk proteins in optimized nutrient conditions
Brief Description of The Accompanying Figures
The accompanying drawings illustrate some of the embodiments of the present disclosure and, together with the descriptions, serve to explain the disclosure. These drawings have been provided by way of illustration and not by way of limitation. The components in the drawings are not necessarily drawn to scale, emphasis instead being placed upon clearly illustrating the principles of the aspects of the embodiments.
Figure 1 – Schematic representation of the single or multiple integrative expression vector.
Figure 2 – Screening & Over expression confirmed using SDS PAGE for Alpha-S1-casein protein
Figure 3 – Screening & Over expression confirmed by SDS PAGE for A2 Beta casein
Figure 4 - HPLC analysis of AS1 protein
Figure 5 - HPLC analysis of A2 Beta casein protein
Detailed Description of the invention
At the very outset of the detailed description, it may be understood that the ensuing description only illustrates a particular form of this invention. However, such a particular form is only exemplary embodiment, and without intending to imply any limitation on the scope of this invention. Accordingly, the description is to be understood as an exemplary embodiment and teaching of invention and not intended to be taken restrictively.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
Definitions:
While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.
The term “milk protein” means a protein that is found in a mammal-produced milk or a protein having a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the sequence of a protein that is found in a mammal-produced milk. Non-limiting examples of milk proteins include: Beta-casein, Kappa-casein, Alpha-S1-casein, Alpha -S2-casein, alpha -lactalbumin, Beta-lactoglobulin, Lactoferrin, Transferrin, and Serum albumin. Additional milk proteins are known in the art.
The term “animal-free milk proteins” is known in the art and means any milk proteins that are not produced by a mammal.
The term "genetically engineered" as used herein relates to an organism that includes exogeneous, non-native nucleic acid sequences either maintained episomally in an expression vector or integrated into the genome of the host organism.
The term “recombinant” refers to nucleic acids or proteins formed by laboratory methods of genetic recombination (e.g., molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome. A recombinant fusion protein is a protein created by combining sequences encoding two or more constituent proteins, such that they are expressed as a single polypeptide. Recombinant fusion proteins may be expressed in vivo in various types of host cells, including plant cells, bacterial cells, fungal cells, mammalian cells, etc. Recombinant fusion proteins may also be generated in vitro.
The term “recombinant” is an art known term. When referring to a nucleic acid (e.g., a gene), the term “recombinant” can be used, e.g., to describe a nucleic acid that has been removed from its naturally occurring environment, a nucleic acid that is not associated with all or a portion of a nucleic acid abutting or proximal to the nucleic acid when it is found in nature, a nucleic acid that is operatively linked to a nucleic acid which it is not linked to in nature, or a nucleic acid that does not occur in nature. The term “recombinant” can be used, e.g., to describe cloned DNA isolates, or a nucleic acid including a chemically synthesized nucleotide analog. When “recombinant” is used to describe a protein, it can refer to, e.g., a protein that is produced in a cell of a different species or type, as compared to the species or type of cell that produces the protein in nature.
As used herein, an endogenous nucleic acid sequence in the genome of an organism (or the encoded protein product of that sequence) is deemed “recombinant” herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous (originating from the same host cell or progeny thereof) or exogenous (originating from a different host cell or progeny thereof). By way of example, a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that this gene has an altered expression pattern. This gene would now become “recombinant” because it is separated from at least some of the sequences that naturally flank it.
A nucleic acid is also considered “recombinant” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered “recombinant” if it contains an insertion, deletion, or a point mutation introduced artificially, e.g., by human intervention. A “recombinant nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.
The term “vector” as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
The term “operably linked” expression control sequences refer to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest.
The term “transfect”, “transfection”, “transfecting,” and the like refer to the introduction of a heterologous nucleic acid into eukaryote, prokaryotic or yeast or fungal cells.
“Transformation” refers to a process by which a nucleic acid is introduced into a cell, either transiently or stably. Transformation may rely on any known method for the insertion of nucleic acid sequences into a prokaryotic or eukaryotic host cell, including Agrobacterium-mediated transformation protocols, viral infection, whiskers, electroporation, heat shock, lipofection, polyethylene glycol treatment, micro-injection, and particle bombardment.
The term “recombinant host cell” (“expression host cell”, “expression host system”, “expression system” or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism. Host cells can be any organism selected from the group consisting of bacteria, yeast and filamentous fungi.
The term “about,” “approximately,” or “similar to” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which can depend in part on how the value is measured or determined, or on the limitations of the measurement system. It should be understood that all ranges and quantities described below are approximations and are not intended to limit the invention. Where ranges and numbers are used these can be approximate to include statistical ranges or measurement errors or variation. In some embodiments, for instance, measurements could be plus or minus 10%.
The phrase “essentially free of” is used to indicate the indicated component, if present, is present in an amount that does not contribute, or contributes only in a de minimus fashion, to the properties of the composition. In various embodiments, where a composition is essentially free of a particular component, the component is present in less than a functional amount. In various embodiments, the component may be present in trace amounts. Particular limits will vary depending on the nature of the component, but may be, for example, selected from less than 10% by weight, less than 9% by weight, less than 8% by weight, less than 7% by weight, less than 6% by weight, less than 5% by weight, less than 4% by weight, less than 3% by weight, less than 2% by weight, less than 1% by weight, or less than 0.5% by weight.
Unless otherwise indicated, and as an example for all sequences described herein under the general format “SEQ ID NO:”, “nucleic acid comprising SEQ ID NO:1” refers to a nucleic acid, at least a portion of which has either (i) the sequence of SEQ ID NO:1, or (ii) a sequence complementary to SEQ ID NO:1. The choice between the two is dictated by the context.
Detailed description:
Bovine milk and dairy products have long traditions in human nutrition. Bovine milk contains the nutrients needed for growth and development of the calf, and is a resource of proteins, lipids, vitamins, amino acids and minerals. Bovine milk also contains growth factors, immunoglobulins, hormones, cytokines, peptides, nucleotides, enzymes, polyamines and other bioactive peptides. Milk proteins are important components for colour, taste and texture of the milk. Milk proteins play a very important role for the production of different kind of dairy products. Milk is consumed as a solo beverage and is also consumed in different forms like cheese, paneer, yogurt, coffee, flavoured drinks etc. Industry also has Ghee as the most consumed value-added dairy product following non-fat milk and butter.

Milk contains two types of protein Casein and Whey. Milk protein composition varies depending on the species (example cow, goat, sheep), breed (example Holstein, Jersey), the animal's feed, and the stage of lactation.
Casein
Casein proteins represents a family of proteins that is present in mammal-produced milk and is capable of self-assembling with other proteins in the family to form micelles and/or precipitate out of an aqueous solution at an acidic pH. Non-limiting examples of casein proteins include Beta-casein, Kappa-casein, Alpha-S1-casein, and Alpha-S2-casein. Non-limiting examples of sequences for casein protein from different mammals are provided herein. Additional sequences for other mammalian caseins are known in the art. In the context of the invention, a "casein protein" refers to a protein at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or completely identical to a natural mammalian casein protein. Preferred casein proteins are bovine casein proteins.
Alpha-S1-casein and Alpha-S2-casein
The Alpha casein fractions also play an important role in casein micelle formation as their presence has a positive effect. Alpha-casein is the largest fraction and includes those phosphoproteins precipitated at low calcium concentrations. The Alpha S1-casein family makes up 40% of this fraction and contains 214 amino acid residues, of which 8.4% are prolyl residues evenly distributed throughout the polypeptide chain. The Alpha S2-casein accounts for 10% of this fraction and is composed of 222 amino acid residues. It is the most hydrophilic of the caseins, with 10 to 13 phosphoryl residues.
Casein kinase
Casein Kinase Fam20C (Fam20C) is a serine/threonine kinase with unique structural features. It contains a catalytic domain typical of serine/threonine kinases, crucial for phosphorylating substrates with Ser-x-Glu/pSer motifs. The catalytic domain of Fam20C exhibits conformational flexibility, allowing it to undergo structural changes during the catalytic cycle, including substrate binding, phosphoryl transfer, and product release. These structural features collectively define the functionality of Fam20C's catalytic domain as a serine/threonine kinase, enabling it to phosphorylate substrates with Ser-x-Glu/pSer motifs and provide desired characteristics to recombinant milk proteins expressed from Bacterial and Yeast systems.
The present disclosure encompasses several embodiments wherein different variants and mutants of Casein kinase are employed to optimize the expression of recombinant milk in host cells. These variants and mutants may be engineered through rational design and protein engineering techniques to enhance their activity, substrate specificity, or stability.
The inventors have also developed a method for selecting a specific variant or mutant of Casein kinase and co-expressing it with the gene encoding the target recombinant milk protein in recombinant host cells. The Casein kinase variant or mutant phosphorylates the recombinant milk proteins more efficiently, leading to increased stability and expression levels. Further, the inventors have also developed a method by optimizing culture conditions such as temperature, pH, and inducer concentration to further enhance the expression of recombinant milk proteins in conjunction with the selected Casein kinase variant or mutant.
The present disclosure also encompasses kits comprising the necessary components for carrying out the disclosed methods, including expression vectors encoding the Casein kinase variants or mutants, recombinant milk proteins and other relevant reagents.
Introducing Nucleic Acids into a Cell
Methods of introducing nucleic acids (e.g., any of the nucleic acids described herein) into a cell to generate a host cell are well-known in the art. Non-limiting examples of techniques that can be used to introduce a nucleic acid into a cell include: calcium phosphate transfection, dendrimer transfection, liposome transfection (e.g., cationic liposome transfection), cationic polymer transfection, electroporation, cell squeezing, sonoporation, optical transfection, protoplast fusion, impalefection, hyrodynamic delivery, gene gun, magnetofection, and viral transduction.
One skilled in the art would be able to select one or more suitable techniques for introducing the nucleic acids into a cell based on the knowledge in the art that certain techniques for introducing a nucleic acid into a cell work better for different types of host cells. Exemplary methods for introducing a nucleic acid into a yeast cell are described in Kawai et al., Bioeng. Bugs 1:395-403, 2010.
Host Cells
In an aspect, the present disclosure provides host cells including any of the nucleic acids (e.g., vectors) described herein. In another aspect, the nucleic acid described herein is stably integrated within the genome (e.g., a chromosome) of the host cell. In other aspects, the nucleic acid described herein is not stably integrated within the genome of the host cell.
In some aspects of the present disclosure, the host cell is a yeast strain or a bacterial strain. an organism selected from the group consisting of bacteria, yeast and filamentous fungi.
Embodiments of the present invention:
An important embodiment of the present disclosure provides a recombinant expression vector for the co-expression of one or more casein kinase along with target animal free milk protein wherein the vector comprises:
i. a polynucleotide sequence encoding a 1st bacterial promoter;
ii. a polynucleotide sequence operably linked to the promoter encoding target animal free milk protein selected from SEQ ID No. 2, or 3,
iii. a polynucleotide sequence encoding the bacterial terminator sequence;
iv. a polynucleotide sequence encoding a 2nd bacterial promoter;
v. a polynucleotide sequence operably linked to the 2nd promoter encoding the one or more mutant or variant casein kinases;
vi. a polynucleotide sequence encoding the bacterial terminator sequence;
vii. a polynucleotide sequence encoding the bacterial antibiotic resistant marker gene.
Another important embodiment of the present disclosure a recombinant expression vector comprising multi locus integration sites for the co-expression of one or more casein kinase along with target animal free milk protein wherein the vector comprises:
i. a first expression cassette comprising
a. a polynucleotide sequence encoding a 1st bacterial promoter;
b. a polynucleotide sequence operably linked to the 1st bacterial promoter encoding target animal free milk protein selected from SEQ ID No. 2, or 3;
c. a polynucleotide sequence encoding the bacterial marker gene;
d. a polynucleotide sequence encoding the bacterial terminator sequence;
ii. a second expression cassette comprising
a. a polynucleotide sequence encoding a 2nd bacterial promoter;
b. a polynucleotide sequence operably linked to the 2nd promoter encoding the one or more mutant or variant casein kinases;
c. a polynucleotide sequence encoding the bacterial marker gene;
d. a polynucleotide sequence encoding the bacterial terminator sequence.

In a preferred embodiment, the casein kinase is a variant or mutant of Casein kinase Fam20C having 90% similarity to SEQ ID No. 1.
In a preferred embodiment, the amino acid sequence of the casein kinase is a truncated sequence. The present disclosure provides serves as the catalytic subunit of a constitutively active serine/threonine-protein kinase complex. It plays a crucial role in phosphorylating a wide range of proteins, particularly those containing acidic residues C-terminal to the target serine or threonine residues. This catalytic unit contributes not only to phosphorylation but also assists in the proper folding and function of target proteins.
In a preferred embodiment, the bacterial resistance marker gene is selected from Zeocin, Kanamycin, Genticin, or Ampicillin.
An embodiment of the present disclosure provides a method for the preparation of the recombinant vector system for the expression of milk proteins, the method comprising the steps of:
i. selecting and designing a plasmid vector containing sequences encoding one or more promoters, one or more variant or mutant casein kinase, bacterial marker sequence and/or terminator sequence;
ii. preparing DNA fragment(s) from a linearized plasmid vector by restriction enzyme digestion or by PCR techniques;
iii. amplifying DNA fragment(s) obtained in step (i) from the insert by PCR techniques while ensuring that the sequence at the end of the fragment is homologous with that of the plasmid;
iv. introducing both DNA fragments are then simultaneously introduced into the host cell;
v. Selecting the positive transformants by identifying the plasmid vector marker;
vi. Recovering the constructed plasmid from the transformed host cell into a second host cell to amplify and identify the structure of the plasmid with restriction enzyme digestion and DNA sequencing.
A preferred embodiment provides that the animal free milk protein selected from any bovine milk proteins selected from the group comprising of Beta-casein, Kappa-casein, Alpha-S1-casein, Alpha-S2-casein.
Another important embodiment of the present disclosure is to provide a process for the production of animal-free milk proteins in recombinant host cells, wherein the method involves culturing in nutrient media host cells with recombinant expression vectors containing target gene sequence as defined in SEQ ID No. 2 or 3 for co-expression of casein kinase and milk proteins.
In some embodiments, the process for the production of animal-free milk proteins comprises the steps of:
i. identifying high quality milk producing cow such as Gir cattle (Bos indicus) by using various bioinformatics techniques and analysis;
ii. performing genetic analysis of identified Gir cattle from step (i)to identify the specific genetic segment which is responsible for production of desired milk proteins;
iii. performing in-silico data analysis and codon optimization of gene sequences for the requisite milk proteins to obtain the gene sequences;
iv. designing recombinant expression vectors for co-expression of casein kinase and milk proteins along with primers for gene cloning procedure to obtain a recombinant expression vector;
v. transforming the recombinant plasmid obtained in step (iv) into host cells by chemical transformation;
vi. isolating and screening multiple clones from each transformed host cells for expression of secreted target proteins;
vii. regrowing the best clones isolated in step (vi) in shake flasks to confirm expression and estimate expression levels of protein expression by SDS-PAGE;
viii. identifying the best strains for further growing in nutrient feed composition in fermenter in to obtain the optimal transformed yeast strain for production of target proteins; and
ix. performing downstream process to purify the animal free protein expressed by the transformed host cells.
In a preferred embodiment, the casein kinase is a variant or mutant of Casein kinase Fam20C having 90% similarity to SEQ ID No. 1.
In a preferred embodiment, the casein kinase is truncated version of Fam20C Casein kinase.
In a preferred embodiment of the present disclosure, the nutrient feed composition has optimized carbon sources, nitrogen sources, vitamins, and minerals.
In a preferred embodiment, the carbon source in the nutrient feed composition is selected from Glucose, Glycerol, Dextrose, Sorbitol, Mannitol or combinations thereof.
In a preferred embodiment, the nitrogen source in the nutrient feed composition is selected from Yeast extract, peptone, Ammonium sulfate, YNB, Soy peptone, Tryptone or combinations thereof.
In a preferred embodiment, the recombinant milk proteins are employed in nutritional supplements, or functional food ingredients.
In a preferred embodiment of the present disclosure, the animal free milk protein are selected from the group comprising of Beta-casein, Kappa-casein, Alpha-S1-casein, Alpha-S2-casein.
A preferred embodiment of the present disclosure, the host cell is selected from bacterial, plant, yeast or fungal cells.
An embodiment of the present disclosure provides the process for the preparation of the milk proteins wherein the downstream purification process in step (ix) can be carried out by any of the methods including centrifugation, column chromatography and/or membrane filtration.
Another important embodiment of the present disclosure provides a process for the isolation and purification of the milk proteins expressed by the host cells.
In a preferred embodiment of the present disclosure, the purification of the milk protein can be carried out by centrifugation, precipitation, dialysis, membrane filtration, column chromatography, spray drying and/or lyophilization.
An embodiment of the present invention provides a recombinant host cell comprising expression vector comprising the nucleic acid sequence encoding the recombinant animal free milk protein.
A preferred embodiment of the present invention, the host cell is selected from bacterial, plant, yeast or fungal cells.
An important embodiment of the present disclosure provides animal-free proteins encoding milk proteins that mimic the naturally occurring milk proteins from Bos indicus.
A preferred embodiment provides that the animal free milk protein selected from the group comprising of Beta-casein, Kappa-casein, Alpha-S1-casein, Alpha-S2-casein.
An important embodiment of the present disclosure provides a food composition comprising the animal free milk protein along with pharmaceutically, nutraceutically acceptable excipients/carriers.
Advantages:
The present invention demonstrates the following advantages:
• Enhanced expression levels of recombinant Caseins in recombinant host cells;
• Increased stability of recombinant milk proteins leading to improved product quality;
• Compatibility with existing industrial processes for protein production;
• Potential cost savings and efficiency gains in the production of recombinant milk proteins.

Experimental data -
Example 1 - Alpha AS1 casein expression using Casein Kinase as Helper molecule in E. coli:

Experimental workflow for verification of synthesized genes in lyophilised form received from GenScript Biotech
Protein Sequence of truncated human casein kinase II (Fam20C) – SEQ ID No. 1

DFSSDPSSNLSSHSLEKLPPAAEPAERALRGRDPGALRPHDPAHRPLLRDPGPRRSESPPGPGGDASLLARLFEHPLYRVAVPPLTEEDVLFNVNSDTRLSPKAAENPDWPHAGAEGAEFLSPGEAAVDSYPNWLKFHIGINRYELYSRHNPAIEALLHDLSSQRITSVAMKSGGTQLKLIMTFQNYGQALFKPMKQTREQETPPDFFYFSDYERHNAEIAAFHLDRILDFRRVPPVAGRMVNMTKEIRDVTRDKKLWRTFFISPANNICFYGECSYYCSTEHALCGKPDQIEGSLAAFLPDLSLAKRKTWRNPWRRSYHKRKKAEWEVDPDYCEEVKQTPPYDSSHRILDVMDMTIFDFLMGNMDRHHYETFEKFGNETFIIHLDNGRGFGKYSHDELSILVPLQQCCRIRKSTYLRLQLLAKEEYKLSLLMAESLRGDQVAPVLYQPHLEALDRRLRVVLKAVRDCVERNGLHSVVDDDLDTEHRAASAR

Alpha-S1-casein – SEQ ID NO. 2
>sp|P02662|CASA1_BOVIN Alpha-S1-casein OS=Bos taurus OX=9913 GN=CSN1S1 PE=1 SV=2
MKLLILTCLVAVALARPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNELSKDIGSESTEDQAMEDIKQMEAESISSSEEIVPNSVEQKHIQKEDVPSERYLGYLEQLLRLKKYKVPQLEIVPNSAEERLHSMKEGIHAQQKEPMIGVNQELAYFYPELFRQFYQLDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSEKTTMPLW

Cloning and Transformation of synthesized gene in pETDuet1 vector
Further, PCR reaction was setup to amplify the AS1 gene fragment and casein kinase gene fragment cloned into pETDuet1 vector and the obtained fragments were cloned into the desired vectors using Gibson assembly by NEBuilder® HiFi DNA Assembly Master mix The amplified products further added in the Gibson assembly mix and incubated for 1 hr at 50° C. Afterwards the Gibson mixture was directly transformed into the E,coli competent cells for the further screening of the clones.
Next day, colony PCR was done for the selected single colonies from transformed plate. The plasmids were isolated using the standard alkaline lysis method from selected colonies. The selected clones were transformed into E. coli host competent cells and the transformed cells were plated on LB agar-ampicillin plates with further incubation at 37°C in an incubator. Shake flask experiment was performed to express AS1 protein using different media.

Figure 2 and 4 showed that the AS1 protein was successfully expressed in E.coli at ~23KDa confirmed by SDS PAGE analysis and HPLC method.

Example 2 – A2 Beta casein expression using Casein Kinase as Helper molecule in E. coli:

Experimental workflow for verification of synthesized genes in lyophilised form received from GenScript Biotech
Protein Sequence of truncated human casein kinase II (Fam20C) – SEQ ID NO. 1
DFSSDPSSNLSSHSLEKLPPAAEPAERALRGRDPGALRPHDPAHRPLLRDPGPRRSESPPGPGGDASLLARLFEHPLYRVAVPPLTEEDVLFNVNSDTRLSPKAAENPDWPHAGAEGAEFLSPGEAAVDSYPNWLKFHIGINRYELYSRHNPAIEALLHDLSSQRITSVAMKSGGTQLKLIMTFQNYGQALFKPMKQTREQETPPDFFYFSDYERHNAEIAAFHLDRILDFRRVPPVAGRMVNMTKEIRDVTRDKKLWRTFFISPANNICFYGECSYYCSTEHALCGKPDQIEGSLAAFLPDLSLAKRKTWRNPWRRSYHKRKKAEWEVDPDYCEEVKQTPPYDSSHRILDVMDMTIFDFLMGNMDRHHYETFEKFGNETFIIHLDNGRGFGKYSHDELSILVPLQQCCRIRKSTYLRLQLLAKEEYKLSLLMAESLRGDQVAPVLYQPHLEALDRRLRVVLKAVRDCVERNGLHSVVDDDLDTEHRAASAR

Protein Sequence of Bovine A2Beta-casein – SEQ ID NO. 3
A2ß-casein
>tr|A0A1U9XAM2|A0A1U9XAM2_BOSIN Beta-casein OS=Bos indicus OX=9915 GN=CSN2 PE=3 SV=1
MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDEL
QDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFTESQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPIIV
No. Amino acid: 209
Molecular Wt.: 23.59 kDa

Cloning and Transformation of Synthesized gene in pETDuet1 vector
Further, PCR reaction was setup to amplify the A2B_CN gene fragment and casein kinase fragment cloned into pETDuet1 vector the obtained fragments were cloned into the desired vectors using Gibson assembly by NEBuilder® HiFi DNA Assembly Master mix. The amplified products further added in the Gibson assembly mix and incubated for 1 hr at 50° C. Afterwards the Gibson mixture was directly transformed into the E,coli competent cells for the further screening of the clones.
Next day, colony PCR was done for the selected single colonies from transformed plate. The plasmids were isolated using the standard alkaline lysis method from selected colonies. The selected clones were transformed into E. coli host competent cells and the transformed cells were plated on LB agar-ampicillin plates with further incubation at 37°C in an incubator. Shake flask experiment was performed to express AS1 protein using different media.
Figure 3 and 5 showed that the A2 Beta casein protein was successfully expressed in E.coli at ~25KDa confirmed by SDS PAGE analysis and HPLC method.
The foregoing broadly outlines the features and technical advantages of the present disclosure in order that the detailed description of the disclosure that follows may be better understood. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying the disclosed methods or for carrying out the same purposes of the present disclosure.
It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "an expression cassette" includes one or more of the expression cassettes disclosed herein and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or sometimes when used herein with the term "having".
When used herein "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms.
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present invention are generally performed according to conventional methods well-known in the art. Generally, nomenclatures used in connection with techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.
The methods and techniques of the present invention are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e. g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001); Ausubel et al., Current Protocols in Molecular Biology, J, Greene Publishing Associates (1992, and Supplements to 2002); Handbook of Biochemistry: Section A Proteins, Vol I 1976 CRC Press; Handbook of Biochemistry: Section A Proteins, Vol II 1976 CRC Press. The nomenclatures used in connection with, and the laboratory procedures and techniques of, molecular and cellular biology, protein biochemistry, enzymology and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. ,CLAIMS:1. A recombinant expression vector comprising single locus or multi-locus integration sites for the co-expression of one or more casein kinase, along with target animal free milk.
2. The recombinant expression vector as claimed in claim 1, wherein the vector containing single locus integration site comprises:
i. a polynucleotide sequence encoding a 1st bacterial promoter;
ii. a polynucleotide sequence operably linked to the promoter encoding target animal free milk protein selected from SEQ ID No. 2, or 3,
iii. a polynucleotide sequence encoding the bacterial terminator sequence;
iv. a polynucleotide sequence encoding a 2nd bacterial promoter;
v. a polynucleotide sequence operably linked to the 2nd promoter encoding the one or more mutant or variant casein kinases;
vi. a polynucleotide sequence encoding the bacterial terminator sequence;
vii. a polynucleotide sequence encoding the bacterial antibiotic resistant marker gene.

3. The recombinant expression vector as claimed in claim 1, wherein the vector containing multi-locus integration sites comprises:
i. a first expression cassette comprising
a. a polynucleotide sequence encoding a 1st bacterial promoter;
b. a polynucleotide sequence operably linked to the 1st bacterial promoter encoding target animal free milk protein selected from SEQ ID No. 2, or 3;
c. a polynucleotide sequence encoding the bacterial marker gene;
d. a polynucleotide sequence encoding the bacterial terminator sequence;
ii. a second expression cassette comprising
a. a polynucleotide sequence encoding a 2nd bacterial promoter;
b. a polynucleotide sequence operably linked to the 2nd promoter encoding the one or more mutant or variant casein kinases;
c. a polynucleotide sequence encoding the bacterial marker gene;
d. a polynucleotide sequence encoding the bacterial terminator sequence.
4. The vector as claimed in claims 1-3, wherein the casein kinase is a variant or mutant of Casein kinase Fam20C having 90% similarity to SEQ ID No. 1.
5. The vector as claimed in claim 1, wherein the casein kinase is a truncated Fam 20C casein kinase.
6. The vector as claimed in claim 1, wherein the bacterial resistance marker gene is selected from Ampicillin, kanamycin, Tetracycline.
7. A method for the preparation of the recombinant vector system as claimed in claim 1 for the expression of milk proteins, the method comprising the steps of:
i. selecting and designing a plasmid vector containing sequences encoding one or more promoters, one or more variant or mutant casein kinase, bacterial marker sequence and/or terminator sequence as claimed in claim 1-3;
ii. preparing DNA fragment(s) from a linearized plasmid vector by restriction enzyme digestion or by PCR techniques;
iii. amplifying DNA fragment(s) obtained in step (i) from the insert by PCR techniques while ensuring that the sequence at the end of the fragment is homologous with that of the plasmid;
iv. introducing both DNA fragments are then simultaneously introduced into the host cell;
v. selecting the positive transformants by identifying the plasmid vector marker;
vi. recovering the constructed plasmid from the transformed host cell into a second host cell to amplify and identify the structure of the plasmid with restriction enzyme digestion and DNA sequencing.
8. A process for the production of animal-free milk proteins in recombinant host cells, wherein the method involves culturing in nutrient media host cells with recombinant expression vectors as claimed in claim 1 containing target gene sequence as defined in SEQ ID No. 2 or 3 for co-expression of casein kinase and milk proteins.

9. The process as claimed in claim 8, wherein the host cell is selected from bacterial, plant, yeast or fungal cells.

10. The process as claimed in claim 9, wherein the host cell is a bacterial cell.

11. The process as claimed in claim 8, wherein the process employs a downstream purification process for the isolation of animal free milk proteins and wherein the purification process can be carried out by centrifugation, heat lysis precipitation, membrane filtration, spray drying and/or lyophilization.

12. A recombinant host cell comprising expression vector comprising the nucleic acid sequence encoding the recombinant animal free milk protein wherein the host cell is selected from bacterial, plant, yeast or fungal cells.

13. The cell as claimed in claim 12, wherein the host cell is a bacterial cell.

14. Animal-free proteins milk proteins as obtained by the process as claimed in claim 8 wherein the proteins mimic the naturally occurring milk proteins from Bos indicus.

15. The protein as claimed in claim 14, wherein the animal free milk protein is selected from the group comprising of Beta-casein, Kappa-casein, Alpha-S1-casein, Alpha-S2-casein.