DVASH OFIR (IL)
THE IP LAW FIRM OF GUY LEVI LLC (US)
WO2021168343A2 | 2021-08-26 | |||
WO2021148620A1 | 2021-07-29 | |||
WO2023133417A2 | 2023-07-13 | |||
WO2020202157A1 | 2020-10-08 |
US10947552B1 | 2021-03-16 | |||
US20210010017A1 | 2021-01-14 |
YONGJIN J. ZHOU, BUIJS NICOLAAS A., ZHU ZHIWEI, QIN JIUFU, SIEWERS VERENA, NIELSEN JENS: "Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories", NATURE COMMUNICATIONS, vol. 7, no. 1, 25 May 2016 (2016-05-25), pages 1 - 9, XP055743962, DOI: 10.1038/ncomms11709
WHAT IS CLAIMED: 1. A system for producing Bubalus bubalis milk product, the system comprising: a. a plurality of bioreactors, each having a proximal end and a distal end, each bioreactor further containing the at least one of recombinant: a yeast, a bacterium, a fungus, and an algae comprising heterologous polynucleotides encoding a Bubalus bubalis polypeptide, wherein each bioreactor is further being in liquid communication with a mixing tank; and b. a plurality of collection receptacle, each collection receptacle associated with a product- specific bioreactor combination. 2. The system of claim 1, wherein the Bubalus bubalis polypeptide is at least one of: a milk protein, a whey protein, and an antimicrobial protein. 3. The system of claim 2, wherein the milk protein is at least one of: αS1-casein (αS1-CN), αS2- casein (αS2-CN), β-casein (β -CN), and κ-casein (κ –CN). 4. The system of claim 2, wherein the whey protein is at least one of: β -lactoglobulin (β -LGB) and α-lactalbumin B (α-LAB). 5. The system of claim 2, wherein the antimicrobial protein is at least one of: lactoferrin, lactoperoxidase and lysozyme C. 6. The system of claim 1, further comprising a bioreactor comprising a carrier having thereon Bubalus bubalis mammary epithelial cells (MECs), adapted to secrete and accumulate fatty acids, or a carrier having thereon a recombinant yeast adapted to overproduce extra-cellular free fatty acids (FFAs), wherein the bioreactor comprising the Bubalus bubalis MECs, or the recombinant yeast is in liquid communication with the mixing tank, and a FFA separator, the FFA separator being in further in liquid communication with the mixing tank. 7. The system of claim 1, further comprising a bioreactor comprising a carrier having thereon a recombinant yeast comprising heterologous polynucleotides encoding at least one human milk oligosaccharide (HMO), wherein the bioreactor comprising the recombinant yeast is in liquid communication with the mixing tank, and a HMO separator, the HMO separator being in further liquid communication with the mixing tank. 8. The system of claim 7, wherein the recombinant yeast adapted to overproduce extracellular free fatty acids (FFAs) comprises a heterologous polynucleotides encoding genes having selective deletions, configured to overexpress extracellular FFAs. 9. The system of claim 8, comprising deletion in at least one of: FAA2, FAA1, FAA4, FAT1, PXA1, and POX1. 10. The system of claim 8, wherein the heterologous polynucleotides is adapted to overexpress at least one of: DGA1 (diacylglycerol acyltransferase) and TGL3 (triacylglycerol lipase). 11. The system of claim 8, wherein the heterologous polynucleotides is adapted to overexpress at least one gene of ATP:citrate lyase (ACL), malic enzyme (ME), limitochondrial citrate transporter (Ctp1), malate dehydrogenase (Mdh3), fatty acid synthase genes (FAS1 and FAS2), a truncated thioesterase (’tesA), and endogenous acetyl-CoA carboxylase (ACC1). 12. The system of claim 7, wherein the recombinant yeast’s heterologous polynucleotide encodes a lactose transporter, a GDP-L fucose synthetic pathway, α-1,2-fucosyltransferase and a 2’-FL transporter. 13. The system of claim 12, wherein the recombinant yeast’s heterologous polynucleotide encodes a deletion of gal80 gene. 14. The system of claim 1, wherein each bioreactor is in further communication with a protein purification module, operable to selectively isolate a predetermined protein. 15. The system of claim 14, wherein the protein purification module comprises at least one of: a size exclusion column, a hollow fiber tangential flow filtration (TFF) column, and an affinity column. 16. The system of claim 1, further comprising an additive container in liquid communication with the mixing tank, operable to selectably provide a predetermined additive into the mixing tank. 17. The system of claim 16, wherein the predetermined additive is at least one of: sugars, vitamins, minerals, plant proteins, amino acids, antioxidants, and plant-source fatty acids. |
Or a sequence having between 80% and 99% homology, its mRNA and cDNA or showing the activity of the encoded proteins. [00025] Moreover, the Bubalus bubalis polypeptide, expressed, isolated and collected using the systems disclosed can be a mature milk protein having antimicrobial activity, that is at least one of: lactoferrin, lactoperoxidase and lysozyme C, encoded by the following sequences:
Or a sequence having between 80% and 99% homology, its mRNA and cDNA or showing the activity of the encoded proteins. [00026] System 10, can optionally further comprise bioreactor 1014 comprising carrier (1044, not shown) having thereon and being operable to support a plurality of Bubalus bubalis mammary epithelial cells (MECs), adapted to secrete and accumulate a fatty acid, or additionally, or alternatively a (bioreactor e.g., with-) carrier 1045 (not shown) having thereon a recombinant yeast adapted to overproduce and overexpress extra-cellular free fatty acids (FFAs), wherein bioreactor 1014 comprising the Bubalus bubalis MECs, or the recombinant yeast is in liquid communication with mixing tank 100, and a FFA separator 302, FFA separator 302 being in further in liquid communication with mixing tank 100. [00027] Mammary epithelial cells (MECs) secrete milk constituents by several routes. Milk lipid is enveloped by a milk fat globule membrane (MFGM) derived from the apical cell surface, and contains some of its constituent proteins. Soluble milk proteins are secreted by exocytosis. MECs can be cultured directly on scaffolding (e.g., carrier 1044), embedded e.g., in a reconstituted basement membrane that can be further cultured a medium containing lactogenic hormones (e.g., estrogen, progesterone and prolactin). [00028] Additionally, or alternatively, genetically modified yeast lines are genetically engineered in an exemplary implementation, to overproduce and/or overexpress extracellular FFAs. In another exemplary implementation, engineered yeast (e.g., s. cerevisiae) line would include deletions in the following genes: FAA2, FAA1, FAA4 and FAT1 (acyl-CoA synthetase), PXA1 (coding for a subunit of the ABC transporter complex Pxa1–Pxa2 that is responsible for importing long chain fatty acids into the peroxisome) and POX1(fatty acyl-CoA oxidase). It would also include overexpression of DGA1 (diacylglycerol acyltransferase) and TGL3 (triacylglycerol lipase). Accordingly, the recombinant yeast adapted to overproduce, and/or overexpress extracellular free fatty acids (FFAs) comprises a heterologous polynucleotides encoding genes having selective deletions, configured to overexpress extracellular FFAs, comprising deletion in at least one of: FAA2, FAA1, FAA4, FAT1, PXA1, and POX1. [00029] As used herein, the term “genetic engineering”, or “genetically engineered” refer to the creation of a non-natural DNA, protein, or organism that would not normally be found in nature and therefore entails applying human intervention. Genetic engineering can be used to produce an engineered DNA, protein, or organism that was conceived of and created in the laboratory using one or more of the techniques of biotechnology such as molecular biology, protein biochemistry, bacterial transformation, transfection, and plant transformation. For example, genetic engineering can be used to create a chimeric gene comprising at least two DNA molecules heterologous to each other using one or more of the techniques of molecular biology, such as gene cloning, DNA ligation, and DNA synthesis, for example, CRISPR-cas9 system. A chimeric gene may consist of two or more heterologous DNA molecules that are operably linked, such as a protein-coding sequence operably linked to a gene expression element such as a transit peptide-coding sequence or a heterologous promoter. Genetic engineering can be used to create an engineered protein whose polypeptide sequence was created using one or more of the techniques of protein engineering, such as protein design using site-directed mutagenesis and directed evolution using random mutagenesis and DNA shuffling. An engineered protein may have one or more deletions, insertions, or substitutions relative to the coding sequence of the wild-type protein and each deletion, insertion, or substitution may consist of one or more amino acids. In another exemplary implementation, an engineered protein may consist of two heterologous peptides that are operably linked, such as an enzyme operably linked to a transit peptide. [00030] In another exemplary implementation, the genetically engineered yeast line can include deletions in acyl-CoA synthetase genes (∆faa1 and ∆faa4) and fatty acyl-CoA oxidase (∆pox1), and overexpression of ATP:citrate lyase (ACL), malic enzyme (ME), limitochondrial citrate transporter (Ctp1), malate dehydrogenase (Mdh3), fatty acid synthase genes (FAS1 and FAS2), a truncated thioesterase (’tesA) and enhanced expression of the endogenous acetyl-CoA carboxylase ACC1 by replacing its native promoter with the TEF1 promoter represented in an exemplary implementation by by the sequence: [00031] In the context of the disclosure, the term "promoter" refers to a region of DNA upstream from the translational start codon and which is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. The term "operably linked" as used herein, refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence (e.g., SEQ ID No.s 1, 3, 5). It is understood that the promoter sequence (e.g., SEQ ID No. 19) also includes transcribed sequences between the transcriptional start and the translational start codon. In an exemplary implementation, various promoters will be selected based on the organism in which the protein is expressed. for example, Tef-1 (see e.g., SEQ ID No. 19) can be used for S.cerevisiae and P.pastoris. [00032] Utilizing oleaginous microorganisms; fungi, yeasts and algae can be adapted in an exemplary implementation to accumulate lipids as much as 20% of their dry cellular weight. Oils/fats accumulated by oleaginous microbes (OM) are gaining significant interest owing to the quality of lipids, which can be used for either food consumption, or fuel purpose. In oleaginous yeasts, lipids are found mainly in the form of neutral lipids, glycolipids, phospholipids, and free fatty acids (FFA). The oleaginous yeasts used in the systems and methods disclosed, can be, for example the genera Yarrowia, Rhodotorula (Rhodosporidium), Lipomyces, Cryptococcus and Trichosporon. Specifically, Yarrowia lipolytica has shown to be a convenient host for industrial processes and as a model organism for investigating lipid synthesis. It is recognized as a generally regarded as safe (GRAS) microorganism, and for this reason Y. lipolytica is used in an exemplary implementation as a host for the production of dietary supplements and nutraceuticals (Caporusso et al., 2021). Wild-type Y. lipolytica grows on a variety of substrates and can accumulate lipids intracellularly to ≥40% of its cell dry weight. Y. lipolytica is known for its pronounced lipolytic and proteolytic activities that is naturally found in foods with high proportions of fat and/or protein, particularly in (fermented) dairy products and meat. [00033] In yet another exemplary implementation, system 10 further comprises bioreactor 1015 comprising carrier 1046 (not shown) having thereon a recombinant yeast comprising heterologous polynucleotides encoding at least one human milk oligosaccharide (HMO), wherein bioreactor 1015 comprising the recombinant yeast is in liquid communication with mixing tank 10, and HMO separator 303, whereby HMO separator 303 is being in further liquid communication with mixing tank 100. For example, the most abundant HMO, 2′-fucosyllactose (2’-FL), can be produced in system 10 disclosed, by genetically engineered S. cerevisiae cell factory. 2’-FL synthesis and secretion in engineered S. cerevisiae, is achieved by transfecting the encoding DNA of the organism with four heterologous metabolic components: a lactose transporter, a GDP-L fucose synthetic pathway, α-1,2- fucosyltransferase and a 2’-FL transporter. In an exemplary implementation heterologous α-1, 2- fucosyltransferase is expressed in Bacillus cereus (FutBc) coding nucleotide and deletion of gal80, where the resulting strain produced 19.56 g/L extracellular and 7.07 g/L intracellular. Accordingly, and in an exemplary implementation, the recombinant yeast’s heterologous polynucleotide encodes a deletion of gal80 gene. [00034] In an exemplary implementation, secreted proteins, and optionally fatty acids or HMOs enter the mixing tank 100 through molecular weight cutoff membranes (5–200 Kda) 102j that will prevent the producing organisms and cells from entering mixing tank 100 as well. This will be achieved via array of valves 103p according to the desired recipe for specific products, i.e., standardized milk, whey or crème. Furthermore, a protein purification step may be implemented, whereby separation is done using for example, size exclusion membrane and/or hollow fiber tangential flow filtration (TFF) and may also include protein isolation by binding to an affinity column. For example, His-tag purification columns (immobilized metal affinity chromatography (IMAC) columns such as, Ni-NTA Agarose column). Accordingly and in an exemplary implementation, each i th bioreactor 1011, 1012, 1013, is in further communication with protein purification module 301, operable to selectively isolate a predetermined protein secreted by each i th bioreactor 1011, 1012, 1013, whereby protein purification module 301 comprises at least one of: a size exclusion column, a hollow fiber tangential flow filtration (TFF) column, and an affinity column 3010. [00035] An additional step may further comprise the extraction of fatty acids and HMOs. As for fatty acids - in the food industry, ethanol and hexane are widely used as low-toxicity solvents for lipid extraction. Green solvents such as bio-derived solvents, ionic liquids and deep eutectic solvents are employed in an exemplary implementation for the extraction of oil from oleaginous microbes. These green solvents are eco-friendly, low in energy and solvent consumptions and display higher efficiency in product formations. Additional green extraction techniques can comprise enzyme assisted extraction (AEE), microwave assisted extraction (MAE) and ultrasound assisted extraction (UAE) (Kumar et al., 2021). Extraction of fatty acids will be followed by their supplement and admixing into mixing tank 100. [00036] Furthermore, a combination of a cationic ion exchanger treatment, an anionic ion exchanger treatment, and a nanofiltration and/or electrodialysis step, allows efficient purification of large quantities of neutral HMOs at high purity and without the need of a chromatographic separation. The purified HMOs may be obtained in solid form post processing by spray drying, as crystalline material or as sterile filtered concentrate. Like the FFAs, separation and purification of HMOs will be followed by their supplement into mixing tank 100. [00037] Collection of purified proteins, as a stand-alone product composed of a mixture of proteins or a single protein according to demand. Purified proteins can also be used as additives and can be added to the mixing tank according to the specificities of each product (i.e., standardized milk, crème or whey). [00038] In an exemplary implementation, system 10 further comprises additive container 500 in liquid communication with mixing tank 100, operable to selectably provide a predetermined additive into the mixing tank. The addition of additives is done according to the requirement per specific product (milk, creme, whey). This may include sugars (plant source including sugar-beet, agave, carrot), vitamins, minerals (including Calcium, phosphorus, sodium and potassium), plant proteins, amino acids, antioxidants, plant-source fatty acids and the like (alternatively or in addition to fatty acids obtained from mammary gland cells or by cell factory yeast lines or oleaginous microorganisms). Moreover, sugar solution used in certain exemplary implementations, can further comprise sugar alcohols, for example: adonitol, allitol, altritol, arabinitol, dulcitol, erythritol, glycerol, iditol, inositol, isomalt, lactitol, maltitol, mannitol, perseitol, ribitol, rhamnitol, sorbitol, threitol or xylitol. Moreover, the sugar solution used in certain exemplary implementations, can comprise indigestible sugars (iS), such as for example: difructose anhydride (DFA) III, fructooligosaccharides (FOS), xylooligosaccharides (XOS), mannanoligosaccharides (MOS), galactooligosaccharides (GOS), and the like. Use of the sugar solutions comprising the sugar alcohols, and iS, in combination with other sugars, can be used to produce low-calorie cultured Buffalo milk. [00039] In additional step, the milk, protein or milk product(s) are collected using collection vessels 601, 602, 603, and finally, vat 800 for post-processing operations. Other post-processing operations can be implemented. The production of dairy products (i.e., pasteurized milk, different cheeses, yogurt, butter etc.) can involve various downstream processes such as, homogenization, pasteurization, fermentation, coagulation etc. Each dairy product will be handled and processed with its own unique set of requirements. In another exemplary implementation production of rennet via yeast/bacteria/fungi/buffalo mammary epithelial cell line can be achived, whereby Buffalo chymosin (rennet) will be accumulated, purified, and used as a coagulation enzyme which is important to the process of (e.g., Mozzarella) cheese making. [00040] The term “homology” as used herein refers to a percentage of identity between two polynucleotides or polypeptide moieties. The homology between sequences from one moiety to another moiety may be determined by known techniques. For example, the homology may be determined by directly aligning parameters of sequence information between two polynucleotide molecules or two polypeptide molecules, such as score, identity, and similarity, etc., using a computer program that sorts sequence information and is readily available (e.g., BLAST 2.0). Further, the homology between polynucleotides may be determined by hybridization of the polynucleotide under a condition in which a stable double strand is formed between homologous regions, followed by degradation by a single-strand-specific nuclease to determine a size of the degraded fragment. [00041] Further, as long as a protein has an activity corresponding to a Buffalo milk protein consisting of the detailed amino acid sequence disclosed, it is possible to add a nonsense sequence before and after the amino acid sequence, or to include a naturally occurring mutation or a silent mutation thereof. In addition, polypeptides having a Buffalo milk protein activity may also be included without limitation as a polypeptide encoded by a polynucleotide that is hybridized with a complementary sequence to all or a part of the nucleotide sequence encoding a probe that is able to be prepared from a known gene sequence, for example, the Buffalo milk nucleotide sequences provided herein, under stringent condition. The term “stringent condition” as used herein means a condition that allows specific hybridization between polynucleotides. The condition depends on a length of the polynucleotide and a degree of complementarity. Parameters thereof are well known in the art and are specifically described in the document (e.g., J. Sambrook et al., supra). For example, the stringent condition may list a condition for hybridizing genes to each other each having high homology of 80%, 90%, 95%, 97%, or 99% or more, a condition for not hybridizing genes to each other each having homology lower than that, or a general washing condition of southern hybridization, i.e., a condition for washing once, specifically two to three times at a salt concentration and a temperature such as 60° C., 1×SSC, 0.1% SDS, specifically, 0° C., 0.1×SSC, 0.1% SDS, and more specifically, 68° C., 0.1×SSC, 0.1% SDS. The probe used in the hybridization may be a part of the complementary sequence of the base sequence. Such a probe may be constructed by a PCR using a gene fragment including the base sequence as a template, by utilizing an oligonucleotide prepared based on the known sequence as a primer. Further, those skilled in the art may adjust the temperature and the salt concentration of the wash solution as needed depending on factors such as a length of the probe (or amplicon). [00042] The processes disclosed, are implemented in an exemplary implementation illustrated for example in FIG 1. Bioreactors 1011 1012,. and 1013 are generally configured for protein production chambers that allow regulated and controlled protein entrance to mixing tank 100 (production organism are unable to enter the main tank due to, e.g., a size exclusion membrane(s) 102j) via an array of valves 103p. Alternatively, in batch processes the proteins secreted by the microorganisms may be separated by stages of centrifugation (following a heat shock, utilizing the expressed proteins’ thermal stability, to remove micro organisms) and protein isolation by binding of protein to His-tag purification columns (whereby the proteins will be adapted to express His-tag). [00043] Collection vessel 601, 602, 603 can each further have stirrer 107n, as well as a plurality of in-line sensors 106q. [00044] Accordingly, the methods implemented using systems 10 disclosed are configured in certain exemplary implementations, with plurality of in-line sensors 106q operable to analyze a plurality of physico-chemical parameters and provide a central processing module (CPM 700, not shown) included in the system, with the parameters in real time. These plurality of physico-chemical parameters can be, for example at least two of: temperature, pressure, pH, dynamic viscosity, complex viscosity, etc.. The real-time measurement can then be used to control flow valves 103j, as well as the residence time of each unit operation and provide simultaneous control. [00045] As further illustrated in FIG. 1, mixing tank 100 can be operated at temperatures of between about 10 °C and about 90 °C. [00046] In the context of the disclosure, the term "operable" means the system and/or the device (e.g., the nutrient dispensing pump) and/or the program, or a certain element, component or step is/are fully functional sized, adapted and calibrated, comprising elements for, having the proper internal dimension to accommodate, and meets applicable operability requirements to perform a recited function when activated, coupled or implemented, regardless of being powered or not, coupled, implemented, effected, actuated, realized or when an executable program is executed by at least one processor associated with the system, method, and/or the device. [00047] The term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives. [00048] All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other. “Combination” is inclusive of blends, mixtures, alloys, reaction products, and the like. The terms “a”, “an” and “the” herein do not denote a limitation of quantity, and are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The suffix “(s)” as used herein is intended to include both the singular and the plural of the term that it modifies, thereby including one or more of that term (e.g., the chamber(s) includes one or more chamber). Reference throughout the specification to “one implementation”, “another implementation”, “an exemplary implementation,”, and so forth, when present, means that a particular element (e.g., feature, structure, and/or characteristic) described in connection with the implementation is included in at least one implementation described herein, and may or may not be present in other implementations. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various implementations. [00049] All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other. Furthermore, the terms “first,” “second,” and the like, herein do not denote any order, quantity, or importance, but rather are used to denote one element from another. [00050] The term “module,” as used herein, means, but is not limited to, a software or hardware component, such as a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC), or o combination of components which are configured, together, to perform certain tasks. A module may advantageously be configured to reside on an addressable storage medium and configured to execute on one or more processors. Thus, a module may include, by way of example, components, such as software components, object-oriented software components, class components and task components, processes, functions, attributes, procedures, subroutines, segments of program code, drivers, firmware, microcode, circuitry, data, databases, data structures, tables, arrays, and variables as well as pumps, conduits, valves and containers. The functionality provided for in the components and modules may be combined into fewer components and modules or further separated into additional components and modules. [00051] Likewise, the term "about" means that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is "about" or "approximate" whether or not expressly stated to be such. For example, “about” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% or at least ±10%, for example at least ±25% of the modified term if this deviation would not negate the meaning of the word it modifies. [00052] Accordingly, and in an exemplary implementation, provided herein is a system for producing Bubalus bubalis milk product, the system comprising: a plurality of bioreactors, each having a proximal end and a distal end, each bioreactor further containing the at least one of recombinant: a yeast, a bacterium, a fungus, and an algae comprising heterologous polynucleotides encoding a Bubalus bubalis polypeptide, wherein each bioreactor is further being in liquid communication with a mixing tank; and a plurality of collection receptacle, each collection receptacle associated with a product-specific bioreactor combination, wherein (i) the Bubalus bubalis polypeptide is at least one of: a milk protein, a whey protein, and an antimicrobial protein (ii) the milk protein being: αS1-casein (αS1-CN), and/or αS2-casein (αS2-CN), and/or β-casein (β -CN), and/or and κ-casein (κ –CN) and/or their combination, (iii) the whey protein being: β -lactoglobulin (β -LGB) and/or α-lactalbumin B (α-LAB), and/or both, (iv) the antimicrobial protein is at least one of: lactoferrin, lactoperoxidase and lysozyme C, wherein (v) the system further comprises a bioreactor comprising a carrier having thereon Bubalus bubalis mammary epithelial cells (MECs), adapted to secrete and accumulate fatty acids, or a carrier having thereon a recombinant yeast adapted to overproduce extra-cellular free fatty acids (FFAs), wherein the bioreactor comprising the Bubalus bubalis MECs, or the recombinant yeast is in liquid communication with the mixing tank, and a FFA separator, the FFA separator being in further in liquid communication with the mixing tank, the system (vi) further comprising a bioreactor comprising a carrier having thereon a recombinant yeast comprising heterologous polynucleotides encoding at least one human milk oligosaccharide (HMO), wherein the bioreactor comprising the recombinant yeast is in liquid communication with the mixing tank, and a HMO separator, the HMO separator being in further liquid communication with the mixing tank, wherein (vii) the recombinant yeast adapted to overproduce extracellular free fatty acids (FFAs, meaning that the amount of FFAs produced from the recombinant yeast will be greater than the amount of FFAs produced from any or all of the wild type yeast) comprises a heterologous polynucleotides encoding genes having selective deletions, configured to overexpress extracellular FFAs, (viii) the heterologous polynucleotides encoding genes being: FAA2, and/or FAA1, and/or FAA4, and/or FAT1, and/or PXA1, and/or POX1, and/or their combination, furthermore (ix) the heterologous polynucleotides is adapted to overexpress: DGA1 (diacylglycerol acyltransferase) and/or TGL3 (triacylglycerol lipase), or (x) the heterologous polynucleotides is adapted to overexpress: gene of ATP:citrate lyase (ACL), and/or malic enzyme (ME), and/or limitochondrial citrate transporter (Ctp1), and/or malate dehydrogenase (Mdh3), and/or fatty acid synthase genes (FAS1 and FAS2), and/or a truncated thioesterase (’tesA), and/or endogenous acetyl-CoA carboxylase (ACC1), and/or their combination, wherein (xi) the recombinant yeast’s heterologous polynucleotide encodes a lactose transporter, a GDP-L fucose synthetic pathway, α-1,2-fucosyltransferase and a 2’- FL transporter, (xii) encoding a deletion of gal80 gene, wherein (xiii) each bioreactor is in further communication (either directly, or through an intermediate member or module) with a protein purification module, operable to selectively isolate a predetermined protein, (xiv) the protein purification module comprises at least one of: a size exclusion column, a hollow fiber tangential flow filtration (TFF) column, and an affinity column, the system (xv) further comprising an additive container in liquid communication with the mixing tank, operable to selectably provide a predetermined additive into the mixing tank, and wherein (xvi) the predetermined additive is sugars, and/or vitamins, and/or minerals, and/or plant proteins, and/or amino acids, and/or antioxidants, and/or plant-source fatty acids, and/or their combination. [00053] Although the foregoing disclosure for methods, systems and compositions for producing Buffalo milk products. More specifically, for continuously, batch-wise and semi- continuously using transformed/transfected yeast and/or fungi and/or bacteria and/or algea to express and/or secrete Bubalus bubalis Casein, Whey protein and additional proteins, collecting the expressed product and using it for producing various products, which has been described in terms of some implementations, other implementations will be apparent to those of ordinary skill in the art from the disclosure herein. Moreover, the described implementations have been presented by way of example only, and are not intended to limit the scope of the inventions. Indeed, the novel methods, programs, libraries and systems described herein may be embodied in a variety of other forms without departing from the spirit thereof. Accordingly, other combinations, omissions, substitutions and modifications will be apparent to the skilled artisan in view of the disclosure herein.
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