Efficient production of recombinant casein

Abstract

The present invention relates to a method for production of recombinant casein, wherein the method comprises the steps of: (a) adding a microbial strain to a first bioreactor, wherein said microbial strain has the capacity to express casein, and said microbial strain comprises means for deactivation of cell division; (b) culturing said microbial strain in the first bioreactor, wherein the cell division is active, thereby forming a continuously growing biomass comprising cells of said microbial strain; (c) deactivating cell division; (d) inducing expression of casein; (e) after sufficient expression of casein, lysing said cells, thereby forming a lysis broth, and separating said lysis broth so that casein can be separated into a casein containing phase; (f) optionally treating the casein containing phase to induce formation of casein micelles; (g) optionally further purifying and / or treating the casein micelles, and (h) optionally producing caseinate. In other aspects, the invention also relates to a bioreactor system, a recombinant casein, and products including a recombinant casein.

Description

[0001] Efficient production of recombinant casein

[0002] Technical field

[0003] The present invention is in the field of biotechnology and fermentation. More specifically, the present invention relates to a method for production of recombinant casein, including configuration of a genetically modified microbial production strain, a bioreactor system for producing recombinant casein, a purification method for the recombinant casein, as well as products and uses of the recombinant casein.

[0004] Technical background

[0005] The demand for food is growing together with the world population and there is a need to develop alternative sources of protein due to the unsustainability of current animal-based food products. Dairy products are a vital part of the food supply and plants-based alternatives struggle to match the functionality, nutrition and flavour. This is to a large extent due to their lack of the milk protein casein. Casein is the main protein in milk making up 80% of the total protein content. Casein is a remarkably versatile protein. It has unique textural and binding properties enabling a wide range of applications in both food and non-food. The combination of nutrition, texture and melting properties of hard cheese is for instance currently not possible to achieve without casein. Thus, efficient production of recombinant casein proteins is an important part in the development of new non-animal based dairy products.

[0006] The current methods for recombinant protein production focus on high value applications and are not commercially viable for producing casein as a commodity. A commercially viable production method for casein must fulfil several criteria; it must use an inexpensive substrate, have a high substrate to product conversion efficiency, have a very high space-time yield in fermentation, inexpensive purification and lastly the production method must work at scale. Fulfilling these criteria is challenging for several reasons.

[0007] There are several production methods currently pursued for recombinant casein production for food applications. Examples of this include the following. A hybrid micelle composition comprising at least two casein proteins (or more) and at least one of the casein proteins is a recombinant protein is disclosed in W02022098835 Al. A food product comprising of recombinant casein wherein the protein is selected from a group of Bos Taurus casein is disclosed in US20190216106 Al. A stimulated human milk casein composition is disclosed in W02023151198 Al. A Bubalus bubalis milk product from heterogenous polynucleotides encoding a Bubalus bubalis polypeptide is disclosed in W02023235555 Al.

[0008] Some approaches appear to have opted for eucaryote productions strains, likely due the track record of these strains for high titer extracellular protein production with native eucaryote protein folding and PTMs. See e.g., W02020223700 and US-B2-11771104. As described further below this technological path is severely limited when the goal is commercially viable production of casein as a commodity.

[0009] Improving the efficiency of the product formation through optimization of the fermentation process has been extensively researched, as seen in Kopp et. al. (2020), where repetitive fed- batch and chemostat cultivation were explored.

[0010] However, there remains a continued strive for improvement of the process of production of suitable proteins for dairy product applications.

[0011] In other cases, methods of improving the efficiency of various stages in the production of specific casein protein have been explored. The sensitive behaviour of casein protein makes the production of the various types of protein complex. Prior art specifically focused on one type of casein protein (Beta casein) and explored methods of processing and production in vivo, see US6071718A, and isolation, see US6121421A. However, more examples with other types of casein being performed in larger scale productions are still needed.

[0012] Further, phosphate depletion is disclosed as a way of stopping the growth media as seen in W02022226507 Al, however any benefits of this action relating to protein production is not explored.

[0013] Additionally, in order to enable efficient utilization of casein protein in dairy products the aggregation of the protein into micelles is essential. The specific use of recombinant casein protein (Alpha and Kappa) in micellar form for application in dairy products have been disclosed in EP3962289A1. However, current commercially mature production methods for casein target laboratory customers at gram or milligram volume and at a cost many orders of magnitude higher than cow milk.

[0014] Thus, existing solutions have not demonstrated a clear path toward scale and cost parity production of casein for dairy product applications with potential for competing in the dairy market.

[0015] Summary of the invention

[0016] The object of the invention is to solve at least one of the drawbacks of the prior art, and to provide an improved production of recombinant casein.

[0017] The present inventors have developed a recombinant protein production method for casein that utilizes a combination of known technology and novel features in order to overcome several of the challenges stated above. The combination of the method steps together with the design of the components and conditions gives rise to a surprisingly effective protein production process.

[0018] In a first aspect, the present disclosure relates to a method for production of recombinant casein, wherein the method comprises the steps of: (a) adding a microbial strain to a first bioreactor, wherein said microbial strain has the capacity to express casein, and said microbial strain comprises means for deactivation of cell division;

[0019] (b) culturing said microbial strain in the first bioreactor, wherein the cell division is active, thereby forming a continuously growing biomass comprising cells of said microbial strain;

[0020] (c) deactivating cell division;

[0021] (d) inducing expression of casein;

[0022] (e) after sufficient expression of casein, lysing said cells, thereby forming a lysis broth, and separating said lysis broth so that casein can be separated into a casein containing phase;

[0023] (f) optionally treating the casein containing phase to induce formation of casein micelles; and

[0024] (g) optionally further purifying and / or treating the casein micelles; and / or

[0025] (h) optionally producing caseinate; wherein part of the biomass obtained in the culturing of step (b) is transferred to a second bioreactor, where at least step (c) and (d) is performed, and wherein the means for deactivation of cell division is a gene switch that is activated by increasing the temperature.

[0026] Based on the above object, the present disclosure takes advantage of the separation of the phases of the protein production process thereby resulting in an improved production of recombinant protein. The improved production of said method is achieved by no longer burdening the cell with the task of cell division thereby allowing more metabolic resources to be available for recombinant protein expression which increases target protein yield.

[0027] The transfer of a part or portion of the biomass from the first bioreactor to the second bioreactor enables a first portion of the biomass to continue in the growth phase in the first bioreactor, while in parallel the second portion in the second bioreactor proceeds to the next phase of the process.

[0028] According to some embodiments, the means for deactivation of cell division is a genetic switch wherein the gene of interest is integrated as a single copy into the microbial strain.

[0029] By "means for deactivation of cell division" is meant a function and / or a gene regulatory element or the like, that may entail a genetic element with the ability to control some part of the function of a cell. If the means for activating cell division or gene regulatory element controls the function of deactivation of the cell division this can have further positive outcomes for the control of the expression of the protein, since the biomass formation of the exponential growth phase can be controlled and therefore separated from the stationary protein expression phase. This enables a more efficient control of the production of the protein. The expression phase can reach higher efficiency since the cell is not metabolically burdened with cell division. The induction of the protein expression can advantageously be started in the same step as the temperature regulated switch or performed separately with a chemical inducer. Further, in some embodiments the genetic switch simultaneously activates protein expression by placing the promoter upstream from the gene of interest. Further, in some embodiments the relocated promoter is constitutive and in some embodiments the promoter is activated by other means such as with a chemical inducer. A genetic switch may be chosen from Switcher Technology (OU Gearbox Biosciences).

[0030] For example, this can be accomplished by using Pop-Out-Plasmid™ technology (OU Gearbox Biosciences). With this technology, cell division is stopped by a temperature induced genetic switch that excises the origin of replication, OriC from the bacterial chromosome. The gene of interest is integrated as a single copy into the host chromosome and therefore is stably maintained during the biomass growth stage at 30°C. At the desired time the production stage can be switched on by temporarily increasing the culture temperature to 37°C. The gene of interest is then together with other genetic elements excised from the chromosome and forms a replicating plasmid. In the context of this disclosure, the "gene of interest" would typically be one or more genes encoding casein protein. The genetic code for these genes is available in public databases. Additional codon optimizations and minor adjustments for fitting the gene in a bacterial setting is added.

[0031] The method enables production of casein and / or caseinate comprising chemical characteristics that enables specific functionalities which are otherwise hard to achieve, and allows for the casein and / or caseinate produced by the method to be suitable for consumer and industrial applications.

[0032] Further, the method enables production of casein and / or caseinate, with or without the step of micellular formation. This allows for tailoring of the production method to accommodate other factors, such as cost of production, level of purity of the casein, level of functionality of the casein, among others. The process of caseinate production produces purified casein at a lower cost than that of micellular formation, whereas micellular formation enables the casein to achieve its full functionality in various applications. Thus, in some embodiments the steps involving micellular formation is performed, without producing caseinate. And in other embodiments the steps involving producing caseinate is performed, whereas micellular formation is not performed. The method may also involve including both micellular formation and caseinate production, as well as neither micellular formation nor caseinate production. According to some embodiments, the microbial strain is an Escherichia coli (E. coli) strain. Bacterial strains have the advantage of having a rate of cell division and biomass formation that is several times faster than that of Eukaryotic strains, making them time and cost effective for protein production.

[0033] In some embodiments, the E. coli strain comprises deletions of proteases. In a preferred embodiment, the E. coli strain comprises the deletions of the proteases Alon AcIpPX. The advantage of deleting these proteases is that casein is sensitive to proteolytic activity and the absence of these genes provides for a more stable environment in the cell cytosol. According to some embodiments, the means for deactivating cell division, also referred to as gene regulatory element, comprises a genetic switch using a gene of interest that is integrated as a single copy into the host chromosome. Thereby, the gene of interest can be stably maintained during the biomass growth phase.

[0034] According to some embodiments the biomass of step (b) exhibits an exponential growth, whereby the environmental factors enable repetitive cell division so the population increases in geometric progression, and limited or essentially no casein expression. Through limited or essentially no casein expression the cells in the first bioreactor is free to use more of the metabolic resources available for the cell division, thereby increasing the biomass production yield of the first bioreactor.

[0035] According to some embodiments a large portion of the biomass formed in step (b) is transferred to the second bioreactor. The transfer of the biomass can advantageously be initiated once an optimal desired yield of biomass has been produced, essentially once the first reactor has been filled to capacity with high density biomass. This allows both the first bioreactor to continue the exponential growth phase without being limited by the already produced biomass, while in parallel the transferred portion of biomass in the second bioreactor can initiate the protein expression phase. The term "optimal desired yield" and "sufficient number of cells" may vary in the different embodiments of the present disclosure depending on factors, such as the defined end product and / or size of the production or any other relevant factor.

[0036] According to some embodiments the transfer of biomass to the second bioreactor is repeated at predetermined intervals enabling a predictable production workflow.

[0037] According to some embodiments the genetic switch is activated by increasing the temperature. This is for example done by increasing temperature to about 37°C at a duration of approximately two hours, whereby the enzymatic switching activity has been completed, and thereafter decreasing the temperature to about 30°C for inducing casein expression. Other methods for activating a genetic switch with similar function may be based on chemically induced coexpression of a bacteriophage-derived polymerase inhibitor peptide. A lower temperature than 37°C cannot activate the promoter to the extent demanded by the timing of production steps and a higher temperature can stress the cell and interfere with the casein expression that follows in the next step. The use of a temperature induced switch can advantageously be used for both turning off cell division and turning on target protein expression and thus saving resources since a further step of careful addition of expensive chemicals is avoided. The use of heat in the production step is also beneficial since the thermal energy can be recaptured by e.g. heat exchangers and heat pumps. In other embodiments a chemical switch is used to turn off cell division e.g. using a bacteriophage system.

[0038] According to some embodiments the lysis of step (d) is performed by heating the fermentation broth. In a preferred embodiment the broth is heated to about 95°C. In some embodiments the broth is heated to between 60°C and 100°C. The use of thermolysis instead of other lysing alternatives, such as high-pressure homogenization, saves resources. The lysis step can thus be performed without adding machinery and transferring the biomass through an additional workflow. The use of heat in the production step is also beneficial since the thermal energy can be recaptured by e.g. heat exchangers and heat pumps. With casein being thermostable it remains soluble in the solution. Thus, in some embodiments, the heating step can serve the triple purpose of cell inactivation, casein release and initial purification.

[0039] In some embodiments the heat lysis is made in a separate vessel. Performing heat lysis outside reactor 2 enables an intermediary step with e.g. washing the cells, concentrating the cells, change the buffer and also do the lysis in a smaller volume.

[0040] According to some embodiments the separation of step (e) is performed by centrifuging the lysis broth to form a pellet and a supernatant, wherein casein is in the supernatant. The separation through centrifugation allows the various components within the pellet and supernatant to be separated based on size, shape, density and / or viscosity which further enables additional utilization of the various components in later steps of the production process and / or in applications outside of the present disclosure. The pellet can be used for other applications outside of the mentioned protein production process.

[0041] According to some embodiments the pellet is valorised. The valorisation can be achieved through the step of hydrolysing the pellet into amino acids and circulating it back into the fermentation. The valorisation can also be performed through the use of industrial digesters. The industrial digesters can transform the pelleted E. coli waste into valuable fertilizer through anaerobic digestion, thus recycling nutrients. This process not only produces nutrient-rich digestate suitable for use as a fertilizer but any further details, features and advantages to add also generates biogas, that can be further used as a renewable energy source. Therefore, implementation of the disclosed method strongly aligns with sustainable waste management practices, under the assumption of adherence to the strict health and safety standards to ensure the final product's quality and safety.

[0042] According to some embodiments the supernatant is ultrafiltrated for further purification. The supernatant will contain contaminants such as heat stable E. coli endogenous protein and lipopolysaccharides (LPS). In some embodiments the ultrafiltration has a cut off at 30kDa thereby letting the casein pass through but filtering out LPS contaminants. Protein contaminants can easily be separated in the purification step downstream but LPS is more difficult to separate out since it shares some of the same functional properties as casein. If the commercial casein application calls for low levels of LPS an ultrafiltration step can be added after the centrifugation. LPS typically has a molecular weight significantly higher than casein in a non-micellar configuration and can thus be separated using ultrafiltration.

[0043] According to some embodiments the micelle induction of step (e) is performed by adding calcium chloride, thereby inducing the casein molecules into forming casein micelles. A peculiarity of casein is that it achieves its full functionality only after aggregating into micelles. It is for instance the casein micelles that aggregate into a cheese curd, the casein molecules absent micellar configuration cannot be coagulated in this manner. The requirement to produce different casein types in the right proportions for micelle aggregation is a significant restraint in any process design involving micellar casein production.

[0044] According to some embodiments the casein micelles are further purified and / or coagulated into a casein curd by e.g. lowering pH and / or are separated by micro-filtration, and / or are further washed or repurified. The different steps of further purification can be used in combination or as called for to get the desired outcome of casein form.

[0045] According to some embodiments the recombinant casein is part of a composition comprising at least two casein types chosen from: Alpha SI, Alpha S2, Beta and Kappa or a combination thereof. In a preferred embodiment the recombinant casein chosen is a combination of Alpha SI and Kappa. Bovine casein micelles contain several casein types. It has been shown that micelles form in the presence of Alpha SI casein and Kappa casein in proportions of 10:1 under certain conditions, Noble and Waugh (1965). It has also been shown that dephosphorylated Alpha SI casein can form micelles with Kappa casein, Bingham (1972). It has furthermore been shown that deglycosylated Kappa casein retains capability to stabilize Alpha SI casein into micelles albeit a higher proportion of Kappa casein is then needed, since the glycosylation provides steric repulsion between micelles, Bonfatti (2014). The combination of Alpha SI and Kappa casein can be expressed at a ratio closely matching the required proportions for unglycosylated Kappa casein at approximately 7:1 Alpha SI and Kappa casein.

[0046] According to some embodiments the expression of Alpha SI and Kappa casein is performed bicistronically under the same promoter, thereby expressing the two types of casein in the same cell. Thereby enabling a major production simplification since parallel production of different casein types is not needed.

[0047] According to some embodiments, the culturing and / or expression steps are repeated with a variation of time durations and volumes for each repeated cycle. Hereby, depending on purpose and desired product and product volume, a series of cycles can be planned and performed.

[0048] According to some embodiments the method is performed with any of the following variations of the micellization process: (i) adding of calcium ions based on other calcium containing salts, (ii) adding of divalent cations based on other metals than calcium, and (iii) adding of phosphate containing reagents together with calcium chloride. Through the micellization process the casein is configured to a size of several orders of magnitude larger than the individual casein molecules. This creates new possibilities for purification. The micelle formation is thereby key not only for casein functionality in food and technical applications but also for protein purification in this production process. According to some embodiments the production of caseinate is performed by (i) precipitating the casein, and (ii) neutralizing the precipitated casein to form a corresponding caseinate.

[0049] According to some embodiments the precipitation of the casein is performed at the isoelectric point of the casein.

[0050] According to some embodiments the precipitation of the casein is performed at low pH.

[0051] According to some embodiments the neutralization of the precipitated casein is performed using an alkali to produce a caseinate salt.

[0052] In a second aspect the present disclosure also relates to a bioreactor system for producing recombinant casein, wherein the bioreactor system is designed for performing the method as described in the previously disclosed, said system comprising:

[0053] (i) a first bioreactor for culturing a microbial strain having the capacity to express casein;

[0054] (ii) a second bioreactor for induction of expression of casein, expression of casein and for lysing cells of the biomass to form a lysis broth;

[0055] (iii) means for separating the lysis broth, such as a centrifuge, wherein the lysis broth is separated into a pellet and a casein containing phase;

[0056] (iv) optionally means for ultrafiltrating the casein containing phase;

[0057] (v) means for treating the casein containing phase in order to induce casein micelle formation;

[0058] (vi) optionally means for further purification and / or treatment of the casein micelles, such as by microfiltration, acid coagulation and / or centrifugation;

[0059] (vii) optionally means for water filtration of water to be provided to the first bioreactor; and

[0060] (viii) means for controlling the system.

[0061] Based on the above object, the present disclosure further takes advantage of the separation of the phases of the protein production process through having multiple cascaded bioreactors. The first bioreactor keeps the cells in a continuous exponential growth phase, with minimal casein expression, while an additional bioreactor (second bioreactor) can be used to receive a transferred part of the biomass after the exponential growth reaches a desired yield. The conditions kept within the first and second bioreactor can be controlled separately by components within each bioreactor thus further allowing for improved production in each of the phases of the protein production process. The growth rate of the bacteria in a bioreactor is controlled by the rate at which new nutrients are supplied, and products are removed, making it a suitable and advantageous setup for industrial-scale production where consistent and controlled bacterial growth is necessary.

[0062] In some embodiments of the present disclosure, the remaining non-casein containing supernatant is filtrated in a water filtration unit so that remaining water can be added to the first bioreactor for another cycle of the production process, thereby enabling further optimization of the resource utilization in the system.

[0063] According to a further aspect, a recombinant casein of a casein composition characterized by that the casein composition comprises at least two types of casein in micellar form, wherein the caseins are chosen from Alpha SI, Alpha S2, Beta and Kappa casein, that has been bicistronically expressed under the same promoter. Hereby, this enables further a major production simplification since parallel production of different casein types is not needed.

[0064] According to a further aspect, a consumer product comprising the recombinant casein as described by the disclosed method and / or process above is chosen from: a dairy product such as cheese, milk, yoghurt, protein supplement powder, a meat analogue product comprising fibres spun from casein, and scaffolds for cultured meat production scaffolds for cultured meat production. Casein is a remarkably versatile protein with its unique textural and binding properties enabling a wide range of applications in food. The combination of nutrition, texture and melting properties of hard cheese is for instance currently not possible to achieve without casein.

[0065] According to a further aspect, an industrial product comprising the recombinant casein as described by the disclosed method and / or process above is chosen from: technical casein or caseinate, for use in glue or paint, such as labelling glue for bottles, biomaterials, scaffolds for cultured meat production, cosmetic and dermatological product, and other industrial products comprising casein or casein derivatives. With the same reasoning as previously, casein is also a highly desirable product for non-food products with its unique textural and binding properties enabling a wide range of applications in both food and non-food.

[0066] Brief description of the drawings

[0067] Figure 1 discloses one example embodiment of the system set-up for performing the method of the disclosure.

[0068] Figure 2 shows SDS-PAGE analysis from a bicistronic expression of casein types of Alpha SI and Kappa using the same promoter, POP-V5 being the strain expressing casein.

[0069] Figure 3 shows SDS-PAGE analysis from a purification step after bicistronic expression.

[0070] Detailed description

[0071] As mentioned, recombinant protein production methods for casein have several challenges. The production method provided here utilizes a combination of known technology and novel features in a way that provides improved efficiency in the production of casein.

[0072] The present disclosure relates, in a first aspect, to a production method for cost efficient production of recombinant casein in micellar form. In a second aspect, the disclosure relates to a system comprising a first and a second bioreactor for use in the production method of the first aspect. In further aspects, the disclosure relates to a recombinant casein, as well as dairy products and industrial products comprising the recombinant casein produced.

[0073] Broadly there are two microbial strain choices available for large scale casein expression: Eucaryote strains, such as Pichia pastoris (yeast) or Aspergillus niger (filamentous fungus), and bacterial strains, such as E. coli (gram negative) and Bacillus subtilis (gram positive). The eucaryote strain route is hampered by casein being an intrinsically disordered protein (IDP) with little tertiary structure. Eucaryote cells have a more complex metabolism where IDPs can be difficult to express. A typical example of this is the unfolded protein response (UPR) in the endoplasmic reticulum (ER) where the unfolded nature of casein can be interpreted by protein sensors as a misfolded protein. The UPR reaction is then to initiate degradation of the "misfolded" protein and halting further translation. Offsetting this reaction by genetic engineering and fermentation optimization is a challenging task due to the complexity of the cellular metabolism. Also, the rate of cell division and biomass formation is several times slower than that of fast-growing bacteria. This affects space-time yield negatively and makes production in continuous manufacturing difficult to achieve due to risk of faster growing contaminant bacteria essentially taking over the bioreactor. Yeast and filamentous fungi also have a more complex genetic makeup, metabolism and cell division behaviour, making them harder to optimize for a particular fermentation application. The bacterial strain route is likewise limited in several ways. On a general note, casein undergoes post-translational modifications in native bovine setting, most notably phosphorylation and glycosylation. This is typically not possible in the same way in bacteria as in eucaryote cells. There is some uncertainty regarding the degree and manner of post-translational modifications (PTMs) in the E. coli strain for Alpha SI and Kappa casein but glycosylation and phosphorylation is typically not happening in the same manner and extent as in native bovine setting. The common strains have different additional limitations. Some examples: Bacillus subtilis has a very high proteolytic activity while the unfolded nature of casein is sensitive to proteolytic degradation, typically making this a less suitable strain choice. E. coli is gram negative and typically it cannot efficiently secrete the target protein into the fermentation broth. Intracellular protein expression necessitates the additional production step of cell lysing which releases cellular content into the fermentation broth typically making protein purification too expensive for commodity protein production.

[0074] Therefore, the present disclosure utilizes a microbial strain, in some embodiments this strain is a E. coli strain, with a gene regulatory element comprising a genetic switch using a gene of interest that is integrated as a single copy into the host chromosome, such as a Pop-Out- Plasmid™ technology licensed from OU Gearbox Biosciences. The present disclosure can also be utilizing similar strains of other origin. The licensed strain features a genetic switch. The switch is triggered by raising temperature from 30°C to 37°C which induces expression of recombinase enzymes that excises a specific site on the E. coli chromosome containing both the gene of interest and the OriC into a plasmid. The cell can then no longer initiate DNA replication and divide. The cells are still metabolically active and may grow in size. Since the cell is no longer burdened by the task of cell division more metabolic resources are thus available for recombinant protein expression which increases target protein yield. The plasmid created contain the gene of interest under a promoter that can either be constitutive or chemically induced. In Pop-Out-Plasmid™ Technology the gene of interest is integrated as a single copy into the host chromosome and therefore is stably maintained during the biomass growth stage at 30^C. At the desired time the production stage can be switched on by temporarily increasing the culture temperature to 37^C.

[0075] According to some embodiments, the casein types Alpha SI and Kappa are expressed bicistronica I ly under the same promoter. The degree and manner of post-translational modifications (PTMs) in the E. coli strain for Alpha SI and Kappa casein may vary, and glycosylation and phosphorylation is typically not happening in the same manner and extent as in native bovine setting. Figure 2 shows an SDS-PAGE analysis from such a bicistronic expression. According to some embodiments, the casein is expressed intracellularly. After completed induction the cells are therefore heated to 95°C for one hour. This lyses the cell so that the casein is released into the fermentation broth and also degrades most of the endogenous E. coli protein rendering it insoluble. Casein is thermostable and remains soluble in the solution. Thus, the heating step serves the triple purpose of cell inactivation, casein release and initial purification. Figure 3 shows an SDS-PAGE analysis of the resulting purification. In some embodiments the cells are lysed for a longer or shorter duration than one hour.

[0076] Based on gel band strength analysis the casein types are expressed at an approximate ratio of 7:1 Alpha SI and Kappa casein, matching the required proportions for unglycosylated Kappa casein. See figure 2.

[0077] The method and system of the present disclosure will be described below with reference to figure 1, showing a repeatable method and corresponding system for efficient casein production. In this embodiment, micellization is performed as part of the method. In other embodiment, micellization may be excluded and / or a caseinate formation step may be included.

[0078] A first bioreactor ("bioreactor 1") is designed for continuous exponential growth of a microbial strain, such as an E. coli strain, having the capacity to express casein, and wherein said microbial strain comprises a gene regulatory element, such as a "genetic switch", for regulation of cell division activity of the microbial strain. Water and substrate are added to the first bioreactor, wherein the added water can be provided by filtrating the supernatant phase from a previous production cycle. Typically, in the first bioreactor, the microbial strain is cultured, thereby producing a continuously growing biomass of cells of the microbial strain. During the culturing phase, essentially no casein expression is performed. The repression of casein expression in the first bioreactor is vital for avoiding evolutionary pressure against the casein gene. After sufficient culturing approximately 80%, percentage can vary depending on optimization and configuration, is transferred to a second bioreactor ("bioreactor 2"). This can be done e.g. twice daily. The remaining biomass is used for starting the subsequent biomass reaccumulating cycle in the first bioreactor. After the transfer of biomass, the cells can go through the step of deactivation of cell division. After the transfer of biomass, the cells can go through the step of induction of protein expression. It is however preferable to utilize a combination of the steps of first deactivating the cell division and then inducing the protein expression is utilized to enable an efficient protein production process. Further there is little to no productivity loss at the mass transfer leading to even further efficiency benefits by having a two-bioreactor system.

[0079] In the second bioreactor, the regulatory element is affected so that cell division is switched off and casein expression is switched on. This can with e.g., be done by raising the temperature from 30°C to 37°C whereby a genetic switch is activated permanently disabling cell division, whereafter the temperature is lowered back down to 30°C and casein expression is induced. The switching at 37°C is highly sensitive to attaining correct temperature as a slightly lower temperature cannot activate the promoter to the extent demanded by the timing of production steps and a higher temperature can stress the cell and interfere with the casein expression that follows in the next step. The induction can be started in the same step as the temperature regulated switch or performed separately with a chemical inducer. After casein expression, the microbial strain cells are lysed, e.g., by raising the temperature to 95°C for one hour, thereby forming a lysis broth.

[0080] In a next step, said lysis broth is separated, e.g., by exhibiting the lysis broth for centrifugation in a centrifuge, into a pellet and supernatant, wherein the casein protein is essentially present in the supernatant. The temperature set during the centrifugation can be altered depending on the desired result of casein degradation. In some embodiments the temperature is kept at 4°C during centrifugation in order to lower proteolytic activity that might degrade the casein. In some embodiments the temperature is kept higher if the workflow is such that significant degradation does not have sufficient time to occur.

[0081] The pellet can be valorised in several ways, e.g. by hydrolysing and recycling amino acid back to the fermentation process or treated by digesters and used as fertilizer.

[0082] The supernatant is exhibited to ultrafiltration (optional step that is typically used to improve purity). In some embodiments the ultrafiltration has a cut off at 30kDa thereby letting the casein pass through but filtering out lipopolysaccharide (LPS) contaminants. In some embodiments the temperature is kept at 4°C during ultrafiltration in order to lower proteolytic activity that might degrade the casein. In some embodiments the temperature is kept higher if the workflow is such that significant degradation does not have sufficient time to occur.

[0083] As the next step, the remaining casein-containing phase is treated to induce casein micelle formation. In some embodiments the micelle induction step is performed by adding calcium chloride (CaCb). A technique that has been described by Noble and Waugh (1965). The resulting micelles contain thousands of casein molecules of the types Alpha SI, Alpha S2, Beta, and Kappa and self-assemble under certain conditions, most notably in the presence of Alpha SI, Beta and Kappa casein and in the presence of divalent cations such as Ca2+.

[0084] The resulting casein micelles are further purified, e.g., by microfiltration, acid coagulation, ultrafiltration and / or centrifugation, resulting in purified casein, such as in micellar form, and remaining supernatant (with essentially no presence of casein).

[0085] The purification process may or may not use an ultrafiltration step and additional purification steps depending on purity demands for various applications. One purification constraint may be that residual LPS contaminants form the E. coli cell walls.

[0086] Coagulation of the casein micelles may be achieved e.g., by lowering pH. The coagulated micelles will precipitate out of the solution and be readily pelleted by centrifugation. Additional steps of washing may be added as called for by the demands of specific commercial applications. Another means of further purification may be trough microfiltration. This filtration will separate the casein from remaining E. coli proteins and other soluble contaminants since these are significantly smaller in size than the casein micelles, otherwise they would have been pelleted in the previous centrifugation.

[0087] The casein produced can be used for commercial applications, such as in dairy products and / or industrial products, whereas the remaining non-casein containing supernatant may be filtrated in a water filtration unit so that remaining water can be added to the first bioreactor for another cycle of the production process.

[0088] The casein may also be purified with or without the step of micellular formation, by production of caseinate. Casein has an isoelectrical point at around pH 4,6. By lowering the pH to about this level, the casein will precipitate. Addition of an alkali to the precipitated casein enables neutralization of the solution and thus stabilisation of the precipitated casein as the insoluble casein is converted into a soluble salt form, a caseinate salt. An alkali may be a sodium alkali, such as sodium hydroxide.

[0089] Method features

[0090] The method steps are initiated by obtaining a microbial strain suitable for the purpose of the production process and setting up the components in the bioreactor system. The system comprises of at least a first and preferably a second bioreactor. Both bioreactors are prepared using the same method steps. The first bioreactor is designed for continuous exponential growth of the added microbial strain, here after referred to as the "culture". The second bioreactor is designed for protein expression of a portion of transferred biomass. The steps of preparing the bioreactors comprise of:

[0091] • setting up the bioreactor containers: Said bioreactor container is adapted to act as the culture vessel, wherein said bioreactor container is of an appropriate size relative to the desired production volume of the culture. Industrial production bioreactors are typically measured in m3and may reach a size of several hundreds of m3, • providing said bioreactor container with means and conditions: said means and conditions enables the continuous exponential growth of the added microbial strain, and

[0092] • autoclaving said bioreactor container; This is done to ensure a sterile environment prior to initiation of the process.

[0093] The means and conditions required for continuous exponential growth comprise installation and calibration of the bioreactor components serving the functions of monitoring and controlling of the continues exponential growth within said bioreactor. The components comprise, but are not limited to; at least one inlet port of said bioreactor that is separate from at least one outlet port of said bioreactor, an agitation system for mixing of cells and medium within said bioreactor, an aeration system for supplying oxygen to said bioreactor and extruding carbon dioxide from said bioreactor, a heating system, and sensors for monitoring; temperature, pH, pressure, nutrient levels, and liquid levels.

[0094] When the first bioreactor is prepared by installation and calibration of bioreactor components and autoclavation, the culture medium can be added into the first bioreactor container and a final sensor calibration is performed. Said final sensor calibration may advantageously be performed by running a blank culture medium sample for up to 24 hours to allow the container to equilibrate, hereafter the culture may be added into the first bioreactor.

[0095] For some embodiments the scaling-up from culture inoculation to the first bioreactor may involve an intermediate smaller bioreactor.

[0096] Similar components serving the functions of monitoring and controlling can be found in the second bioreactor.

[0097] The environmental conditions kept in the first and second bioreactor can be controlled separately by the components within each bioreactor. The environmental conditions are set to reflect the parameters of optimal conditions for the culture in each bioreactor at the phase of the process the culture is currently in. Different cultures may require different conditions at the various phases. For the first bioreactor the parameters of the conditions are set to reflect an optimal environment for cultivation. In the second bioreactor the parameters of the conditions are set to reflect an optimal environment for protein expression.

[0098] Parameters affecting the optimal conditions for the bioreactors comprise of but not limited to; temperature, pH, oxygen levels, substrate composition and timing and movement of impellers.

[0099] In such a system, the growth rate of the bacteria is controlled by the rate at which new nutrients are supplied and products are removed, making it an ideal setup for industrial-scale production where consistent and controlled bacterial growth is necessary. In some embodiments, the culturing and / or expression steps are repeated with a variation of time durations and volumes for each repeated cycle. In some embodiments, a portion of the microbial strain cultured in the first bioreactor may be kept in the first bioreactor for subsequent cycles. In some embodiments, the time (duration), volume and other parameters may be varied from cycle to cycle, in order to obtain a variation of product volumes and depending on purpose and desired outcome. The method of the present disclosure is thus suitable for such cascaded production methods including variations of production parameters such as volume and duration (time) of each cycle.

[0100] The continuous culture in the first bioreactor is operating under conditions that maintain E. coli cells in a state of exponential growth phase. This is achieved by carefully regulating critical parameters such as nutrient supply, pH, temperature, and oxygen availability to ensure that the bacterial cells divide at a constant rate, thus exhibiting logarithmic or exponential increase in cell number over time. The system is typically designed to continuously supply fresh medium. This mode of operation enables high-density cultivation of E. coli, optimizing the production of biomass.

[0101] The temperature is typically measured by the temperature sensor comprised within the second bioreactor.

[0102] After completed thermolysis the temperature is lowered to approximately 30°C, or in some case as low as 4°C if needed to prevent proteolytic activity, and transferred out of the bioreactor for downstream processing. The next step is centrifugation to separate the soluble casein from the insoluble components mainly consisting of cell wall and membrane fragments and denatured E. coli endogenous protein. By centrifugation the insoluble components are pelleted while the soluble casein is retained in the supernatant.

[0103] The solids retained in the centrifugation phase are preferably valorised as described earlier.

[0104] After centrifugation and the optional phase of ultrafiltration follows micellization. In this step calcium chloride is added in one aliquot for a final concentration of O,lmM followed by gentle stirring. In some embodiments the final concentration is higher or lower than O,lmM. Additional reagents such as buffers and phosphates may be added. This step is typically performed at 37°C. The addition of calcium ions forces the Alpha SI casein and Kappa casein into a micellar configuration. The casein micelle contains thousands of casein molecules and is typically 150nM in diameter though the diameter is known to vary significantly. The casein is now in a configuration with a size several orders of magnitude larger than the individual casein molecules. This creates new possibilities for purification. The micelles can be purified with microfiltration. This filtration will separate the casein from remaining E. coli proteins and other soluble contaminants since these are significantly smaller in size than the casein micelles, otherwise they would have been pelleted in the previous centrifugation. This purification step can also include coagulation of the casein micelles e.g. by lowering pH. The coagulated micelles will precipitate out of the solution and be readily pelleted by centrifugation. Additional steps of washing may be added as called for by the demands of specific commercial applications.

[0105] A caseinate may be produced by performing the steps of precipitating the casein, and neutralizing the precipitated casein to form a corresponding caseinate. The precipitation of the casein may be performed at the isoelectric point of the casein. The precipitation of the casein may be performed at low pH. A low pH would generally constitute a pH below 7. It may preferably be a pH in the interval of pH 3-6, even more preferably a pH in the interval of pH 4-5, most preferable a pH close to 4,6. A low pH is selected to correspond with the isoelectric point of the caseinate where the casein has minimal colloidal stability and tends to precipitate out of solution.

[0106] The neutralization of the precipitated casein may be performed using an alkali to produce a caseinate salt. The neutralisation converts the insoluble casein into a soluble salt form, a caseinate salt. The neutralized casein solution, comprising said casein salt, may further be dried to produce a powdered form of sodium caseinate.

[0107] System features

[0108] In a second aspect, the present disclosure relates to a system for producing recombinant casein, comprising the following components:

[0109] (i) a first bioreactor for culturing a microbial strain having the capacity to express casein;

[0110] (ii) a second bioreactor for induction of expression of casein, expression of casein and for lysing cells of the biomass to form a lysis broth;

[0111] (iii) means for separating the lysis broth, such as a centrifuge, wherein the lysis broth is separated into a pellet and a casein containing phase;

[0112] (iv) optionally means for ultrafiltrating the casein containing phase;

[0113] (v) means for treating the casein containing phase in order to induce casein micelle formation;

[0114] (vi) optionally means for further purification and / or treatment of the casein micelles, such as by microfiltration, acid coagulation and / or centrifugation;

[0115] (vii) optionally means for water filtration of water to be provided to the first bioreactor; and

[0116] (viii) means for controlling the system.

[0117] In some embodiments, the system further comprises means for transferring material between different components of the system, such as from the first to the second bioreactor.

[0118] In some embodiments, the means for controlling the system may include a processor and / or a computer program product or the like, for control and variation of production parameters such as time, pH, temperature and volume of method steps, as well as controlling means for transferring material from a first to a second bioreactor or the like. In some embodiments, the means for controlling the system supports the use of cascaded methods. The details of the ingoing components are further as discussed above in the present disclosure in relation to the method aspect of the present disclosure.

[0119] Recombinant casein features

[0120] In a third aspect, the present disclosure relates to a recombinant casein, produced by the method of the present disclosure, wherein the casein comprises at least two types of casein that has been bicistronically expressed under the same promoter.

[0121] In some embodiments, the recombinant casein is in the form of casein micelles. In some embodiments, the recombinant casein is chosen from Alpha SI, Alpha S2, Beta and Kappa casein, or combinations thereof.

[0122] A recombinant casein may be a casein composition. Said casein composition may be formed by combining at least two types of casein.

[0123] Further details of the recombinant casein are as discussed above in the present disclosure.

[0124] Product features

[0125] In a fourth aspect, the present disclosure relates to a consumer product comprising the recombinant casein of the present disclosure and / or as produced by the method of the present disclosure, chosen from a dairy product such as cheese, milk, yoghurt, protein supplement powder, a meat analogue product comprising fibres spun from casein, and scaffolds for cultured meat production.

[0126] In a fifth aspect, the present disclosure relates to an industrial product comprising the recombinant casein of the present disclosure and / or as produced by the method of the present disclosure, chosen from technical casein or caseinate, for use in glue or paint, such as labelling glue for bottles, cosmetic and dermatological product, biomaterials; scaffolds for cultured meat production; and other industrial applications of casein or casein derivates. Technical casein is a known commodity used in various non-food applications such as glue, paint and fibres. Further, various medical products are conceivable. Casein may e.g. be used in various medical applications such as oral drug delivery component and speciality wound healing materials. Further details of products comprising recombinant casein of this disclosure are as discussed above in the present disclosure.

[0127] The invention will now be described in further detail in the following examples. Examples

[0128] In practice, the casein is expressed intracellularly in the cytosol. After completed induction the cells are therefore heated to 95°C for one hour. This lyses the cell so that the casein is released into the fermentation broth and also degrades most of the endogenous E. coli protein rendering it insoluble. Casein is thermostable and remains soluble in the solution. Thus, the heating step serves the triple purpose of cell inactivation, casein release and initial purification. Figure 3 shows an SDS-PAGE analysis of the resulting purification where the casein is still soluble and present in the supernatant while most of the endogenous E. coli protein is removed following heat lysis at 95°C for 60 minutes and 120 minutes. In some embodiments the cells are lysed for a longer or shorter duration than one hour.

[0129] Example 1 - Small scale SDS PAGE analysis from a bicistronic expression of casein types Alpha SI and Kappa using the same promoter of an inducible or constitutive type.

[0130] An Escherichia coli strain expressing Alpha SI and Kappa casein was provided, and was allowed to express the casein proteins under the same promoter.

[0131] The protein expression was estimated in 2xYT-M9gluc medium consisting of:

[0132] 31 g / L Difco 2xYT (Yeast Extract Tryptone) Medium, 6.78 g / L Na2HPO4, 3 g / L KH2PO4, 1 g / L NH4CI, 0.5 g / L NaCI, 4 mM MgSO4, 0.2 mM CaCI2, 3 g / L glucose; pH=7.

[0133] 1) Overnight culture of POP-V3 was diluted to an OD600 of 0.005 into 1 L flask containing 150 mL of 2xYT-M9gluc media supplemented with 1 pM HSL (final concentration).

[0134] 2) The cultures were grown at 30°C and shaking at 180 rpm for 4 hours.

[0135] 3) Thereafter the temperature was increased to 37°C for 2 hours to induce the switch.

[0136] 4) Thereafter the temperature was lowered back to 30°C to allow the synthesis of caseins.

[0137] 5) 6.5 hours later the culture was collected: a) For measuring cell dry weight (CDW) 2 mL of cell culture was transferred to 2 mL tube (preheated at 65°C for 12 hours and weighed) and pelleted at 5000 x g for 5 minutes, supernatant was removed, and the pellet was washed with 1 mL of MilliQ water to remove residual salts and media components. The cells were pelleted at 5000 x g for 5 minutes and after removing the supernatant the pellets were dried at 65°C for 24 hours. The weight of the tubes was determined and the amount of CDW calculated. The CDW measurement was performed in triplicates. b) Cells for casein analysis was collected from 100 mL of culture by centrifugation at 5,000 x g for 5 minutes at 4°C and suspended in of 5 mL of ice-cold PBS. The final volume of suspension increased approx, to 6 mL because of the pellets. This sample is referred as "Suspension" on Figure 3. c) The 6 mL cell suspension was heated at 95°C in a 15 mL centrifuge tube in water bath. At timepoint 1 or 2 hours of heating, a 2 mL aliquot of the heated sample was transferred to a new tube and allowed to cool on ice. Soluble and insoluble proteins were separated by centrifugation at 16,000 x g for 5 minutes at 4°C and the sample of soluble proteins (supernatant; sample is referred as "Super" on Figure 3) was taken for SDS-PAGE and Qubit Protein Assay (ThermoFisher Scientific).

[0138] Results:

[0139] Cell dry weight in the used growth conditions (QD600 at harvest was ~8) was measured 2.4 ± 0.1 g / L

[0140] Heating samples at 95°C for 1 hour successfully releases caseins from the cells. Extending the heating time did not increase the amount caseins released (Figure 3). The total protein content was measured 4.0 mg / mL in the supernatant of the 1 h heated sample. To estimate the ratio between Alpha SI and Kappa caseins in the heat-extracted supernatant, the said sample was diluted and visualized on SDS-PAGE. The densitometric analysis of the bands as well as visual inspection reveal that the ratio of Alpha SI and Kappa caseins is in the range of 7:1 to 10:1. It is difficult to get an exact number as the bands on the gel do not fall in linear range within the same dilution. As a rough estimate 55-75% of the total proteins in the supernatant are caseins according to densitometric analysis of protein bands on SDS-PAGE.

[0141] Figure 2 shows an SDS-PAGE analysis from the bicistronic expression. Based on gel band strength analysis the casein types are expressed at an approximate ratio of 7:1 Alpha SI and Kappa casein, closely matching the required proportions for unglycosylated Kappa casein. See figure 2.

[0142] Example 2 - Small scale test of micellization and coagulation.

[0143] Biomass from cultivation according to protocol in example 1 is used. The addition of CaCb. changes appearance of the solution from yellow into a milky white. Subsequent lowering of pH creates visible coagulation that precipitates out of the solution. SDS-PAGE analysis confirm that casein is no longer present in the supernatant.

[0144] • 4.45 g of wet cell weight was solubilized in 13.35 mL of Tris-HCI, 50 mM, pH 7.0 and heat lysed for lh at 95°C.

[0145] • 1.2 mL (~ 55.4 mg total protein) of the concentrated lysate obtained after 3 kDa-UF was used.

[0146] • Sample incubated at 37°C for 10 min

[0147] • First addition of lOmM CaCb

[0148] • lh incubation at 37°C

[0149] • Centrifugation 5000g 5min

[0150] • Second addition of 10mM CaCb

[0151] • pH adjusted in range 5-5,5

[0152] o hftt tphse: / A / dmoei.roicragn / 1 C0h.1e0m2i1ca / jla S0o1c0ie8t8ya,08276(10), 2246-2257. https: / / doi.org / 10.1021 / ja01088a026

[0153] References

[0154] Kopp, J., Kittier, S., Slouka, C., Herwig, C., Spadiut, O., & Wurm, D. J. (2020). Repetitive Fed- Batch: A Promising Process Mode for Biomanufacturing With E. coli. Frontiers in Bioengineering and Biotechnology, 8. https: / / doi.org / 10.3389 / fbioe.2020.573607 Bonfatti, V., Chiarot, G., Garnier, P., (2014) Glycosylation of K-casein: Genetic and nongenetic variation and effects on rennet coagulation properties of milk. https: / / www.journalofdairyscience.org / article / S0022-0302(14)00094- 0 / fulltexthttps: / / doi.org / 10.1038 / s41572-020-0196-7

[0155] Waugh, D. F., & Noble, R. W. (1965b). Casein Micelles. Formation and Structure. 111. Journal https: / / doi.org / 10.1021 / bi00763a010Bingham, E. W., Farrell, H. M., & Carroll, R. J. (1972b). Properties of dephosphorylated asl-casein. Precipitation by calcium ions and micelle formation. Biochemistry, 11(13), 2450-2454. https: / / doi.org / 10.1021 / bi00763a010

Claims

Claims1. A method for production of recombinant casein, comprising the steps of:(a) adding a microbial strain to a first bioreactor, wherein said microbial strain has the capacity to express casein, and said microbial strain comprises means for deactivation of cell division;(b) culturing said microbial strain in the first bioreactor, wherein the cell division is active, thereby forming a continuously growing biomass comprising cells of said microbial strain;(c) deactivating cell division;(d) inducing expression of casein;(e) after sufficient expression of casein, lysing said cells, thereby forming a lysis broth, and separating said lysis broth so that casein can be separated into a casein containing phase;(f) optionally treating the casein containing phase to induce formation of casein micelles; and(g) optionally further purifying and / or treating the casein micelles; and(h) optionally producing caseinate; wherein part of the biomass obtained in the culturing of step (b) is transferred to a second bioreactor, in which at least step (c) and (d) is performed, and wherein the means for deactivation of cell division is a genetic switch that is activated by increasing the temperature.

2. The method according to claim 1, wherein the genetic switch comprises a gene of interest, which is integrated as a single copy into the microbial strain.

3. The method according to any of the preceding claims, wherein the microbial strain is an E. coli strain.

4. The method according to claim 3, wherein the E. coli strain includes deletions of proteases Alon AcIpPX.

5. The method according to claim 4, wherein the E. coli strain comprises a promoter upstream of the gene of interest, downstream of the gene of interest, or a bidirectional promoter.

6. The method according to any of the preceding claims, wherein the biomass of step (b) exhibits an exponential growth, and limited or essentially no casein expression.

7. The method according to any one the preceding claims, wherein the transfer of biomass to the second bioreactor is repeated at predetermined intervals.

8. The method according to any of the preceding claims, wherein the genetic switch is activated by increasing the temperature to about 37°C at a duration of about two hours and thereafter decreasing the temperature to about 30°C for inducing casein expression.

9. The method according to any one of the preceding claims, wherein the lysis of step (d) is performed by heating the fermentation broth to about 95°C.

10. The method according to any one of the preceding claims, wherein the separation of step (e) is performed by centrifuging the lysis broth to form a pellet and a supernatant, wherein casein is in the supernatant.

11. The method according to claim 10, wherein the pellet is valorised.

12. The method according to claim 10 or 11, wherein the supernatant is ultrafiltrated for further purification.

13. The method according to any of the preceding claims, wherein the micelle induction of step (e) is performed by adding calcium chloride, thereby forming casein micelles.

14. The method according to any of the preceding claims, wherein the casein micelles are further purified and / or treated by coagulation into a casein curd by lowering pH and / or are separated by micro-filtration, and / or are further washed or repurified.

15. The method according to any of the preceding claims, wherein the recombinant casein is part of a composition comprising at least two casein types chosen from: Alpha SI, Alpha S2, Beta and Kappa, or any combination thereof.

16. The method according to any of the preceding claims, wherein the expression is performed bicistronica I ly under the same promoter, thereby expressing two types of casein.

17. The method according to any of the preceding claims, wherein the culturing and / or expression steps are repeated with a variation of time durations and volumes for each repeated cycle.

18. The method according to any of the preceding claims, wherein any of the following variations of the micellization process can be performed: (i) adding of calcium ions based on other calcium containing salts, (ii) adding of divalent cations based on othermetals than calcium, and (iii) adding of phosphate containing reagents together with calcium chloride.

19. The method according to any of the preceding claims, wherein the step of production of caseinate is performed by (i) precipitating the casein, followed by (ii) neutralizing the precipitated casein to form a corresponding caseinate.

20. The method according to claim 19, wherein the precipitation of the casein is performed at the isoelectric point of the casein.

21. The method according to any of claims 19-20, wherein the precipitation of the casein is performed at low pH.

22. The method according to any of claims 19-21, wherein the neutralization of the precipitated casein is performed using an alkali to produce a caseinate salt.

23. A bioreactor system for producing recombinant casein, wherein the bioreactor system is designed for performing the method of claims 1-22, comprising: a. a first bioreactor for culturing a microbial strain having the capacity to express casein; b. a second bioreactor for induction of expression of casein, expression of casein and for lysing cells of the biomass to form a lysis broth; c. means for separating the lysis broth, such as a centrifuge, wherein the lysis broth is separated into a pellet and a casein containing phase; d. optionally means for ultrafiltrating the casein containing phase; e. means for treating the casein containing phase in order to induce casein micelle formation; f. optionally means for further purification and / or treatment of the casein micelles, such as by microfiltration, acid coagulation and / or centrifugation; g. optionally means for water filtration of water to be provided to the first bioreactor; and h. means for controlling the system, i. means of deactivating the cell division, which is a genetic switch activated by increasing the temperature, and j. means for transferring biomass from the first to the second bioreactor.

24. A recombinant casein of a casein composition, characterized by that the casein composition comprises at least two types of casein in micellar form, wherein the caseins are chosen from Alpha SI, Alpha S2, Beta and Kappa casein, that has been bicistronically expressed under the same promoter.

25. A consumer product comprising the recombinant casein of claim 24 or as produced by the method according to claims 1-22, chosen from:(i) a dairy product such as cheese, milk, yoghurt, and protein supplement powder;(ii) a meat analogue product comprising fibres spun from casein;(iii) a cosmetic and dermatological product;(iv) a scaffold for cultured meat production; and(v) other consumer products comprising casein or casein derivatives.

26. An industrial product comprising the recombinant casein of claim 24, or as produced by the method according to claims 1-22, chosen from:(i) technical casein or caseinate, for use in glue or paint, labelling adhesives, such as labelling glue or labelling adhesives for bottles;(ii) biomaterials;(iii) a scaffold for cultured meat production; and(iv) other industrial applications of casein or casein derivates.

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