Glycosome for milk-N-trisaccharide

Abstract

The invention is in the technical field of synthetic biology, metabolic engineering and cell culture. The present invention provides cells for the production of one or more biological products, where the cells are capable of producing one or more precursors used in the production of the one or more biological products, and where the cells are genetically engineered to express a sugar input that internalizes at least one of the one or more precursors. The present invention further provides a cell genetically engineered to express or overexpress a sugar input body having an uptake activity with respect to a milk-N-trisaccharide (LN3, GlcNAc-beta 1, 3-Gal-beta 1, 4-Glc). The invention also provides an application of the cell in culture or incubation. Also described are methods for producing one or more biological products by using any of the sugar input bodies and purifying the biological products. The present invention further relates to sugar input bodies having ingestion activity with respect to LN3 and to the use of any one of said sugar input bodies for the production of one or more biological products.

Description

Technical Field

[0001] The present invention is in the technical fields of synthetic biology, metabolic engineering, and cell culture. The present invention provides cells for producing one or more biological products, wherein the cells are capable of producing one or more precursors used in the production of the one or more biological products, and wherein the cells are genetically modified to express a sugar importer that internalizes at least one of the one or more precursors. The present invention further provides cells that are genetically modified to express or overexpress a sugar importer having uptake activity with respect to lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc). The present invention also provides the use of the cells in culture or incubation. The present invention also describes methods for producing one or more biological products and purifying the biological products by using any of the sugar importers. The present invention further relates to a sugar importer having uptake activity with respect to LN3 and the use of any of the sugar importers for producing one or more biological products. Background Art

[0002] Sugars are widely distributed in nature and play roles in many vital phenomena such as differentiation, development, and biorecognition processes associated with the generation and progression of fertilization, embryogenesis, inflammation, immune processes, metastasis, and host-pathogen adhesion. An important group of sugars can be found in milk. Sugars in mammalian milk, such as mammalian milk oligosaccharides (MMO) (including sugars in human milk, such as human milk oligosaccharides (HMO)), regulate key developmental and immune processes in early life. HMOs are, for example, milk bioproducts known to improve the immediate and long-term health and development of infants by shaping the infant gut microbiome, influencing the infant immune system, protecting the infant from intestinal and immunological disorders, and by involving proper brain development and cognition. Additionally, HMOs have been reported to have a role in enhancing adult health (Azad et al., (2018), J. Nutr. 148, 1733-1742; Bode (2015), Early Hum. Dev. 1-4; Etzold and Bode (2014), Curr. Opin. Virol. 7, 101-107; Kellman et al., (2022), Nat. Commun. 13, 2455; Pérez-Escalante et al., (2022), Crit. Rev. Food Sci. Nutr. 62, 181-214; Reily et al., (2019), Nat. Rev. Nephrol. 15, 346-366; Varki (2017), Glycobiology 27, 3-49; Walsh et al., (2020), J. Funct. Foods 72, 104074).

[0003] Due to their positive impact on animal and human health, there is a great scientific and commercial interest in sugars, especially sugars in milk (e.g., MMO and HMO); however, their availability is limited. Today, many sugars (including oligosaccharides) are synthesized in the following ways: chemically, by in vitro glycosylation reactions enzymatically, by chemoenzymatic synthesis, by fermentation methods, and / or by chemical, physical, and / or biodegradation of polysaccharides. In particular, fermentation methods for producing oligosaccharides have been successful. However, the synthesis of sugars or mixtures of different sugars is often hampered by the production of unwanted compounds (e.g., by-products and intermediates), thereby reducing the purity of the desired sugar and / or reducing the yield of the synthesis. SUMMARY OF THE INVENTION Summary of the Invention

[0005] The object of the present invention is to provide tools and methods by means of which one or more biological products can be produced, preferably in an efficient, timely, and cost-effective manner, and the tools and methods produce a large amount of the desired biological products.

[0006] According to the present invention, this and other objects are achieved by providing cells for producing one or more biological products, wherein the cells are capable of producing, preferably producing, more preferably genetically modified to produce one or more precursors used in the production of the one or more biological products, and wherein the cells are genetically modified to express, preferably overexpress a sugar importer that internalizes at least one of the one or more precursors. The present invention further provides sugar importers having uptake activity with respect to lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc), each of which can be used in a method for producing one or more biological products. Further, any of the sugar importers can be used in cells for producing one or more biological products. The present invention also provides methods and cells for producing one or more biological products, and methods for separating and purifying the one or more biological products.

[0007] Definition

[0008] The vocabulary used in this specification to describe the present invention and its various embodiments should be understood not only in its ordinary defined meaning, but also to include structures, materials, or actions that exceed the scope of the ordinary defined meaning by special definitions in this specification. Thus, if an element can be understood to include more than one meaning in the context of this specification, its use in the claims must be understood to be generic to all possible meanings supported by this specification and by the vocabulary itself.

[0009] The various aspects and embodiments of the invention disclosed herein should be understood not only in the order and context specifically described in this specification, but also in any order and any combination thereof. Each of the embodiments identified herein can be combined together, unless otherwise stated. All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0010] Whenever the context requires, unless otherwise specifically stated, all terms used in the singular shall be considered to include the plural and vice versa. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the laboratory procedures described herein in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization are those well known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, enzymatic reactions and purification steps are carried out according to the manufacturer's instructions.

[0011] In this specification, embodiments of the invention have been disclosed, and although specific terms have been used, these terms are used in a descriptive sense only and not for purposes of limitation. The scope of the invention is set forth in the following claims. It must be understood that the illustrative embodiments have been set forth for purposes of example only and should not be regarded as limiting the invention. It will be apparent to those skilled in the art that changes, other embodiments, improvements, details, and uses can be made consistent with the letter and spirit of the disclosure herein and within the scope of this disclosure, the scope of which is limited only by the claims, interpreted in accordance with patent law, including the doctrine of equivalents. In the following claims, the reference characters used to designate claim steps are provided for convenience of description only and are not intended to imply any particular order for performing the recited steps, unless otherwise specifically stated.

[0012] Throughout the application, unless otherwise expressly stated, the features "synthesis" and "production" are used interchangeably. Throughout the application, unless otherwise expressly stated, the expression "able to..." is preferably replaced by the active voice of the verb, and vice versa. For example, the expression "able to express" is preferably replaced by "express", and vice versa, i.e., "express" is preferably replaced by "able to express". In this document and in its claims, the verbs "comprise", "have" and "include" and their inflections are used in their non-limiting sense to mean including the item(s) following the word, but not excluding items not specifically mentioned. Throughout the application, the verb "comprise (include)" can be replaced by "consist of" or "consist essentially of", and vice versa. Additionally, the verb "consist of" can be replaced by "consist essentially of", which means that the composition as defined herein may contain additional components other than those specifically identified, and such additional components do not change the unique features of the invention. Throughout this document and in its claims, unless otherwise specifically stated, the verbs "comprise", "have" and "include" and their inflections can preferably be replaced by "consist of" (and its inflections) or "consist essentially of" (and its inflections), and vice versa. Additionally, the reference to an element by the indefinite article "a" or "an" does not exclude the possibility of there being more than one of said element, unless the context clearly requires there to be only one of said element. Thus, the indefinite article "a" or "an" often means "at least one (kind)". Throughout the application, unless otherwise expressly stated, the articles "a" and "an" are preferably replaced by "at least two (kinds)", more preferably by "at least three (kinds)", even more preferably by "at least four (kinds)", even more preferably by "at least five (kinds)", even more preferably by "at least six (kinds)", and most preferably by "at least two (kinds)". When used in association with a numerical value (e.g., "about 10") or with a range (e.g., "about x to about y"), the word "about" or "approximately" preferably means that the value or range is interpreted as being as accurate as the method used to measure it. If no error margin is specifically specified, then when used in association with a numerical value, the expression "about" or "approximately" is interpreted as having the same rounding as the given value. Throughout this document and its claims, unless otherwise stated, the expression "x to y" (where x and y represent numerical values) refers to a numerical range, where x is the lower limit value of the range and y is the upper limit value of the range. Herein, x and y are also included in the said range.

[0013] In accordance with the present invention, the term "polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, but are not limited to: single-stranded and double-stranded DNA; DNA as a mixture of single-stranded and double-stranded regions or single-stranded, double-stranded, and triple-stranded regions; single-stranded and double-stranded RNA; and RNA as a mixture of single-stranded and double-stranded regions; hybrid molecules that contain DNA and RNA, which may be single-stranded or more typically double-stranded or triple-stranded regions or a mixture of single-stranded and double-stranded regions. Additionally, as used herein, "polynucleotide" refers to a triple-stranded region that contains RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically only involve some of the molecules. One of the molecules of the triple-helical region is often an oligonucleotide. As used herein, the term "polynucleotide" also includes the above-described DNA or RNA that contains one or more modified bases. Thus, DNA or RNA having a backbone modified for stability or for other reasons is a "polynucleotide" according to the present invention. In addition, it should be understood that DNA or RNA containing rare bases (e.g., inosine) or modified bases (e.g., tritylated bases) is encompassed by the term "polynucleotide". It will be appreciated that a very wide variety of modifications have been made to DNA and RNA, which serve many useful purposes known to those skilled in the art. As employed herein, the term "polynucleotide" encompasses such chemically modified, enzymatically modified, or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells (including, for example, simple and complex cells). The term "polynucleotide" also encompasses short polynucleotides that are often referred to as oligonucleotides.

[0014] "Polypeptide" means any peptide or protein that comprises two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide" refers to both short chains (commonly referred to as peptides, oligopeptides, and oligomers) and longer chains (commonly referred to as proteins). Polypeptides can contain amino acids other than the 20 genetically encoded amino acids. "Polypeptide" includes those that are modified by natural processes such as processing and other post-translational modifications, but also includes those that are modified by chemical modification techniques. Such modifications are described fully in basic textbooks and in more detailed monographs, as well as in multivolume research literature, and they are well known to the person skilled in the art. The same type of modification can be present at several sites in a given polypeptide, either in the same or varying degrees. Further, a given polypeptide can contain many types of modifications. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of pyroglutamic acid, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, lipid attachment, sulfation, γ-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation, selenylation, addition of amino acids to proteins mediated by transfer-RNA, such as arginylation, and ubiquitination. Polypeptides can be branched or cyclic, with or without branches. Cyclic, branched, or branched-cyclic polypeptides can arise from post-translational natural processes and can also be prepared by entirely synthetic methods.

[0015] As used herein, the term "polynucleotide encoding a polypeptide" encompasses such polynucleotides that include a sequence encoding a polypeptide of the present invention. The term also encompasses such polynucleotides that include a single contiguous region or discontiguous regions (e.g., interrupted by integrated phage or insertion sequences or editing) encoding the polypeptide, along with additional regions that may also contain coding and / or non-coding sequences.

[0016] "Isolated" means changed from its natural state "by the hand of man", i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that occurs naturally in a living organism is not "isolated", but the same polynucleotide or polypeptide separated from the materials with which it coexists in its natural state is "isolated", as the term is used herein. Similarly, as the term is used herein, a "synthetic" sequence means any sequence that is generated synthetically rather than directly isolated from a natural source. As the term is used herein, "synthesized" means any sequence that is generated synthetically rather than directly isolated from a natural source.

[0017] "Recombinant" means DNA that has been genetically engineered and is prepared by transplanting or splicing a gene from one species into the cells of a host organism of a different species. Such DNA becomes part of the host's genetic makeup and is replicated.

[0018] The terms "recombinant" or "transgenic" or "metabolically engineered" or "genetically engineered" as used herein in relation to a cell or host cell are used interchangeably and indicate that the cell replicates a heterologous nucleic acid or expresses a peptide or protein encoded by a heterologous nucleic acid (i.e., a sequence that is "foreign to the cell" or "foreign to the location or environment in the cell"). Such cells are described as transformed with at least one heterologous or exogenous gene or as transformed by the introduction of at least one heterologous or exogenous gene. Recombinant or genetically engineered cells can contain genes not found in the natural (non-recombinant) form of the cell. Recombinant cells can also contain genes found in the natural form of the cell, where the genes have been modified by artificial means and reintroduced into the cell. The term also encompasses cells that contain nucleic acid endogenous to the cell that has been modified or whose expression or activity has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, promoter replacement, site-specific mutagenesis, CrispR, riboswitch, recombination engineering, ssDNA mutagenesis, transposon mutagenesis, and related techniques known to those of skill in the art. Thus, a "recombinant polypeptide" is a polypeptide produced by a recombinant cell. The term also encompasses cells that have been modified by removing nucleic acid endogenous to the cell (e.g., knocking out a gene) by means well known to those of skill in the art.

[0019] As used herein, "heterologous sequence" or "heterologous nucleic acid" is a sequence or nucleic acid that is derived from a source that is foreign to a particular cell (e.g., from a different species), or if from the same source, has been modified from its original form or location in the genome. Thus, a heterologous nucleic acid operably linked to a promoter is from a source different from the source from which the promoter is derived, or if from the same source, has been modified from its original form or location in the genome. A heterologous sequence can be stably introduced into the genome of a host microbial cell, for example, by transfection, transformation, conjugation, or transduction, where techniques will depend on the cell and the sequence to be introduced. Various techniques are known to those skilled in the art and are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). The terms "mutant" or "modified" cell or microorganism as used within the context of the present invention refer to a cell or microorganism that has been genetically modified.

[0020] Within the context of the present disclosure, the term "endogenous" refers to any polynucleotide, polypeptide, or protein sequence that is a natural part of a cell and occurs at its natural location in the cell chromosome. The term "exogenous" refers to any polynucleotide, polypeptide, or protein sequence that is derived from outside of the cell under study and is not a natural part of that cell, or that does not occur at its natural location in the cell chromosome or plasmid.

[0021] When used in reference to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme, the term "heterologous" refers to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is from or derived from a source other than the host biological species. In contrast, a "homologous" polynucleotide, gene, nucleic acid, polypeptide, or enzyme is used herein to refer to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is derived from the host biological species. When referring to a gene regulatory sequence or accessory nucleic acid sequence (e.g., a promoter, 5' untranslated region, 3' untranslated region, poly A addition sequence, intron sequence, splice site, ribosome binding site, internal ribosome entry sequence, genomic homology region, recombination site, etc.) used to maintain or manipulate a gene sequence, "heterologous" means that the regulatory sequence or accessory sequence is not naturally associated with the gene to which the regulatory or accessory nucleic acid sequence is juxtaposed in a construct, genome, chromosome, or episome. Thus, a promoter that is operably linked to a gene to which it is not operably linked in its natural state (i.e., in the genome of an ungenetically modified organism) is referred to herein as a "heterologous promoter", even if the promoter may be from the same species (or in some cases, the same organism) as the gene to which it is linked.

[0022] The term "modified expression" of a gene refers to a change in expression compared to the wild-type expression of the gene at any stage in the production process of a desired biological product. The modified expression is either lower or higher expression compared to the wild type, where the term "higher expression" is also defined as "overexpression" of the gene in the case of an endogenous gene, or as "expression" in the case of a heterologous gene not present in the wild-type strain. Lower expression is obtained by means of well-known techniques common to those skilled in the art (such as using siRNA, CrispR, CrispRi, riboswitches, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutant genes, knockout genes, transposon mutagenesis, etc.), which are used to alter the gene in such a way that the gene is "less able" (i.e., statistically significantly "less able" compared to a functional wild-type gene) or completely unable (e.g., a knocked-out gene) to produce a functional end product. The term "riboswitch" as used herein is defined as a portion of messenger RNA that folds into a complex structure that blocks expression by interfering with translation. Binding of an effector molecule induces a conformational change, thus allowing post-transcriptional regulation of expression. In addition to altering the gene of interest as described above to obtain lower expression, lower expression can also be obtained by altering the transcription unit, promoter, untranslated region, ribosome binding site, Shine-Dalgarno sequence, or transcription terminator. Lower expression or reduced expression can be obtained, for example, by mutating one or more base pairs in the promoter sequence, or by completely changing the promoter sequence to a constitutive promoter with lower expression intensity compared to the wild type, or an inducible promoter that results in regulated expression, or a repressible promoter that results in regulated expression. Overexpression or expression is obtained by means of well-known techniques common to those skilled in the art (e.g., using artificial transcription factors, de novo design of promoter sequences, ribosome engineering, introducing or reintroducing an expression module at euchromatin, using high-copy number plasmids), where the gene is part of an "expression cassette", which refers to any sequence in which there is a promoter sequence, untranslated region sequence (including ribosome binding sequence, Shine-Dalgarno sequence, or Kozak sequence), coding sequence (e.g., sugar transporter gene sequence), and optionally a transcription terminator and which results in the expression of a functionally active protein. The expression is constitutive or conditional or regulated or coordinatable.

[0023] The term "constitutive expression" is defined as not being regulated by other transcription factors (such as bacterial σ factors, e.g., σ 70 、σ 54Or related σ-factors, and the yeast mitochondrial RNA polymerase specific factor MTF1, which associates with the RNA polymerase core enzyme) for regulated expression. Non-limiting examples of such transcription factors are CRP, LacI, ArcA, Cra, IclR in Escherichia coli (E. coli), or Aft2p, Crz1p, Skn7 in Saccharomyces cerevisiae, or DeoR, GntR, Fur in Bacillus subtilis (B. subtilis). These transcription factors bind to specific sequences and can block or enhance expression under certain growth conditions. RNA polymerase is the catalytic machinery for synthesizing RNA from a DNA template. RNA polymerase binds to specific DNA sequences to initiate transcription, for example via σ-factors in prokaryotic hosts or via MTF1 in yeast. Constitutive expression provides a constant level of expression without the need for induction or repression.

[0024] The term "regulated expression" is defined as expression regulated under certain growth conditions by other transcription factors (e.g., bacterial σ-factors) in addition to the subunits of RNA polymerase. Examples of such transcription factors are described above. Generally, expression regulation is achieved by means of inducers such as, but not limited to, IPTG, arabinose, rhamnose, fucose, allolactose or pH shift or temperature shift or carbon depletion or substrate or the produced bioproduct.

[0025] The term "control sequence" refers to a sequence recognized by the cell's transcription and translation systems that permits the transcription and translation of a polynucleotide sequence into a polypeptide. Thus, such DNA sequences are essential for the expression of an operably linked coding sequence in a particular host cell, cell or organism. Such control sequences can be, but are not limited to, promoter sequences, ribosome binding sequences, Shine Dalgarno sequences, Kozak sequences, transcription terminator sequences. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator gene sequence, and a ribosome binding site. It is known that eukaryotic cells utilize promoters, polyadenylation signals and enhancers. DNA regarding a presequence or a secretion leader sequence can be operably linked to DNA regarding a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer can be operably linked to a coding sequence if it affects the transcription of said sequence; or a ribosome binding site can be operably linked to a coding sequence if it affects the transcription of said sequence; or a ribosome binding site can be operably linked to a coding sequence if it is positioned so as to facilitate translation. Further, the control sequence can be controlled by external chemicals (e.g., but not limited to IPTG, arabinose, lactose, allolactose, rhamnose or fucose) via an inducible promoter or via a genetic circuit that induces or represses the transcription or translation of the polynucleotide into a polypeptide.

[0026] Generally, "operably linked" means that the DNA sequences being linked are adjacent and, in the case of a secretory leader sequence, are adjacent and in reading phase. However, enhancers do not have to be adjacent.

[0027] The term "wild type" refers to the generally known genetic or phenotypic situation as it occurs in nature.

[0028] As used herein, the term "modified expression of a protein" refers to: i) higher expression or overexpression of an endogenous protein, ii) expression of a heterologous protein, iii) expression and / or overexpression of a variant protein having higher activity compared to the wild-type (i.e., native in the expression host) protein, iv) reduced expression of an endogenous protein, or v) expression and / or overexpression of a variant protein having reduced activity compared to the wild-type (i.e., native in the expression host) protein. Preferably, as used herein, the term "modified expression of a protein" refers to: i) higher expression or overexpression of an endogenous protein, ii) expression of a heterologous protein, or iii) expression and / or overexpression of a variant protein having higher activity compared to the wild-type (i.e., native in the expression host) protein.

[0029] The term "modified activity" of a protein relates to the non-natural activity of the protein at any stage in the production process of the desired biological product. As used herein in relation to the activity of a protein, the term "non-natural" indicates that the protein has been modified to have an activity that is abrogated, impaired, reduced, delayed, higher, accelerated, or improved compared to the native activity of the protein. Modified activity of a protein is obtained by modified expression of the protein or by expression of a modified (i.e., mutant) form of the protein. The mutant form of the protein can be obtained by expression of a mutant form of the gene encoding the protein, which, for example, contains a deletion, insertion, and / or mutation of one or more nucleotides compared to the native gene sequence. Mutant forms of genes can be obtained by techniques well known to those skilled in the art (such as, but not limited to, site-directed mutagenesis; CrispR; riboswitches; recombineering; ssDNA mutagenesis; transposon mutagenesis).

[0030] As used herein in relation to a cell producing one or more biological products, the term "non-natural" indicates that the one or more biological products i) are not produced naturally, or ii) when produced naturally, are not produced by the cell in the same amounts; and the cell has been genetically engineered to be capable of producing the one or more biological products or to have higher production of the one or more biological products.

[0031] As used herein, the term "mammary cell" generally refers to mammalian mammary epithelial cells, mammalian mammary epithelial luminal cells, or mammalian epithelial acinar cells, or any combination thereof. As used herein, the term "mammary-like cell" generally refers to a mammalian cell having a phenotype / genotype similar (or substantially similar) to that of a native mammalian mammary cell but derived from a mammalian non-mammary cell source. Such mammalian mammary-like cells can be engineered to remove at least one undesired genetic component and / or include at least one predetermined genetic construct typical of mammalian mammary cells. Non-limiting examples of mammalian mammary-like cells can include: mammalian mammary epithelial-like cells, mammalian mammary epithelial luminal-like cells, mammalian non-mammary cells that exhibit one or more characteristics of cells of the mammalian mammary cell lineage, or any combination thereof. Further non-limiting examples of mammalian mammary-like cells can include mammalian cells having a phenotype similar (or substantially similar) to that of a native mammalian mammary cell, or more particularly, a phenotype similar (or substantially similar) to that of a native mammalian mammary epithelial cell. Mammalian cells having a phenotype similar (or substantially similar) to that of a native mammalian mammary cell or mammalian mammary epithelial cell, or exhibiting at least one characteristic similar (or substantially similar) to that of a native mammalian mammary cell or mammalian mammary epithelial cell, can include mammalian cells that naturally exhibit or have been engineered to be capable of expressing at least one milk component (e.g., those derived from a mammary cell lineage or a non-mammary cell lineage).

[0032] As used herein, the term "non-mammary cell" generally can include any mammalian cell of non-mammary lineage. In the context of the present invention, a non-mammary cell can be any mammalian cell capable of being engineered to express at least one milk component. Non-limiting examples of such non-mammary cells include: hepatocytes, blood cells, kidney cells, umbilical cord blood cells, epithelial cells, epidermal cells, muscle cells, fibroblasts, mesenchymal cells, or any combination thereof. In some cases, molecular biology and genome editing techniques can be engineered to simultaneously eliminate, silence, or attenuate numerous genes.

[0033] With respect to a polynucleotide, a "fragment" refers to a clone or any portion of a polynucleotide molecule, particularly a portion of a polynucleotide that retains the useful functional characteristics of the full-length polynucleotide molecule. Useful fragments include oligonucleotides and polynucleotides that can be used in hybridization or amplification techniques or in the regulation of replication, transcription, or translation. A "polynucleotide fragment" refers to any subsequence of a polynucleotide SEQ ID NO, typically comprising or consisting of at least about 9, 10, 11, 12 consecutive nucleotides from said polynucleotide SEQ ID NO, such as at least about 30 nucleotides or at least about 50 nucleotides of any of the polynucleotide sequences provided herein. Exemplary fragments can additionally or alternatively include fragments that comprise, consist essentially of, or consist of a region that encodes a conserved family domain of a polypeptide. Exemplary fragments can additionally or alternatively include fragments that comprise a conserved domain of a polypeptide. Thus, a fragment of a polynucleotide SEQ ID NO preferably refers to a nucleotide sequence that comprises or consists of said polynucleotide SEQ ID NO, wherein no more than about 200, 150, 100, 50, or 25 consecutive nucleotides are missing, preferably no more than about 50 consecutive nucleotides are missing, and that retains the useful functional characteristics (e.g., activity) of the full-length polynucleotide molecule, which can be evaluated by one of ordinary skill in the art by routine experimentation. Alternatively, a fragment of a polynucleotide SEQ ID NO preferably refers to a nucleotide sequence that comprises or consists of a certain amount of consecutive nucleotides from said polynucleotide SEQ ID NO, wherein the amount of consecutive nucleotides is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100% of the full length of said polynucleotide SEQ ID NO, preferably at least 80%, more preferably at least 85%, even more preferably at least 87%, even more preferably at least 90%, even more preferably at least 95%, most preferably at least 97%, and that retains the useful functional characteristics (e.g., activity) of the full-length polynucleotide molecule, which can be routinely evaluated by one of ordinary skill in the art.Thus, a fragment of a polynucleotide SEQ ID NO preferably means a nucleotide sequence that comprises or consists of the polynucleotide SEQ ID NO, where a certain amount of consecutive nucleotides is missing and where the amount is not more than 50%, 40%, 30% of the full length of the polynucleotide SEQ ID NO, preferably not more than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5% of the full length of the polynucleotide SEQ ID NO, more preferably not more than 15%, even more preferably not more than 10%, even more preferably not more than 5%, most preferably not more than 2.5%, and where the fragment retains the available functional properties (e.g., activity) of the full-length polynucleotide molecule, which can be routinely evaluated by a person skilled in the art.

[0034] Regarding a polypeptide, a "fragment" refers to a subsequence of the polypeptide that performs at least one biological function of the complete polypeptide in substantially the same manner as or to a similar extent as the complete polypeptide. A "subsequence of a polypeptide" or "segment of amino acid residues" as described herein refers to a sequence of contiguous amino acid residues derived from the polypeptide. For example, a polypeptide fragment can contain a recognizable structural motif or functional domain, such as a DNA binding site or a domain that binds to a DNA promoter region, an activation domain, or a domain for protein-protein interaction, and can initiate transcription. Fragments can vary in size from as few as 3 amino acid residues of the complete polypeptide to the full length, e.g., having a length of at least about 10 amino acid residues, e.g., having a length of at least about 20 amino acid residues, e.g., having a length of at least about 30 amino acid residues, e.g., having a length of at least about 100 amino acid residues, e.g., having a length of at least about 150 amino acid residues, e.g., having a length of at least about 200 amino acid residues. Thus, a fragment of a polypeptide SEQ ID NO (or UniProt ID) preferably refers to such a polypeptide sequence that contains or consists of the polypeptide SEQ ID NO (or UniProt ID), wherein no more than about 200, 150, 125, 100, 80, 60, 50, 40, 30, 20, or 15 contiguous amino acid residues are missing, preferably no more than about 100 contiguous amino acid residues are missing, more preferably no more than about 50 contiguous amino acid residues are missing, even more preferably no more than about 40 contiguous amino acid residues are missing, and performs at least one biological function of the complete polypeptide in substantially the same manner as, preferably to a similar or greater extent than, the complete polypeptide, which can be routinely evaluated by a person skilled in the art.Alternatively, a fragment of a polypeptide SEQ ID NO (or UniProtID) preferably means a polypeptide sequence that comprises or consists of a certain amount of contiguous amino acid residues from said polypeptide SEQ ID NO (or UniProtID), wherein the amount of contiguous amino acid residues is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100% of the full length of said polypeptide SEQ ID NO (or UniProt ID), preferably at least 80%, more preferably at least 85%, even more preferably at least 87%, even more preferably at least 90%, even more preferably at least 95%, most preferably at least 97%, and which performs at least one biological function of the full-length polypeptide in substantially the same manner as the full-length polypeptide, preferably to a similar or greater extent, which can be routinely evaluated by the person skilled in the art. Thus, a fragment of a polypeptide SEQ ID NO (or UniProt ID) preferably means a polypeptide sequence that comprises or consists of said polypeptide SEQ ID NO (or UniProtID), wherein a certain amount of contiguous amino acid residues are missing and wherein the amount is not more than 50%, 40%, 30% of the full length of said polypeptide SEQ ID NO (or UniProtID), preferably not more than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5% of the full length of said polypeptide SEQ ID NO (or UniProtID), more preferably not more than 15%, even more preferably not more than 10%, even more preferably not more than 5%, most preferably not more than 2.5%, and which performs at least one biological function of the full-length polypeptide in substantially the same manner as the full-length polypeptide, preferably to a similar or greater extent, which can be routinely evaluated by the person skilled in the art.

[0035] Throughout the application, the sequence of a polypeptide may be represented by a SEQ ID NO or alternatively by a UniProtID. Thus, the terms "polypeptide SEQ ID NO" and "polypeptide UniProt ID" may be used interchangeably, unless otherwise expressly stated.

[0036] Homologs can be identified by analyzing nucleotide and polypeptide sequence alignments. For example, a search of a nucleotide or polypeptide sequence database can identify homologs of a nucleotide or polypeptide of interest. Sequence analysis can involve BLAST, reciprocal BLAST, or PSI-BLAST analysis of a non-redundant database, using the amino acid sequence of a reference polypeptide sequence. In some cases, the amino acid sequence is deduced from the nucleotide sequence. Typically, those polypeptides in the database that have greater than 40% sequence identity to the polypeptide of interest are candidates for further evaluation as homologous polypeptides. Amino acid sequence similarity allows conservative amino acid substitutions, such as one hydrophobic residue for another, or one polar residue for another, or one acidic amino acid for another, or one basic amino acid for another, and so on. Preferably, conservative substitutions are intended to represent combinations such as the following: glycine is substituted by alanine and vice versa; valine, isoleucine, and leucine are substituted by methionine and vice versa; aspartic acid is substituted by glutamic acid and vice versa; asparagine is substituted by glutamine and vice versa; serine is substituted by threonine and vice versa; lysine is substituted by arginine and vice versa; cysteine is substituted by methionine and vice versa; and phenylalanine and tyrosine are substituted by tryptophan and vice versa. If desired, a manual inspection of such candidates can be performed to reduce the number of candidates to be further evaluated.

[0037] Domains can be characterized, for example, by the following tools: Pfam (El-Gebali et al., Nucleic Acids Res. 47 (2019) D427-D432), IPR (InterPro domain) (http: / / ebi.ac.uk / interpro) (Mitchell et al., Nucleic Acids Res. 47 (2019) D351-D360), Protein Fingerprint Domains (PRINTS) (Attwood et al., Nucleic Acids Res. 31 (2003) 400-402), SUBFAM domain (Gough et al., J. Mol. Biol. 313 (2001) 903-919), TIGRFAM domain (Selengut et al., Nucleic Acids Res. 35 (2007) D260-D264), Conserved Domain Database (CDD) nomenclature (https: / / www.ncbi.nlm.nih.gov / cdd) (Lu et al., Nucleic Acids Res. 48 (2020) D265-D268), PTHR domain (http: / / www.pantherdb.org) (Mi et al., Nucleic Acids.Res. 41 (2013) D377-D386; Thomas et al., Genome Research 13 (2003) 2129-2141) or PATRIC identifier or PATRIC DB full-family domain (https: / / www.patricbrc.org / ) (Davis et al., Nucleic Acids Res. 48 (D1) (2020) D606-D612). Protein or polypeptide sequence information and functional information can be provided by extensive resources on protein sequences and annotation data such as the Universal Protein Resource (UniProt) (www.uniprot.org) (Nucleic Acids Res. 2021, 49 (D1), D480-D489). UniProt contains a curated and richly managed protein database called UniProt Knowledgebase (UniProtKB), along with UniProt Reference Clusters (UniRef) and UniProt Archive (UniParc). The UniProt identifier (UniProtID) is unique for each protein present in the database. Throughout the application, the sequence of a polypeptide is represented by SEQ ID NO or UniProtID.Unless otherwise stated, the UniProtID of the described protein corresponds to its sequence version 01 as present in the UniProt database (www.uniprot.org) version 2021_03 and accessed on June 9, 2021. InterPro provides protein function analysis by classifying them into families and predicting domains and important sites. To classify proteins in this way, InterPro uses prediction models called signatures, which are provided by several different databases that make up the InterPro consortium, called member databases. The protein signatures from these member databases are combined into a single searchable resource, which, in its own right, produces a powerful integrated database and diagnostic tool.

[0038] The terms "IPR001927", "PDOC00680", "NA_GALACTOSIDE_SYMP", "PS00872" are used interchangeably and refer to the sodium:galactoside symporter family signature (Pourcher et al., 1991, Biochem. Biophys. Res. Commun. 178, 1176-1181; Reizer et al., 1994, Biochim. Biophys. Acta 1197, 133-166).

[0039] Those skilled in the art should understand that for the databases used herein, including InterPro 90.0 (released on August 4, 2022), PFAM 32.0 (released in September 2018), PANTHER 18.0 (released on September 17, 2023), Conserved Domain Database CDD 3.20 (released in September 2022), the content of each database is fixed at each release and will not change. When the content of a particular database is changed, that particular database accepts a new release version with a new release date. All release versions of each database with their corresponding release dates and the specific content annotated at these specific release dates are available and known to those skilled in the art.

[0040] In the context of two or more nucleic acid or polypeptide sequences, the terms "identical" or "percent identity" or "identity %" refer to two or more sequences or subsequences that are the same or have a specified percentage of identical nucleotide or amino acid residues when compared or aligned for maximum correspondence, as measured by using a sequence comparison algorithm or by visual inspection. For sequence comparison, one sequence acts as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the designated program parameters. The percent sequence identity can preferably be determined by aligning the two sequences and identifying the number of positions with identical residues and dividing by the number of residues in the shorter of the sequences x 100. The percent identity can be calculated overall over the full-length sequence of a given SEQ ID NO (i.e., the reference sequence), resulting in an overall percent identity score. Alternatively, the percent identity can be calculated over a partial sequence of the reference sequence, resulting in a local percent identity score. A partial sequence preferably means at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94% or 95% of the full-length reference sequence. In another preferred embodiment, a partial sequence of a reference polypeptide sequence means a segment having at least 150 amino acid residues up to the total number of amino acid residues of the reference polypeptide sequence. In another more preferred embodiment, a partial sequence of a reference polypeptide sequence means a segment having at least 200 amino acid residues up to the total number of amino acid residues of the reference polypeptide sequence. Using the full-length of the reference sequence in a local sequence alignment results in an overall percent identity score between the test sequence and the reference sequence.

[0041] The percent identity can be determined by using different algorithms such as BLAST and PSI-BLAST (Altschul et al., 1990, J Mol Biol 215:3, 403-410; Altschul et al., 1997, Nucleic Acids Res 25:17, 3389-402), the Clustal Omega method (Sievers et al., 2011, Mol. Syst. Biol. 7:539), the MatGAT method (Campanella et al., 2003, BMC Bioinformatics, 4:29) or EMBOSS Needle.

[0042] As used herein, a polypeptide comprising or consisting of an amino acid sequence having 25% or more sequence identity over a segment of at least 50 amino acid residues of a reference polypeptide sequence will be understood to have 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% sequence identity over a segment of at least 50 amino acid residues of the reference polypeptide sequence.

[0043] As used herein, an amino acid sequence that has 25% or more sequence identity over a segment of at least 100 amino acid residues of a reference polypeptide sequence, or a polypeptide consisting thereof, will be understood to have 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% sequence identity over a segment of at least 100 amino acid residues of the reference polypeptide sequence.

[0044] As used herein, a polypeptide comprising or consisting of an amino acid sequence having 25% or more sequence identity over a segment of at least 150 amino acid residues of a reference polypeptide sequence will be understood to have 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% sequence identity over a segment of at least 150 amino acid residues of the reference polypeptide sequence.

[0045] As used herein, a polypeptide comprising or consisting of an amino acid sequence having 25% or more sequence identity over a segment of at least 200 amino acid residues of a reference polypeptide sequence will be understood to have 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% sequence identity over a segment of at least 200 amino acid residues of the reference polypeptide sequence.

[0046] As used herein, a polypeptide comprising or consisting of an amino acid sequence having 25% or more sequence identity over a segment of at least 250 amino acid residues of a reference polypeptide sequence will be understood to have 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% sequence identity over a segment of at least 250 amino acid residues of the reference polypeptide sequence.

[0047] As used herein, a polypeptide comprising or consisting of an amino acid sequence having 25% or more sequence identity to the full-length sequence of a reference polypeptide sequence will be understood to have 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% sequence identity to the amino acid sequence of the full-length reference polypeptide sequence.

[0048] Throughout the application, unless otherwise expressly stated, a polypeptide comprising, consisting of, or having an amino acid sequence that has 25% or more sequence identity to the full-length amino acid sequence of a reference polypeptide (usually designated by a SEQ ID NO or UniProt ID) is preferably 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the full-length reference sequence, more preferably at least 50%, even more preferably at least 55%, even more preferably at least 60%, even more preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, and most preferably at least 90% sequence identity. Additionally, unless otherwise expressly stated, a polynucleotide sequence comprising, consisting of, or having a nucleotide sequence that is 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the full-length reference sequence is more preferably at least 50%, even more preferably at least 55%, even more preferably at least 60%, even more preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, and most preferably at least 90% sequence identity.

[0049] For the purposes of the present invention, the percent identity is determined by using MatGAT2.01 (Campanella et al., 2003, BMC Bioinformatics 4:29). The following default parameters for proteins are used: (1) Gap cost Existence: 12, and Extension: 2; (2) The matrix employed is BLOSUM50.

[0050] As used herein, the term "sugar importer" refers to a polypeptide that can internalize or absorb sugar from the outside of a cell into the inside of the cell. The sugar can be a monosaccharide, an activated monosaccharide, a phosphorylated monosaccharide, a disaccharide, an oligosaccharide, or a polysaccharide as defined herein.

[0051] As used herein, the term "major facilitator superfamily (MFS) of transporters" refers to the largest known superfamily of secondary active transporters that is commonly found in all living organisms. MFS transporters are responsible for transporting a broad spectrum of substrates either down or up their concentration gradients by using the energy stored in an electrochemical gradient. MFS transporters have a typical topology that consists mainly of 12 transmembrane (TM) domains organized into two 6-TM bundles that are connected via long and flexible intracellular loops; MFS transporters with more than 12 TM domains have also been identified (Chang et al., 2004, Mol. Membr. Biol. 21(3), 171-181; Drew et al., 2021, Chem. Rev. 121(9), 5289-5335; Pao et al., 1998, Microbiol. Mol. Biol. Rev. 62, 1-34; Reddy et al., 2012, FEBS J. 279, 2022-2035; Wang et al., 2020, Biochim. Biophys. Acta Biomembr. 1862(9), 183277).

[0052] As used herein, the terms "transmembrane domain" or "TM domain" are used interchangeably and refer to a protein domain that spans a membrane. The domain covers the α-helical transmembrane region and the region of a β-barrel transmembrane protein that spans the membrane. An α-helical transmembrane domain typically has an α-helical topology conformation, where the amino acid residues are often hydrophobic. The presence of transmembrane domains in a given polypeptide sequence can be determined, for example, by X-ray diffraction or predicted based on a hydrophobicity scale, for example, via an algorithm based on a deep learning protein language model, such as DeepTMHMM (Hallgren et al., 2022, bioRxiv 2022.04.08.487609), DMCTOP (Yang et al., 2022, IEEE / ACM Trans. Comput. Biol. Bioinform. 19, 295-304), PureseqTM (Wang et al., 2019, bioRxiv, 627307), BetAware-Deep (Madeo et al., 2021, J. Mol. Biol. 433, 166729).

[0053] As used herein, the term "non-TM helix" refers to a helical polypeptide sequence that is not present in the TM domain as defined herein.

[0054] As used herein, the term "glycosyltransferase" refers to an enzyme that is capable of catalyzing the transfer of a sugar moiety of a donor to a specific acceptor to form a glycosidic bond. The donor may be a precursor as defined herein. Glycosyltransferases and related proteins that utilize nucleotide diphosphate-sugars, nucleotide monophosphate-sugars, and sugar phosphates have been described as belonging to different sequence-based families (Campbell et al., Biochem. J. 326, 929-939 (1997)) and are available on the CAZy (CArbohydrate-Active EnZymes) website (www.cazy.org).

[0055] As used herein, a glycosyltransferase may be selected from the list consisting of, but not limited to: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase, N-acetylmannosaminyltransferase, xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosaminyltransferase, N-glycolylneuraminyltransferase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosaminyltransferase, UDP-N-acetylglucosamine enolpyruvate transferase, and fucosaminyltransferase.

[0056] As used herein, the term "monosaccharide" refers to a sugar that cannot be decomposed into simpler sugars by hydrolysis, is classified as an aldose or a ketose, and contains one or more hydroxyl groups / molecules. Monosaccharides are carbohydrates that contain only one simple sugar. Examples of monosaccharides include: hexoses, D-glucopyranose, D-galactofuranose, D-galactopyranose, L-galactopyranose, D-mannopyranose, D-allopyranose, L-altropyranose, D-gulopyranose, L-idopyranose, D-talopyranose, D-ribofuranose, D-ribopyranose, D-arabinofuranose, D-arabinopyranose, L-arabinofuranose, L-arabinopyranose, D-xylopyranose, D-lyxopyranose, D-erythrofuranose, D-threofuranose, heptoses, L-glycero-D-manno-pyranheptose (LDmanHep), D-glycero-D-manno-pyranheptose (DDmanHep), 6-deoxy-L-altropyranose, 6-deoxy-D-gulopyranose, 6-deoxy-D-talopyranose, 6-deoxy-D-galactopyranose, 6-deoxy-L-galactopyranose, 6-deoxy-D-mannopyranose, 6-deoxy-L-mannopyranose, 6-deoxy-D-glucopyranose, 2-deoxy-D-arabino-hexose, 2-deoxy-D-erythro-pentose, 2,6-dideoxy-D-arabino-pyranose, 3,6-dideoxy-D-arabino-pyranose, 3,6-dideoxy-L-arabino-pyranose, 3,6-dideoxy-D-xylopyranose, 3,6-dideoxy-D-ribo-pyranose, 2,6-dideoxy-D-ribo-pyranose, 3,6-dideoxy-L-xylopyranose, 2-amino-2-deoxy-D-glucopyranose, 2-amino-2-deoxy-D-galactopyranose, 2-amino-2-deoxy-D-mannopyranose, 2-amino-2-deoxy-D-allopyranose, 2-amino-2-deoxy-L-altropyranose, 2-amino-2-deoxy-D-gulopyranose, 2-amino-2-deoxy-L-idopyranose, 2-amino-2-deoxy-D-talopyranose, 2-acetamido-2-deoxy-D-glucopyranose, 2-acetamido-2-deoxy-D-galactopyranose, 2-acetamido-2-deoxy-D-mannopyranose, 2-acetamido-2-deoxy-D-allopyranose, 2-acetamido-2-deoxy-L-altropyranose, 2-acetamido-2-deoxy-D-gulopyranose, 2-acetamido-2-deoxy-L-idopyranose, 2-acetamido-2-deoxy-D-talopyranose, 2-acetamido-2,6-dideoxy-D-galactopyranose, 2-acetamido-2,6-dideoxy-L-galactopyranose, 2-acetamido-2,6-dideoxy-L-mannopyranose, 2-acetamido-2,6-dideoxy-D-glucopyranose, 2-acetamido-2,6-dideoxy-L-altropyranose, 2-acetamido-2,6-deoxy-D-talopyranose, D-glucuronic acid, D-galacturonic acid, D-mannuronic acid, D-alluronic acid, L-alturonic acid, D-guluronic acid, L-guluronic acid, L-iduronic acid, D-taluronate, sialic acid, 5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid, 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid, 5-hydroxyacetamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid, erythritol, arabitol, xylitol, ribitol, glucitol, galactitol, mannitol, D-ribo-hex-2-ulopyranose, D-arabino-hex-2-ulose (D-fructofuranose), D-arabino-hex-2-ulopyranose, L-xylulo-hex-2-ulopyranose, D-xylulo-hex-2-ulopyranose, D-threo-pent-2-ulopyranose, D-altro-hept-2-ulopyranose, 3-C-(hydroxymethyl)-D-erythrofuranose, 2,4,6-trideoxy-2,4-diamino-D-glucopyranose, 6-deoxy-3-O-methyl-D-glucose, 3-O-methyl-D-rhamnose, 2,6-dideoxy-3-methyl-D-ribo-hexose, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucopyranose, 2-acetamido-3-O-[(R)-carboxyethyl]-2-deoxy-D-glucopyranose, 2-hydroxyacetamido-3-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucopyranose, 3-deoxy-D-xylo-hept-2-ulopyranosonic acid, 3-deoxy-D-manno-oct-2-ulopyranosonic acid, 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid, 5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulopyranosonic acid, 5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-L-altro-non-2-ulopyranosonic acid, 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid, 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulopyranosonic acid, 2-acetamido-2,6-dideoxy--L-arabino-4-hexulose, 2-acetamido-2,6-deoxy-L-lyxo-4-hexulose, N-acetyl-L-rhamnosamine, N-acetyl-D-fucosamine, N-acetyl-L-pneumosamine, N-acetylmuramic acid, N-acetyl-L-quinovosamine, glucose (Glc), galactose (Gal), N-acetylglucosamine (GlcNAc), glucosamine (Glcn), mannose (Man), xylose (Xyl), N-acetylmannosamine (ManNAc), N-glycolylneuraminic acid, N-acetylgalactosamine (GalNAc), galactosamine (Galn), fucose (Fuc), rhamnose (Rha), glucuronic acid, gluconic acid, fructose (Fru), and polyols. The term "polyol" means an alcohol containing multiple hydroxyl groups. For example, glycerol, sorbitol, or mannitol.,

[0057] As used herein, the term "phosphorylated monosaccharide" refers to one of the monosaccharides listed above that has been phosphorylated. Examples of phosphorylated monosaccharides include, but are not limited to: glucose-1-phosphate, glucose-6-phosphate, glucose-1,6-diphosphate, galactose-1-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, fructose-1-phosphate, glucosamine-1-phosphate, glucosamine-6-phosphate, N-acetylglucosamine-1-phosphate, mannose-1-phosphate, mannose-6-phosphate, or fucose-1-phosphate. Some (but not all) of these phosphorylated monosaccharides are precursors or intermediates for the production of activated monosaccharides.,

[0058] The terms "activated monosaccharide", "nucleotide-activated sugar", "nucleotide-sugar", "activated sugar", "nucleoside" or "nucleotide donor" are used interchangeably herein and refer to the activated form of a monosaccharide. Examples of activated monosaccharides include, but are not limited to: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), UDP-glucuronic acid, UDP-galacturonic acid, UDP-2-acetamido-2,6-dideoxy-α-L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy-α-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose, CMP-sialic acid (CMP-Neu5Ac or CMP-N-acetylneuraminic acid), GDP-fucose (GDP-Fuc), GDP-rhamnose, and UDP-xylose. Nucleotide-sugars act as sugar donors in glycosylation reactions. Glycosylation reactions are reactions catalyzed by glycosyltransferases.

[0059] As used herein, the term "disaccharide" refers to a sugar polymer containing two simple sugars (i.e., monosaccharides). Such disaccharides preferably contain monosaccharides selected from the list of monosaccharides used above herein. Examples of disaccharides include lactose (Gal-β1,4-Glc), lacto-N-biose (Gal-β1,3-GlcNAc), N-acetyllactosamine (Gal-β1,4-GlcNAc), LacDiNAc (GalNAc-β1,4-GlcNAc), N-acetylgalactosaminylglucose (GalNAc-β1,4-Glc), Neu5Ac-α2,3-Gal, Neu5Ac-α2,6-Gal, fucopyranosyl-(1-4)-N-glycolylneuraminic acid (Fuc-(1-4)-Neu5Gc), sucrose (Glc-α1,2-Fru), maltose (Glc-α1,4-Glc), and melibiose (Gal-α1,6-Glc).

[0060] As the term is used herein and as is commonly understood in the prior art, the term "oligosaccharide" refers to a sugar polymer that contains a small number, typically three to twenty, preferably three to ten, simple sugars, i.e., monosaccharides. Preferably, the oligosaccharides described herein contain monosaccharides selected from the list used above herein. The oligosaccharides used in the present invention can be of linear structure or can contain branches. The bond between two sugar units (e.g., glycosidic bond, galactosidic bond, glucosidic bond, etc.) can be represented, for example, as 1,4, 1->4, or (1-4), which are used interchangeably herein. For example, the terms "Gal-β1,4-Glc", "Gal-β1,4-Glc", "β-Gal-(1->4)-Glc", "β-Gal-(1->4)-Glc", "Galβ1-4-Glc", "Gal-β(1-4)-Glc", and "Gal-β(1-4)-Glc" have the same meaning, i.e., a β-glycosidic bond connects carbon-1 of galactose (Gal) to carbon-4 of glucose (Glc). Each monosaccharide can be in a cyclic form (e.g., pyranose or furanose form). The bonds between individual monosaccharide units can include α1->2, α1->3, α1->4, α1->6, α2->1, α2->3, α2->4, α2->6, β1->2, β1->3, β1->4, β1->6, β2->1, β2->3, β2->4, and β2->6. Oligosaccharides can contain both α- and β-glycosidic bonds, or can contain only α-glycosidic bonds or only β-glycosidic bonds. The term "polysaccharide" refers to a compound composed of a large number, typically more than twenty, monosaccharides linked by glycosidic bonds.

[0061] Examples of oligosaccharides include, but are not limited to: milk oligosaccharides; mammalian milk oligosaccharides or MMO; human milk oligosaccharides or HMO; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; fucosylated oligosaccharides containing N-acetyllactosamine; non-fucosylated oligosaccharides containing N-acetyllactosamine; fucosylated oligosaccharides containing lacto-N-biose; non-fucosylated oligosaccharides containing lacto-N-biose; negatively charged oligosaccharides containing N-acetyllactosamine; negatively charged oligosaccharides containing lacto-N-biose; O-antigens; enterobacterial common antigen (ECA); glycan chains present in lipopolysaccharides (LPS); oligosaccharide repeats present in capsular polysaccharides; peptidoglycan (PG); amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans.

[0062] As used herein, "sialylated oligosaccharide" is understood to mean an oligosaccharide containing a negatively charged sialic acid, i.e., an oligosaccharide having a sialic acid residue. It has acidic properties. Some examples are 3-SL (3'-sialyllactose or 3'SL or Neu5Ac-α2,3-Gal-β1,4-Glc), 3'-sialyllactosamine, 6-SL (6'-sialyllactose, 6'-sialyllactose or 6'SL or Neu5Ac-α2,6-Gal-β1,4-Glc), 8-SL (8'-sialyllactose, 8'-sialyllactose or 8'SL or Neu5Ac-α2,8-Gal-β1,4-Glc), 3,6-disialyllactose (Neu5Ac-α2,3-(Neu5Ac-α2,6)-Gal-β1,4-Glc), 6,6'-disialyllactose (Neu5Ac-α2,6-Gal-β1,4-(Neu5Ac-α2,6)-Glc), 8,3-disialyllactose (Neu5Ac-α2,8-Neu5Ac-α2,3-Gal-β1,4-Glc), 6'-sialyllactosamine, oligosaccharides containing 6'-sialyllactose (also known as 6'-sialyllactose, 6'SL and 6'-SL), SGG hexose (Neu5Acα-2,3Galβ-1,3GalNacβ-1,3Galα-1,4Galβ-1,4Gal), sialylated tetrasaccharide (Neu5Acα-2,3Galβ-1,4GlcNacβ-14GlcNAc), sialylated lacto-N-triose, sialylated lacto-N-tetraose, sialyl lacto-N-neotetraose, LSTa, LSTb, LSTc, LSTd, monosialyl lacto-N-hexose, disialyl lacto-N-hexose I, monosialyl lacto-N-neohexose I, monosialyl lacto-N-neohexose II, disialyl lacto-N-neohexose, disialyl lacto-N-tetraose, disialyl lacto-N-hexose II, sialyl lacto-N-tetraose a, disialyl lacto-N-hexose I, sialyl lacto-N-tetraose b, 3'-sialyl-3-fucosyllactose, fucodisialyl lacto-N-hexose, disialomonofucosyl lacto-N-neohexose, monofucosyl monosialyl lacto-N-octose (sialyl Lea), sialyl lacto-N-fucopentaose II, disialyl lacto-N-fucopentaose II, monofucosyl disialyl lacto-N-tetraose and oligosaccharides carrying one or several sialic acid residues, including but not limited to the oligosaccharide moieties of gangliosides selected from the following: GM3 (3'-sialyllactose, Neu5Acα-2,3Galβ-4Glc) and oligosaccharides containing this GM3 moiety, GD3 (Neu5Acα-2,8Neu5Acα-2,3Galβ-1,4Glc), GT3 (Neu5Acα-2,8Neu5Acα-2,8Neu5Acα-2,3Galβ-1,4Glc), GM2 (GalNAcβ-1,4(Neu5Acα-2,3)Galβ-1,4Glc), GM1 (Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,3)Galβ-1,4Glc), GD1a (Neu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,3)Galβ-1,4Glc), GT1a (Neu5Acα-2,8Neu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,3)Galβ-1,4Glc), GD2 (GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc), GT2,

[0063] (GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc), GD1b (Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc), GT1b

[0064] (Neu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc), GQ1b (Neu5Acα-2,8Neu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc), GT1c (Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc), GQ1c (Neu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc), GP1c (Neu5Acα-2,8Neu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc), GD1a (Neu5Acα-2,3Galβ-1,3(Neu5Acα-2,6)GalNAcβ-1,4Galβ-1,4Glc), fucosyl-GM1 (Fucα-1,2Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,3)Galβ-1,4Glc); all of which can be extended to produce the corresponding gangliosides by reacting the above oligosaccharide moiety with ceramide or synthesizing the above oligosaccharide on ceramide.

[0065] The terms "LNT II", "LNT-II", "LN3", "lacto-N-triose II", "lacto-N-triose II", "lacto-N-triose", "lacto-N-triose" or "GlcNAcβ1-3Galβ1-4Glc" used in the present invention are used interchangeably.

[0066] The terms "LN3-derived oligosaccharide", "LN3 - derived oligosaccharide", or "oligosaccharide derived from LN3" as used in the present invention are used interchangeably and refer to an oligosaccharide having a degree of polymerization of at least 4, which comprises an LN3 core that has been extended by at least one additional monosaccharide subunit described herein. The oligosaccharide can be a linear or branched oligosaccharide. The oligosaccharide can comprise α - glycosidic bonds and / or β - glycosidic bonds. Examples include but are not limited to: LNT, LNnT, LSTa, LSTb, LSTc, LSTd, DSLNnT, DSLNT, LNFP - I, LNFP - II, LNFP - III, LNFP - V, LNFP - VI, lacto - N - neofucopentaose I, lacto - N - difucosylhexose I (LDFH I), lacto - N - difucosylhexose II (LDFH II), monofucosyl lacto - N - hexose III (MFLNHIII), difucosyl lacto - N - hexose (DFLNHa), difucosyl - lacto - N - neohexose, 3'-sialyl - 3 - fucosyllactose, disialylmonofucosyl lacto - N - neohexose, monofucosylmonosialyl lacto - N - octose (sialyl Lea), sialyl lacto - N - fucosylhexose II, disialyl lacto - N - neofucopentaose II, monofucosyldisialyl lacto - N - tetrasaccharide, GalNAc - LNFP - I, LNnDFH II, LNDFH I, LNDFHII, LNH, p - LNH, LNnH, p - LNnH, F - LNH I, F - LNH - II, DF - LNH I, DF - LNH II, DFLNH c, DF - LNnH, DF - p - LNH, DF - p - LNnH, TF - LNH, F - LSTa, F - LSTb, F - LSTc, FS - LNH, FS - LNnH I and FDS - LNH II.

[0067] The terms "LNT", "lacto - N - tetrasaccharide", "lacto - N - tetrasaccharide", or "Galβ1 - 3GlcNAcβ1 - 3Galβ1 - 4Glc" as used in the present invention are used interchangeably.

[0068] The terms "LNnT", "lacto - N - neotetrasaccharide", "lacto - N - neotetrasaccharide", "neo - LNT", or "Galβ1 - 4GlcNAcβ1 - 3Galβ1 - 4Glc" as used in the present invention are used interchangeably.

[0069] The terms "LSTa", "LS - tetrasaccharide a", "sialyl - lacto - N - tetrasaccharide a", "sialyl lacto - N - tetrasaccharide a", or "Neu5Ac - a2,3 - Gal - b1,3 - GlcNAc - b1,3 - Gal - b1,4 - Glc" as used in the present invention are used interchangeably.

[0070] In the present invention, the terms "LSTb", "LS-tetrasaccharide b", "sialyl-lacto-N-tetraose b", "sialyl lacto-N-tetraose b", or "Gal-β1,3-(Neu5Ac-α2,6)-GlcNAc-β1,3-Gal-β1,4-Glc" are used interchangeably.

[0071] In the present invention, the terms "LSTc", "LS-tetrasaccharide c", "sialyl-lacto-N-tetraose c", "sialyl lacto-N-tetraose c", "sialyl lacto-N-neotetraose c", or "Neu5Ac-α2,6-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc" are used interchangeably.

[0072] In the present invention, the terms "LSTd", "LS-tetrasaccharide d", "sialyl-lacto-N-tetraose d", "sialyl lacto-N-tetraose d", "sialyl lacto-N-neotetraose d", or "Neu5Ac-α2,3-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc" are used interchangeably.

[0073] The terms "DSLNnT" and "disialyl lacto-N-neotetraose" are used interchangeably and refer to Neu5Ac-α2,6-Gal-β1,4-GlcNAc-β1,3-[Neu5Ac-α2,6]-Gal-β1,4-Glc.

[0074] The terms "DSLNT", "DS-LNT", and "disialyl lacto-N-tetraose" are used interchangeably and refer to Neu5Ac-α2,3-Gal-β1,3-[Neu5Ac-α2,6]-GlcNAc-β1,3-Gal-β1,4-Glc.

[0075] "Charged oligosaccharides" are oligosaccharide structures that contain one or more negatively charged monosaccharide subunits (including N-acetylneuraminic acid (Neu5Ac), commonly known as sialic acid, N-glycolylneuraminic acid (Neu5Gc), glucuronic acid, and galacturonic acid). Charged oligosaccharides are also referred to as acidic oligosaccharides. Sialic acid belongs to the family of derivatives of neuraminic acid (5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid). Neu5Gc is a derivative of sialic acid that is formed by hydroxylation of the N-acetyl group at C5 of Neu5Ac. In contrast, neutral oligosaccharides are non-sialylated oligosaccharides and thus do not contain acidic monosaccharide subunits. Neutral oligosaccharides include uncharged fucosylated oligosaccharides that contain one or more fucose subunits in their glycan structure, as well as uncharged non-fucosylated oligosaccharides that lack any fucose subunits. Other examples of charged oligosaccharides are sulfated chitosan and deacetylated chitosan.

[0076] As used herein, the terms "neutral oligosaccharide" and "uncharged oligosaccharide" are used interchangeably and, as commonly understood in the art, refer to oligosaccharides that do not have a negative charge from a carboxylic acid group. Examples of such neutral oligosaccharides are 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 2',3-difucosyllactose (diFL), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucosylhexose I, lacto-N-difucosylhexose II, 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexose, lacto-N-neohexose, para-lacto-N-hexose, para-lacto-N-neohexose, difucosyl-lacto-N-hexose, and difucosyl-lacto-N-neohexose.

[0077] As used herein and as commonly understood in the art, "fucosylated oligosaccharide" is an oligosaccharide carrying fucose residues. Such fucosylated oligosaccharides are sugar structures comprising at least three monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of the monosaccharide subunits is fucose. Fucosylated oligosaccharides may contain more than one fucose residue, for example two, three or more. Fucosylated oligosaccharides may be neutral or charged oligosaccharides, which may also contain sialic acid structures, for example. Fucose may be linked to other monosaccharide subunits (including glucose, galactose, GlcNAc) via α-glycosidic bonds (including α-1,2, α-1,3, α-1,4, α-1,6 bonds). Examples include 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), difucosyllactose (diFL), lacto-N-fucopentaose I (LNFPI), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), lacto-N-fucopentaose VI (LNFP VI), lacto-N-neofucopentaose I, lacto-N-difucosylhexose I (LDFHI), lacto-N-difucosylhexose II (LDFH II), monofucosyllacto-N-hexose III (MFLNHIII), difucosyllacto-N-hexose (DFLNHa), difucosyl-lacto-N-neohexose, 3'-sialyl-3-fucosyllactose, disialylmonofucosyllacto-N-neohexose, monofucosylmonosialyllacto-N-octose (sialyl Lea), sialyllacto-N-fucopentaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose.

[0078] The terms "LNFP-I", "lacto-N-fucopentaose I", "LNFP I", "LNFPI", "LNF IOH type I determinant", "LNF I", "LNF1", "LNF 1", and "H blood group antigen pentasaccharide type 1" are used interchangeably and refer to Fuc-α1,2-Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc. The terms "GalNAc-LNFP-I" and "A blood group antigen hexasaccharide type I" are used interchangeably and refer to GalNAc-α1,3-(Fuc-α1,2)-Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc. The terms "LNFP-II" and "lacto-N-fucopentaose II" are used interchangeably and refer to Gal-β1,3-[Fuc-α1,4]-GlcNAc-β1,3-Gal-β1,4-Glc. The terms "LNFP-III", "LNFP III", "LNFPIII", and "lacto-N-fucopentaose III" are used interchangeably and refer to Gal-β1,4-(Fuc-α1,3)-GlcNAc-β1,3-Gal-β1,4-Glc. The terms "LNFP-V", "LNFPV", "LNFPV", and "lacto-N-fucopentaose V" are used interchangeably and refer to Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-(Fuc-α1,3)-Glc. The terms "LNFP-VI", "LNFP VI", "LNnFP V", and "lacto-N-neofucopentaose V" are used interchangeably and refer to Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-(Fuc-α1,3)-Glc. The terms "LNnFP I" and "lacto-N-neofucopentaose I" are used interchangeably and refer to Fuc-α1,2-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc. The terms "LNDFH I", "lacto-N-difucosylhexose I", "LNDFH-I", "LDFH I", "Le bThe terms "-lactose" and "Lewis-b hexose" are used interchangeably and refer to Fuc-α1,2-Gal-β1,3-[Fuc-α1,4]-GlcNAc-β1,3-Gal-β1,4-Glc. The terms "LNDFH II", "lacto-N-difucohexose II", "LNDFH-II", "Lewis a-Lewis x", and "LDFH II" are used interchangeably and refer to Gal-β1,3-[Fuc-α1,4]-GlcNAc-β1,3-Gal-β1,4-(Fuc-α1,3)-Glc. The terms "LNnDFH II", "lacto-N-neodifucohexose II", "LNDFH III", "Lewis x hexose", and "LeX hexose" are used interchangeably and refer to Gal-β1,4-(Fuc-α1,3)-GlcNAc-β1,3-Gal-β1,4-(Fuc-α1,3)-Glc. The terms "α-tetrasaccharide" and "A-tetrasaccharide" are used interchangeably and refer to GalNAc-α1,3-(Fuc-α1,2)-Gal-β1,4-Glc. The terms "LNH" and "lacto-N-hexose" are used interchangeably and refer to Gal-β1,3-GlcNAc-β1,3-(Gal-β1,4-GlcNAc-β1,6)-Gal-β1,4-Glc. The terms "p-LNH", "pLNH", and "p-lacto-N-hexose" are used interchangeably and refer to Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc. The terms "LNnH" and "lacto-N-neohexose" are used interchangeably and refer to Gal-β1,4-GlcNAc-β1,3-[Gal-β1,4-GlcNAc-β1,6]-Gal-β1,4-Glc. The terms "p-LNnH", "pLNnH", and "p-lacto-N-neohexose" are used interchangeably and refer to Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc.

[0079] The terms "F-LNH I", "FLNH I", and "fucosyl lacto-N-hexose I" are used interchangeably and refer to Fuc-α1,2-Gal-β1,3-GlcNAc-β1,3-[Gal-β1,4-GlcNAc-β1,6]-Gal-β1,4-Glc. The terms "F-LNH-II", "FLNH II", and "fucosyl lacto-N-hexose II" are used interchangeably and refer to Gal-β1,3-GlcNAc-β1,3-[Gal-β1,4-[Fuc-α1,3]-GlcNAc-β1,6]-Gal-β1,4-Glc. The terms "DF-LNH I", "difucosyl lacto-N-hexose I", "DF-LNH a", "DFLNH a", "difucosyl lacto-N-hexose a", and "2,3-difucosyl lacto-N-hexose" are used interchangeably and refer to Fuc-α1,2-Gal-β1,3-GlcNAc-β1,3-[Gal-β1,4-[Fuc-α1,3]-GlcNAc-β1,6]-Gal-β1,4-Glc. The terms "DF-LNH II", "DF-LNH b", "DFLNH b", and "difucosyl lacto-N-hexose II" are used interchangeably and refer to Gal-β1,3-[Fuc-α1,4]-GlcNAc-β1,3-[Gal-β1,4-[Fuc-α1,3]-GlcNAc-β1,6]-Gal-β1,4-Glc. The terms "DFLNH c", "DF-LNH c", and "difucosyl lacto-N-hexose c" are used interchangeably and refer to Fuc-α1,2-Gal-β1,3-[Fuc-α1,4]-GlcNAc-β1,3-[Gal-β1,4-GlcNAc-β1,6]-Gal-β1,4-Glc. The terms "DF-LNnH" and "difucosyl lacto-N-neohexose" are used interchangeably and refer to Gal-β1,4-[Fuc-α1,3]-GlcNAc-β1,3-[Gal-β1,4-[Fuc-α1,3]-GlcNAc-β1,6]-Gal-β1,4-Glc.

[0080] The terms "DF-p-LNH", "DF-p-LNH", "DF-pLNH" and "difucosyl-p-lacto-N-hexose" are used interchangeably and refer to Gal-β1,3-[Fuc-α1,4]-GlcNAc-β1,3-Gal-β1,4-[Fuc-α1,3]-GlcNAc-β1,3-Gal-β1,4-Glc. The terms "DF-p-LNnH", "DF-p-LNnH" and "difucosyl-p-lacto-N-neohexose" are used interchangeably and refer to Gal-β1,4-[Fuc-α1,3]-GlcNAc-β1,3-Gal-β1,4-[Fuc-α1,3]-GlcNAc-β1,3-Gal-β1,4-Glc. The terms "TF-LNH" and "trifucosyl lacto-N-hexose" are used interchangeably and refer to Fuc-α1,2-Gal-β1,3-[Fuc-α1,4]-GlcNAc-β1,3-[Gal-β1,4-[Fuc-α1,3]-GlcNAc-β1,6]-Gal-β1,4-Glc.

[0081] The terms "F-LSTa", "F-LSTa", "S-LNF II" and "fucosyl-sialyl lacto-N-tetraose a" are used interchangeably and refer to Neu5Ac-α2,3-Gal-β1,3-[Fuc-α1,4]-GlcNAc-β1,3-Gal-β1,4-Glc. The terms "F-LSTb", "F-LSTb", "S-LNF I" and "fucosyl-sialyl lacto-N-tetraose b" are used interchangeably and refer to Fuc-α1,2-Gal-β1,3-(Neu5Ac-α2,6)-GlcNAc-β1,3-Gal-β1,4-Glc. The terms "F-LSTc", "F-LSTc" and "fucosyl-sialyl lacto-N-neotetraose" are used interchangeably and refer to Neu5Ac-α2,6-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-[Fuc-α1,3]-Glc.

[0082] The term "FS-LNH" and "fucosyl-sialyl lacto-N-hexose" are used interchangeably and refer to Fuc-α1,2-Gal-β1,3-GlcNAc-β1,3-(Neu5Ac-α2,6-Gal-β1,4-GlcNAc-β1,6)-Gal-β1,4-Glc.

[0083] The terms "FS-LNnH I" and "fucosyl-sialyl-lacto-N-neohexose I" are used interchangeably and refer to Neu5Ac-α2,6-Gal-β1,4-GlcNAc-β1,3-[Gal-β1,4-[Fuc-α1,3]-GlcNAc-β1,6]-Gal-β1,4-Glc.

[0084] The terms "FDS-LNH II" and "fucosyldisialyl-lacto-N-hexose II" are used interchangeably and refer to Neu5Ac-α2,3-Gal-β1,3-[Neu5Ac-α2,6]-GlcNAc-β1,3-[Gal-β1,4-[Fuc-α1,3]-GlcNAc-β1,6]-Gal-β1,4-Glc.

[0085] Mammalian milk oligosaccharides include oligosaccharides present in milk (including colostrum) found at any stage during lactation from humans and mammals, including but not limited to cattle (Bos taurus), sheep (Ovis aries), goats (Capra aegagrus hircus), Bactrian camels (Camelus bactrianus), horses (Equus ferus caballus), pigs (Sus scropha), dogs (Canis lupus familiaris), Hokkaido brown bears (Ursus arctos yesoensis), polar bears (Ursus maritimus), Japanese black bears (Ursus thibetanus japonicus), striped skunks (Mephitis mephitis), hooded seals (Cystophora cristata), Asian elephants (Elephas maximus), African elephants (Loxodonta africana), giant anteaters (Myrmecophaga tridactyla), bottlenose dolphins (Tursiops truncatus), minke whales (Balaenoptera acutorostrata), tammar wallabies (Macropus eugenii), red kangaroos (Macropus rufus), common brushtail possums (Trichosurus vulpecula), koalas (Phascolarctos cinereus), eastern quolls (Dasyurus viverrinus), platypuses (Ornithorhynchus anatinus).As used herein, "mammalian milk oligosaccharide" or MMO refers to oligosaccharides such as, but not limited to, the following: 3-fucosyllactose, 2'-fucosyllactose, 6-fucosyllactose, 2',3-difucosyllactose, 2',2-difucosyllactose, 3,4-difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, 3,6-disialyllactose, 6,6'-disialyllactose, 8,3-disialyllactose, 3,6-disialyllacto-N-tetraose, lactodifucotetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, sialyllacto-N-tetraose c, sialyllacto-N-tetraose b, sialyllacto-N-tetraose a, lacto-N-difucosylhexose I, lacto-N-difucosylhexose II, lacto-N-hexose, lacto-N-neohexose, para-lacto-N-hexose, monofucosylmonosialyllacto-N-tetraose c, monofucosyl-para-lacto-N-hexose, monofucosyllacto-N-hexose III, isofucosylated lacto-N-hexose III, isofucosylated lacto-N-hexose I, sialyllacto-N-hexose, sialyllacto-N-neohexose II, difucosyl-para-lacto-N-hexose, difucosyllacto-N-hexose, difucosyllacto-N-hexose a, difucosyllacto-N-hexose c, galactosylated chitosan, fucosylated oligosaccharide, neutral oligosaccharide, and / or sialylated oligosaccharide.

[0086] The term "human milk oligosaccharide" or "HMO" refers to oligosaccharides found in human breast milk (including preterm human milk, colostrum, and term human milk). HMOs include fucosylated oligosaccharides, non-fucosylated neutral oligosaccharides, and sialylated oligosaccharides (see, e.g., Chen X., Chapter Four: Human Milk Oligosaccharides (HMOS): Structure, Function, and Enzyme-Catalyzed Synthesis in Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). Examples of HMOs include 3-fucosyllactose, 2'-fucosyllactose, 2',3-difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, LN3, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, sialyllacto-N-tetraose c, sialyllacto-N-tetraose b, sialyllacto-N-tetraose a, difucosyllacto-N-tetraose, lacto-N-hexose, lacto-N-difucosylhexose I, lacto-N-difucosylhexose II, disialyllacto-N-tetraose, fucosyllacto-N-hexose, difucosyllacto-N-hexose, fucodisialyllacto-N-hexose, disialyllacto-N-hexose.

[0087] The term "cultivation" refers to the culture medium in which cells are cultured or fermented, the cells themselves, and one or more biological products produced by the cells in the whole culture broth, i.e., inside the cells (intracellularly) and outside the cells (extracellularly).

[0088] The term "incubation" refers to a mixture in which one or more biological products are produced. The mixture may contain one or more enzymes, one or more precursors, and one or more acceptors (which are present in a buffer solution) as defined herein, and is incubated at a certain temperature for a certain time, so that one or more biological products can be catalytically produced from the one or more precursors and the one or more acceptors by the one or more enzymes in the mixture. The mixture may also contain: i) cells obtained after cultivation or incubation, optionally subjecting the cells to cell lysis; ii) a buffer solution or a culture or incubation medium in which the cells are cultured or fermented; and iii) one or more biological products produced by the cells in the whole culture broth, i.e., inside the cells (intracellularly) and outside the cells (extracellularly). The incubation may also be the cultivation as defined herein.

[0089] The terms "reactor" and "incubator" refer to a vessel filled with the culture or incubation. Examples of reactors or incubators include, but are not limited to, microfluidic devices, well plates, tubes, shake flasks, fermenters, bioreactors, process vessels, cell culture incubators, CO2 incubators. The reactors and incubators can each vary in size from laboratory scale to large-scale industrial scale.

[0090] As used herein, the term "cell productivity index (CPI)" refers to the mass of the biological product produced by the cells divided by the mass of the cells produced in the culture.

[0091] The term "purified" refers to a material that is substantially or essentially free of components that interfere with the activity of the biomolecule. For cells, carbohydrates, nucleic acids, and polypeptides, the term "purified" refers to a material that is substantially or essentially free of the components that normally accompany the material (as found in its native state). Typically, the purified carbohydrates, oligosaccharides, proteins, or nucleic acids of the present invention are at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% pure, usually at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure, as measured by band intensity on a silver-stained gel or other methods for determining purity. Purity or homogeneity can be specified by many means well known in the art, such as polyacrylamide gel electrophoresis of a protein or nucleic acid sample, followed by visualization after staining. For some purposes, high resolution will be required and HPLC or similar purification means will be employed. For disaccharides and oligosaccharides, purity can be determined by methods such as, but not limited to, thin layer chromatography, gas chromatography, NMR, HPLC, capillary electrophoresis, or mass spectrometry. Further herein, the terms "contaminant" and "impurity" preferably mean particles, cells, cell components, metabolites, cell debris, proteins, peptides, amino acids, nucleic acids, glycolipids, and / or endotoxins that may be present in an aqueous medium (e.g., a culture or incubation medium).

[0092] The term "clarification" as used herein refers to the act of treating an aqueous medium (e.g., a culture or incubation medium) to remove suspended particles and contaminants from the production process, such as cells, cell components, insoluble metabolites, and debris, which can interfere with the final purification of the one or more biological products. Such treatment can be carried out in a conventional manner by centrifugation, flocculation, flocculation with optional sonication, gravity filtration, microfiltration, foam separation, or vacuum filtration (e.g., through a ceramic filter, which may include Celite TM filter aid).

[0093] As used herein, the term "precursor" refers to a substance that is taken up or synthesized by a cell for the specific production of one or more biological products according to the invention. In this sense, a precursor can be an acceptor as defined herein, but can also be another substance, a metabolite, that is first modified within the cell as part of the biochemical synthesis pathway of one or more biological products. The term "precursor" as used herein will also be understood to mean a chemical compound that participates in a chemical or enzymatic reaction to produce another compound (e.g., an intermediate or acceptor as defined herein) (as part of the metabolic pathway of one or more biological products). The term "precursor" as used herein will also be understood to mean a donor that is used by a glycosyltransferase to modify an acceptor as defined herein with a sugar moiety via a glycosidic bond as part of the metabolic pathway of one or more biological products. Examples of such precursors include acceptors as defined herein, and / or dihydroxyacetone, glucosamine, N-acetylglucosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, galactosyllactose, phosphorylated sugars or sugar phosphates, such as but not limited to glucose-1-phosphate, galactose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, mannose-6-phosphate, mannose-1-phosphate, glycerol-3-phosphate, glyceraldehyde-3-phosphate, dihydroxyacetone-phosphate, glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, N-acetylmannosamine-6-phosphate, N-acetylglucosamine-1-phosphate, N-acetylneuraminic acid-9-phosphate and nucleotide-activated sugars, such as nucleotide diphosphate-sugars and nucleotide monophosphate-sugars as defined herein, such as UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, CMP-sialic acid, GDP-mannose, GDP-4-dehydro-6-deoxy-α-D-mannose, GDP-fucose.

[0094] Optionally, the cell is transformed to comprise and express at least one nucleic acid sequence encoding a protein selected from the group consisting of a lactose transporter, an N-acetylneuraminic acid transporter, a fucose transporter, a glucose transporter, a galactose transporter, a transporter for nucleotide-activated sugars, wherein the transporter internalizes a precursor added to the medium for the synthesis of one or more biological products of the invention.

[0095] As used herein, the term "acceptor" refers to a monosaccharide, disaccharide or oligosaccharide that can be modified by a glycosyltransferase. Examples of such acceptors include: glucose, galactose, fructose, glycerol, sialic acid, fucose, mannose, maltose, sucrose, lactose, lacto-N-triose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-pentaose (LNP), lacto-N-neopentaose, para-lacto-N-pentaose, para-lacto-N-neopentaose, lacto-N-neonataopentaose I, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), para-lacto-N-neohexaose (pLNnH), para-lacto-N-hexaose (pLNH), lacto-N-heptaose, lacto-N-neoheptaose, para-lacto-N-neoheptaose, para-lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N-neooctaose, isolacto-N-octaose, para-lacto-N-octaose, isolacto-N-neooctaose, neonato-lacto-N-neooctaose, para-lacto-N-neooctaose, isolacto-N-nonose, neonato-lacto-N-nonose, lacto-N-nonose, lacto-N-decaose, isolacto-N-decaose, neonato-lacto-N-decaose, lacto-N-neodecaose, and oligosaccharides containing one or more N-acetyl lactosamine units and / or one or more lacto-N-disaccharide units, or intermediates that become oligosaccharides, their fucosylated and sialylated forms, ceramides, N-acylated sphingoids, glucosylceramides, lactosylceramides, lactosylceramides, sphingosine, phytosphingosine, sphingosine synthons, peptide backbones with β-GlcNAc-Asn residues, glycoproteins with terminal GlcNAc and Gal residues, immunoglobulins. Detailed Description of the Invention

[0097] According to a first aspect, the present invention provides a cell for producing one or more biological products, wherein the cell is capable of producing, preferably producing, one or more precursors used in the production of at least one of the one or more biological products, and wherein the cell is genetically modified to express, preferably overexpress, a polynucleotide sequence encoding a sugar importer that internalizes at least one of the one or more precursors into the cell.

[0098] Within the scope of the present invention, the one or more biological products described herein are selected from the list comprising the following: sugars; monosaccharides; activated monosaccharides; phosphorylated monosaccharides; disaccharides; oligosaccharides; polysaccharides; sugars in milk; sugars in mammalian milk; mammalian milk oligosaccharides (MMO); sugars in human milk; human milk oligosaccharides (HMO); neutral (uncharged) sugars; negatively charged sugars; fucosylated sugars; sialylated sugars; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; O-antigens; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; LN3; lacto-N-tetraose (LNT); lacto-N-neotetraose (LNnT); oligosaccharides derived from LN3; 3'-sialyllactose (3'SL); 6'-sialyllactose (6'SL); sialyllacto-N-tetraose a (LSTa); sialyllacto-N-tetraose b (LSTb); sialyllacto-N-tetraose c (LSTc); sialyllacto-N-tetraose d (LSTd); lacto-N-fucopentaose I; lacto-N-neofucopentaose; lacto-N-fucopentaose II; lacto-N-fucopentaose III; lacto-N-fucopentaose V; lacto-N-neofucopentaose V; lacto-N-difucosylhexose I; lacto-N-neodifucosylhexose; lacto-N-difucosylhexose II; monofucosyllacto-N-hexose III; difucosyllacto-N-hexose a; 6'-galactosyllactose; 3'-galactosyllactose; lacto-N-hexose; lacto-N-neohexose; 2'-fucosyllactose (2'FL); 3-fucosyllactose (3-FL); difucosyllactose (DiFL); chitosan; oligosaccharides containing chitosan; heparosan; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate; acetylhyaluronic acid; hyaluronic acid; and keratan sulfate. In a preferred embodiment of the present invention, the one or more biological products are LN3 as defined herein and / or one or more LN3-derived oligosaccharides.

[0099] In a preferred embodiment of the present invention, the one or more precursors described herein are selected from the list comprising: sugars; monosaccharides; activated monosaccharides; phosphorylated monosaccharides; disaccharides; oligosaccharides; polysaccharides; sugars in milk; sugars in mammalian milk; mammalian milk oligosaccharides (MMO); sugars in human milk; human milk oligosaccharides (HMO); neutral (uncharged) sugars; negatively charged sugars; fucosylated sugars; sialylated sugars; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; O-antigens; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; LN3; lacto-N-tetraose (LNT); lacto-N-neotetraose (LNnT); oligosaccharides derived from LN3; 3'-sialyllactose (3'SL); 6'-sialyllactose (6'SL); sialyllacto-N-tetraose a (LSTa); sialyllacto-N-tetraose b (LSTb); sialyllacto-N-tetraose c (LSTc); sialyllacto-N-tetraose d (LSTd); lacto-N-fucopentaose I; lacto-N-neofucopentaose; lacto-N-fucopentaose II; lacto-N-fucopentaose III; lacto-N-fucopentaose V; lacto-N-neofucopentaose V; lacto-N-difucosylhexose I; lacto-N-neodifucosylhexose; lacto-N-difucosylhexose II; monofucosyllacto-n-hexose III; difucosyllacto-N-hexose a; 6'-galactosyllactose; 3'-galactosyllactose; lacto-N-hexose; lacto-N-neohexose; 2'-fucosyllactose (2'FL); 3-fucosyllactose (3-FL); difucosyllactose (DiFL); chitosan; oligosaccharides containing chitosan; heparosan; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate; acetylhyaluronic acid; hyaluronic acid; and keratan sulfate. In a more preferred embodiment of the present invention, the one or more precursors are LN3 and / or one or more LN3-derived oligosaccharides as defined herein. In an even more preferred embodiment, one of the precursors is LN3.

[0100] In a preferred embodiment of the cells and / or methods of the present invention, the cells produce a bioproduct. In another preferred embodiment of the cells and / or methods of the present invention, the cells produce more than one bioproduct. In another preferred embodiment of the cells and / or methods of the present invention, the cells produce a mixture comprising at least one bioproduct described herein. In a more preferred embodiment, the cells produce LN3. In another and / or additional preferred embodiment, the cells produce LN3-derived oligosaccharides. In another and / or additional preferred embodiment, the cells produce LNT and / or LNnT. In another and / or additional preferred embodiment, the cells produce a mixture of LN3-derived oligosaccharides.

[0101] In another preferred embodiment of the cells and / or methods of the present invention, the cells produce a bioproduct and a precursor used in the production of the bioproduct, wherein the precursor is present extracellularly by any one or more of the following: passive transport outside the cell during and / or at the end of culturing and / or incubating the cells, active transport outside the cell, secretion, excretion, and / or cell lysis, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes the precursor into the cell to be used for producing the bioproduct.

[0102] In another preferred embodiment of the cells and / or methods of the present invention, the cells produce a bioproduct and two precursors used in the production of the bioproduct, wherein one of the precursors is present extracellularly by any one or more of the following: passive transport outside the cell during and / or at the end of culturing and / or incubating the cells, active transport outside the cell, secretion, excretion, and / or cell lysis, and wherein the other of the two precursors is present intracellularly, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes the extracellular precursor into the cell to be used for producing the bioproduct.

[0103] In another preferred embodiment of the cells and / or methods of the present invention, the cells produce a bioproduct and two precursors used in the production of the bioproduct, wherein both of the precursors are present extracellularly by any one or more of the following means: passive transport outside the cells during and / or at the end of culturing and / or incubating the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes one of the extracellular precursors into the cells to be used for producing the bioproduct. In a more preferred embodiment, the cells produce a bioproduct and two precursors used in the production of the bioproduct, wherein both of the precursors are present extracellularly by any one or more of the following means: passive transport outside the cells during and / or at the end of culturing and / or incubating the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes both of the extracellular precursors into the cells to be used for producing the bioproduct. In another more preferred embodiment, the cells produce a bioproduct and two precursors used in the production of the bioproduct, wherein both of the precursors are present extracellularly by any one or more of the following means: passive transport outside the cells during and / or at the end of culturing and / or incubating the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the cells are genetically engineered to (over)express two polynucleotide sequences each encoding a different sugar importer, wherein the first sugar importer internalizes one of the extracellular precursors and the second sugar importer internalizes the other of the extracellular precursors into the cells to be used for producing the bioproduct.

[0104] In another preferred embodiment of the cells and / or methods of the present invention, the cells produce a bioproduct and three or more precursors used in the production of the bioproduct, wherein one of the precursors is present extracellularly by any one or more of the following means: passive transport outside the cells during and / or at the end of culturing and / or incubating the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the other precursors are present intracellularly, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes the extracellular precursor into the cells to be used for producing the bioproduct.

[0105] In another preferred embodiment of the cells and / or methods of the present invention, the cells produce a bioproduct and three or more precursors used in the production of the bioproduct, wherein two of the precursors are present extracellularly by any one or more of the following means: passive transport outside the cells during and / or at the end of culturing and / or incubating the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the other precursors are present intracellularly, and the cells are genetically modified to (over)express a polynucleotide sequence encoding a sugar importer that internalizes one of the extracellular precursors into the cells to be used for producing the bioproduct. In a more preferred embodiment, the cells produce a bioproduct and three or more precursors used in the production of the bioproduct, wherein two of the precursors are present extracellularly by any one or more of the following means: passive transport outside the cells during and / or at the end of culturing and / or incubating the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the other precursors are present intracellularly, and the cells are genetically modified to (over)express a polynucleotide sequence encoding a sugar importer that internalizes both of the extracellular precursors into the cells to be used for producing the bioproduct. In another more preferred embodiment, the cells produce a bioproduct and three or more precursors used in the production of the bioproduct, wherein two of the precursors are present extracellularly by any one or more of the following means: passive transport outside the cells during and / or at the end of culturing and / or incubating the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the other precursors are present intracellularly, and the cells are genetically modified to (over)express two polynucleotide sequences each encoding a different sugar importer, wherein the first sugar importer internalizes one of the extracellular precursors and the second sugar importer internalizes the other of the extracellular precursors into the cells to be used for producing the bioproduct.

[0106] In another preferred embodiment of the cells and / or methods of the present invention, the cells produce a bioproduct and three or more precursors used in the production of the bioproduct, wherein at least three of the precursors are present extracellularly by any one or more of the following: passive transport outside the cells during and / or at the end of the culturing and / or incubation of the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and any other precursors (if present) are present intracellularly, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes one of the extracellular precursors into the cells to be used for the production of the bioproduct. In a more preferred embodiment, the cells produce a bioproduct and three or more precursors used in the production of the bioproduct, wherein at least three of the precursors are present extracellularly by any one or more of the following: passive transport outside the cells during and / or at the end of the culturing and / or incubation of the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and any other precursors (if present) are present intracellularly, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes two of the extracellular precursors into the cells to be used for the production of the bioproduct. In an even more preferred embodiment, the cells produce a bioproduct and three or more precursors used in the production of the bioproduct, wherein at least three of the precursors are present extracellularly by any one or more of the following: passive transport outside the cells during and / or at the end of the culturing and / or incubation of the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and any other precursors (if present) are present intracellularly, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes three or more (if present) of the extracellular precursors into the cells to be used for the production of the bioproduct. In another more preferred embodiment, the cells produce a bioproduct and three or more precursors used in the production of the bioproduct, wherein at least three of the precursors are present extracellularly by any one or more of the following: passive transport outside the cells during and / or at the end of the culturing and / or incubation of the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and any other precursors (if present) are present intracellularly, and the cells are genetically engineered to (over)express two or more polynucleotide sequences encoding two or more different sugar importers that internalize three or more (if present) of the extracellular precursors into the cells to be used for the production of the bioproduct.

[0107] In another preferred embodiment of the cells and / or methods of the present invention, the cells produce two bioproducts and a precursor used in the production of one or both of the bioproducts, wherein the precursor is present extracellularly by any one or more of the following: passive transport outside the cells during and / or at the end of culturing and / or incubating the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes the precursor into the cells to be used for producing one or both of the bioproducts.

[0108] In another preferred embodiment of the cells and / or methods of the present invention, the cells produce two bioproducts and two precursors each used in the production of one or both of the bioproducts, wherein one of the precursors is present extracellularly by any one or more of the following: passive transport outside the cells during and / or at the end of culturing and / or incubating the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and wherein the other of the two precursors is present intracellularly, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes the extracellular precursor into the cells to be used for producing one or both of the bioproducts.

[0109] In another preferred embodiment of the cells and / or methods of the present invention, the cells produce two bioproducts and two precursors each used in the production of one or both of the bioproducts, wherein both of the precursors are present extracellularly by any one or more of the following: passive transport outside the cells during and / or at the end of culturing and / or incubating the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes one of the extracellular precursors into the cells to be used for the production of one or both of the bioproducts. In a more preferred embodiment, the cells produce two bioproducts and two precursors each used in the production of one or both of the bioproducts, wherein both of the precursors are present extracellularly by any one or more of the following: passive transport outside the cells during and / or at the end of culturing and / or incubating the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes both of the extracellular precursors into the cells to be used for the production of one or both of the bioproducts. In another more preferred embodiment, the cells produce one bioproduct and two precursors used in the production of the bioproduct, wherein both of the precursors are present extracellularly by any one or more of the following: passive transport outside the cells during and / or at the end of culturing and / or incubating the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the cells are genetically engineered to (over)express two polynucleotide sequences each encoding a different sugar importer, wherein the first sugar importer internalizes one of the extracellular precursors and the second sugar importer internalizes the other of the extracellular precursors into the cells to be used for the production of the bioproduct.

[0110] In another preferred embodiment of the cells and / or methods of the present invention, the cells produce two bioproducts and three or more precursors each used in the production of one or both of the bioproducts, wherein one of the precursors is present extracellularly by any one or more of the following: passive transport outside the cells during and / or at the end of culturing and / or incubating the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the other precursors are present intracellularly, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes the extracellular precursor into the cells to be used for the production of one or both of the bioproducts.

[0111] In another preferred embodiment of the cells and / or methods of the present invention, the cells produce two bioproducts and three or more precursors each used in the production of one or both of the bioproducts, wherein two of the precursors are present extracellularly by any one or more of the following means: passive transport outside the cells during and / or at the end of culturing and / or incubating the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the other precursors are present intracellularly, and the cells are genetically modified to (over)express a polynucleotide sequence encoding a sugar importer that internalizes one of the extracellular precursors into the cells to be used for the production of one or both of the bioproducts. In a more preferred embodiment, the cells produce two bioproducts and three or more precursors each used in the production of one or both of the bioproducts, wherein two of the precursors are present extracellularly by any one or more of the following means: passive transport outside the cells during and / or at the end of culturing and / or incubating the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the other precursors are present intracellularly, and the cells are genetically modified to (over)express a polynucleotide sequence encoding a sugar importer that internalizes both of the extracellular precursors into the cells to be used for the production of one or both of the bioproducts. In another more preferred embodiment, the cells produce two bioproducts and three or more precursors each used in the production of one or both of the bioproducts, wherein two of the precursors are present extracellularly by any one or more of the following means: passive transport outside the cells during and / or at the end of culturing and / or incubating the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the other precursors are present intracellularly, and the cells are genetically modified to (over)express two polynucleotide sequences each encoding a different sugar importer, wherein the first sugar importer internalizes one of the extracellular precursors and the second sugar importer internalizes the other of the extracellular precursors into the cells to be used for the production of the bioproducts.

[0112] In another preferred embodiment of the cells and / or methods of the present invention, the cells produce two bioproducts and three or more precursors each used in the production of one or both of the bioproducts, wherein at least three of the precursors are present outside the cells by any one or more of the following means: passive transport outside the cells during and / or at the end of the culturing and / or incubation of the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the other precursors (if any) are present inside the cells, and the cells are genetically modified to (over)express a polynucleotide sequence encoding a sugar importer that internalizes one of the extracellular precursors into the cells to be used for the production of one or both of the bioproducts. In a more preferred embodiment, the cells produce two bioproducts and three or more precursors each used in the production of one or both of the bioproducts, wherein at least three of the precursors are present outside the cells by any one or more of the following means: passive transport outside the cells during and / or at the end of the culturing and / or incubation of the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the other precursors (if any) are present inside the cells, and the cells are genetically modified to (over)express a polynucleotide sequence encoding a sugar importer that internalizes two of the extracellular precursors into the cells to be used for the production of one or both of the bioproducts. In an even more preferred embodiment, the cells produce two bioproducts and three or more precursors each used in the production of one or both of the bioproducts, wherein at least three of the precursors are present outside the cells by any one or more of the following means: passive transport outside the cells during and / or at the end of the culturing and / or incubation of the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the other precursors (if any) are present inside the cells, and the cells are genetically modified to (over)express a polynucleotide sequence encoding a sugar importer that internalizes three or more (if any) of the extracellular precursors into the cells to be used for the production of one or both of the bioproducts. In another more preferred embodiment, the cells produce two bioproducts and three or more precursors each used in the production of one or both of the bioproducts, wherein at least three of the precursors are present outside the cells by any one or more of the following means: passive transport outside the cells during and / or at the end of the culturing and / or incubation of the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the other precursors (if any) are present inside the cells, and the cells are genetically modified to (over)express two or more polynucleotide sequences encoding two or more different sugar importers that internalize three or more (if any) of the extracellular precursors into the cells to be used for the production of one or both of the bioproducts.

[0113] In another preferred embodiment of the cells and / or methods of the present invention, the cells produce three or more biological products and a precursor used in the production of one, two, three, more than three (if present) or all of the biological products, wherein the precursor is present outside the cells by any one or more of the following: passive transport outside the cells during and / or at the end of the culturing and / or incubation of the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes the precursor into the cells to be used in the production of one, two, three, more than three (if present) or all of the biological products.

[0114] In another preferred embodiment of the cells and / or methods of the present invention, the cells produce three or more biological products and two precursors used in the production of one, two, three, more than three (if present) or all of the biological products, wherein one of the precursors is present outside the cells by any one or more of the following: passive transport outside the cells during and / or at the end of the culturing and / or incubation of the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and wherein the other of the two precursors is present inside the cells, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes the extracellular precursor into the cells to be used in the production of one, two, three, more than three (if present) or all of the biological products.

[0115] In another preferred embodiment of the cells and / or methods of the present invention, the cells produce three or more biological products and two precursors used in the production of one, two, three, more than three (if present), or all of the biological products, wherein the two precursors are present extracellularly by any one or more of the following means: passive transport outside the cells during and / or at the end of the culturing and / or incubation of the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes one of the extracellular precursors into the cell for use in the production of one, two, three, more than three (if present), or all of the biological products. In a more preferred embodiment, the cells produce three or more biological products and two precursors used in the production of one, two, three, more than three (if present), or all of the biological products, wherein the two precursors are present extracellularly by any one or more of the following means: passive transport outside the cells during and / or at the end of the culturing and / or incubation of the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes both of the extracellular precursors into the cell for use in the production of one, two, three, more than three (if present), or all of the biological products. In another more preferred embodiment, the cells produce three or more biological products and two precursors used in the production of one, two, three, more than three (if present), or all of the biological products, wherein the two precursors are present extracellularly by any one or more of the following means: passive transport outside the cells during and / or at the end of the culturing and / or incubation of the cells, active transport outside the cells, secretion, excretion, and / or cell lysis, and the cells are genetically engineered to (over)express two polynucleotide sequences each encoding a different sugar importer, wherein the first sugar importer internalizes one of the extracellular precursors and the second sugar importer internalizes the other of the extracellular precursors into the cell for use in the production of one, two, three, more than three (if present), or all of the biological products.

[0116] In another preferred embodiment of the cells and / or methods of the present invention, the cells produce three or more biological products and three or more precursors used in the production of one, two, three, more than three (if present) or all of the biological products, wherein one of the precursors is present outside the cell by any one or more of the following means: passive transport outside the cell during and / or at the end of the cultivation and / or incubation of the cells, active transport outside the cell, secretion, excretion and / or cell lysis, and the other precursors are present inside the cell, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes the extracellular precursor into the cell to be used for the production of one, two, three, more than three (if present) or all of the biological products.

[0117] In another preferred embodiment of the cell and / or method of the present invention, the cell produces three or more biological products and three or more precursors used in the production of one, two, three, more than three (if any), or all of the biological products, wherein two of the precursors are present outside the cell by any one or more of the following means: passive transport outside the cell during and / or at the end of culturing and / or incubating the cell, active transport outside the cell, secretion, excretion, and / or cell lysis, and the other precursors are present inside the cell, and the cell is genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes one of the extracellular precursors into the cell to be used in the production of one, two, three, more than three (if any), or all of the biological products. In a more preferred embodiment, the cell produces three or more biological products and three or more precursors used in the production of one, two, three, more than three (if any), or all of the biological products, wherein two of the precursors are present outside the cell by any one or more of the following means: passive transport outside the cell during and / or at the end of culturing and / or incubating the cell, active transport outside the cell, secretion, excretion, and / or cell lysis, and the other precursors are present inside the cell, and the cell is genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes both of the extracellular precursors into the cell to be used in the production of one, two, three, more than three (if any), or all of the biological products. In another more preferred embodiment, the cell produces three or more biological products and three or more precursors used in the production of one, two, three, more than three (if any), or all of the biological products, wherein two of the precursors are present outside the cell by any one or more of the following means: passive transport outside the cell during and / or at the end of culturing and / or incubating the cell, active transport outside the cell, secretion, excretion, and / or cell lysis, and the other precursors are present inside the cell, and the cell is genetically engineered to (over)express two polynucleotide sequences each encoding a different sugar importer, wherein the first sugar importer internalizes one of the extracellular precursors and the second sugar importer internalizes the other of the extracellular precursors into the cell to be used in the production of one, two, three, more than three (if any), or all of the biological products.

[0118] In another preferred embodiment of the cells and / or methods of the present invention, the cells produce three or more biological products and three or more precursors used in the production of one, two, three, more than three (if present) or all of the biological products, wherein at least three of the precursors are present extracellularly by any one or more of the following means: passive transport outside the cells during and / or at the end of the culturing and / or incubation of the cells, active transport outside the cells, secretion, excretion and / or cell lysis, and the other precursors (if present) are present intracellularly, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes one of the extracellular precursors into the cells to be used in the production of one, two, three, more than three (if present) or all of the biological products. In a more preferred embodiment, the cells produce three or more biological products and three or more precursors used in the production of one, two, three, more than three (if present) or all of the biological products, wherein at least three of the precursors are present extracellularly by any one or more of the following means: passive transport outside the cells during and / or at the end of the culturing and / or incubation of the cells, active transport outside the cells, secretion, excretion and / or cell lysis, and the other precursors (if present) are present intracellularly, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes two of the extracellular precursors into the cells to be used in the production of one, two, three, more than three (if present) or all of the biological products. In an even more preferred embodiment, the cells produce three or more biological products and three or more precursors used in the production of one, two, three, more than three (if present) or all of the biological products, wherein at least three of the precursors are present extracellularly by any one or more of the following means: passive transport outside the cells during and / or at the end of the culturing and / or incubation of the cells, active transport outside the cells, secretion, excretion and / or cell lysis, and the other precursors (if present) are present intracellularly, and the cells are genetically engineered to (over)express a polynucleotide sequence encoding a sugar importer that internalizes three or more (if present) of the extracellular precursors into the cells to be used in the production of one, two, three, more than three (if present) or all of the biological products.In another more preferred embodiment, the cell produces three or more biological products and three or more precursors used in the production of one, two, three, more than three (if present), or all of the biological products, wherein at least three of the precursors are present extracellularly by any one or more of the following: passive transport outside the cell during and / or at the end of the culturing and / or incubation of the cell, active transport outside the cell, secretion, excretion, and / or cell lysis, and the other precursors (if present) are present intracellularly, and the cell is genetically engineered to (over)express two or more polynucleotide sequences encoding two or more different sugar importers that internalize three or more (if present) of the extracellular precursors into the cell for use in the production of one, two, three, more than three (if present), or all of the biological products.

[0119] Preferably, the cell is genetically engineered to produce at least one of the one or more precursors used in the production of at least one of the one or more biological products. More preferably, the cell is genetically engineered to produce all of the one or more precursors used in the production of at least one of the one or more biological products.

[0120] In a particular embodiment of the invention, the cell is genetically engineered to express, preferably overexpress, a polynucleotide sequence encoding a sugar importer that internalizes at least one of the one or more precursors into the cell. In a preferred embodiment of the cells and / or methods of the invention, the sugar importer described herein internalizes at least two of the one or more precursors into the cell. In a more preferred embodiment, the sugar importer described herein internalizes all of the one or more precursors into the cell.

[0121] In another preferred embodiment of the cells and / or methods of the invention, the sugar importer has uptake activity with respect to lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc). The LN3 can be synthesized in the following ways: chemically, enzymatically by an in vitro glycosylation reaction, by chemoenzymatic synthesis, by fermentation methods, and / or by chemical, physical, and / or biological degradation of polysaccharides, as is well known to those skilled in the art. Preferably, the LN3 is produced by a cell. More preferably, the LN3 is produced by the cells of the invention.

[0122] In another and / or additional preferred embodiment of the cells and / or methods of the invention, one of the one or more precursors produced by the cell is LN3.

[0123] In another and / or further preferred embodiment of the cells and / or methods of the present invention, one of the one or more precursors produced by the cells is LN3, and the sugar importer encoded by the polynucleotide sequence has uptake activity with respect to the LN3 produced by the cells in the cells genetically engineered to express, preferably overexpress, the polynucleotide sequence.

[0124] According to a second aspect, the present invention provides cells for producing one or more biological products described herein, wherein the cells are genetically engineered to express, preferably overexpress, a polynucleotide sequence encoding a sugar importer having uptake activity with respect to lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc).

[0125] In a preferred embodiment of the cells and / or methods of the present invention, the sugar importer described herein is derived from the major facilitator superfamily (MFS) of transporters.

[0126] In another and / or further preferred embodiment of the cells and / or methods of the present invention, the sugar importer described herein comprises a polypeptide sequence comprising IPR domains selected from the list comprising: IPR001927, IPR002178, IPR016152, IPR018043, IPR020846, IPR036259 and IPR039672 as defined by InterPro 90.0 released on August 4, 2022. In a more preferred embodiment of the cells and / or methods of the present invention, the sugar importer described herein comprises a polypeptide sequence comprising the IPR001927 domain as defined by InterPro 90.0 released on August 4, 2022.

[0127] In another and / or further preferred embodiment of the cells and / or methods of the present invention, the sugar importer described herein comprises a polypeptide sequence comprising the PF13347 domain and / or the PF00359 domain as defined by PFAM 32.0 released in September 2018.

[0128] In another and / or further preferred embodiment of the cells and / or methods of the present invention, the sugar importer described herein comprises a polypeptide sequence comprising PANTHER domains selected from the list comprising: PTHR11328, PTHR11328:SF24, PTHR11328:SF36 and PTHR11328:SF39 as defined by PANTHER 18.0 released on September 17, 2023.

[0129] In another and / or additional preferred embodiment of the cells and / or methods of the present invention, the sugar importer described herein comprises a polypeptide sequence that comprises the cd17332 domain and / or the cd00211 domain as defined by the Conserved Domain Database CDD 3.20 released in September 2022.

[0130] In another and / or additional preferred embodiment of the cells and / or methods of the present invention, the sugar importers described herein comprise a polypeptide sequence that is at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% identical to any one of the polypeptide sequences shown by SEQ ID NO: 04, 12, 02, 01, 03, 09, 16, 19, 20 or 21 over a segment of at least 50 amino acid residues, at least 100 amino acid residues, at least 150 amino acid residues, at least 200 amino acid residues, at least 250 amino acid residues.In a more preferred embodiment, the sugar importer of the present invention comprises a polypeptide sequence which is at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to any one of the polypeptide sequences shown by SEQ ID NO:04, 12, 02, 01, 03, 09, 16, 19, 20 or 21 on a segment of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues.

[0131] In another and / or additional preferred embodiment of the cells and / or methods of the present invention, the sugar importer described herein comprises a polypeptide sequence that is at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to any of the full-length polypeptide sequences shown by SEQ ID NO:04, 12, 02, 01, 03, 09, 16, 19, 20 or 21, and has the sugar importer activity described herein.

[0132] In another and / or additional preferred embodiment of the cells and / or methods of the present invention, the sugar importer described herein comprises a polypeptide sequence shown by any of SEQ ID NO:04, 12, 02, 01, 03, 09, 16, 19, 20 or 21.

[0133] In another and / or further preferred embodiment of the cells and / or methods of the present invention, the sugar importer described herein has uptake activity with respect to LN3 and comprises a polypeptide sequence having a deletion, insertion, and / or mutation of one or more amino acid residues at the following positions: i) the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO: 12, 02, 01, 09, 19, 20, or 21, and / or ii) the C-terminus of the last TM domain of any one of SEQ ID NO: 12, 02, 01, 09, 19, 20, or 21. In a more preferred embodiment, the sugar importer has uptake activity with respect to LN3 and comprises a polypeptide sequence having a deletion, insertion, and / or mutation of one or more amino acid residues at the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO: 12, 02, 01, 09, 19, 20, or 21, wherein the deletion, insertion, and / or mutation of the one or more amino acid residues is in a non-TM helix or at the N-terminus of a non-TM helix.

[0134] In another and / or further more preferred embodiment, the sugar importer of the present invention has uptake activity with respect to LN3 and comprises a polypeptide sequence having a deletion, insertion, and / or mutation of one or more amino acid residues at the C-terminus of the last TM domain of any one of SEQ ID NO: 12, 02, 01, 09, 19, 20, or 21, wherein the deletion, insertion, and / or mutation of the one or more amino acid residues is in a non-TM helix or at the C-terminus of a non-TM helix.

[0135] Preferably, a variant sugar importer having a deletion, insertion, and / or mutation of one or more amino acid residues at i) the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO: 12, 02, 01, 09, 19, 20, or 21 and / or ii) the C-terminus of the last TM domain of any one of SEQ ID NO: 12, 02, 01, 09, 19, 20, or 21 has a higher, faster, and / or more efficient (i.e., lower energy-consuming) uptake ratio of the sugar compared to the native sugar importer lacking the deletion, insertion, and / or mutation.

[0136] In another preferred embodiment of the cells and / or methods of the present invention, cells for producing one or more of the biological products described herein and expressing a variant sugar importer produce a higher titer of the biological product, exhibit a higher purity of the biological product, have a higher production rate, have a higher cell performance index, have a higher specific productivity, and / or have a higher growth rate compared to cells having the same genetic composition but expressing any one of native SEQ ID NO:12, 02, 01, 09, 19, 20 or 21, wherein the variant sugar importer has i) a deletion, insertion and / or mutation of one or more amino acid residues at the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO:12, 02, 01, 09, 19, 20 or 21 and / or ii) at the C-terminus of the last TM domain of any one of SEQ ID NO:12, 02, 01, 09, 19, 20 or 21.

[0137] In a more preferred embodiment of the present invention, the sugar importer described herein comprises, consists of or consists essentially of a polypeptide sequence having SEQ ID NO:03 and has uptake activity with respect to LN3; SEQ ID NO:03 is a mutant polypeptide of the sugar importer having SEQ ID NO:02, wherein SEQ ID NO:03 has a deletion of 39 amino acid residues at the N-terminus of the first TM domain of SEQ ID NO:02 and an insertion of 19 amino acid residues at the N-terminus of the first TM domain of SEQ ID NO:02. The first TM domain of SEQ ID NO:02 starts at Ser at position 40. Both polypeptides having SEQ ID NO:02 and 03 have uptake activity with respect to LN3. In an even more preferred embodiment, the sugar importer described herein is a polypeptide having SEQ ID NO:03 and has uptake activity with respect to LN3.

[0138] In another more preferred embodiment of the present invention, the sugar importer described herein comprises, consists of, or consists essentially of a polypeptide sequence having SEQ ID NO:04 and has an uptake activity with respect to LN3; SEQ ID NO:04 is a mutant polypeptide of the sugar importer having SEQ ID NO:02, wherein said SEQ ID NO:04 has a deletion of 39 amino acid residues at the N-terminus of the first TM domain of SEQ ID NO:02 and an insertion of 19 amino acid residues at the N-terminus of the first TM domain of SEQ ID NO:02. The first TM domain of SEQ ID NO:02 starts at Ser at position 40. Both the polypeptides having SEQ ID NO:02 and 04 have an uptake activity with respect to LN3. In a more preferred embodiment, the sugar importer described herein is a polypeptide having SEQ ID NO:04 and has an uptake activity with respect to LN3.

[0139] In another more preferred embodiment of the present invention, the sugar importer has an uptake activity with respect to LN3 and comprises a polypeptide sequence having any one of SEQ ID NO:16, which comprises a mutation of one or more amino acid residues at the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO:09. SEQ ID NO:16 is a mutant polypeptide of the sugar importer having SEQ ID NO:09, wherein said SEQ ID NO:16 has a deletion of 23 amino acid residues at the N-terminus of the first TM domain of SEQ ID NO:09 and an insertion of 20 amino acid residues at the N-terminus of the first TM domain of SEQ ID NO:09. The first TM domain of SEQ ID NO:09 starts at Val at position 24. In a more preferred embodiment, the sugar importer described herein is a polypeptide having SEQ ID NO:16 and has an uptake activity with respect to LN3.

[0140] A polypeptide sequence having one or more amino acid residue deletions, insertions, and / or mutations compared to a reference polypeptide sequence can be obtained by expressing a mutant form of a polynucleotide sequence encoding the reference polypeptide sequence. The mutant form of the polynucleotide sequence can contain one or more nucleotide deletions, one or more nucleotide insertions, a frameshift of one or more nucleotides in the coding sequence, and / or one or more single nucleotide polymorphisms (SNPs) compared to the native polynucleotide sequence. The mutant form of the polynucleotide sequence can be obtained by techniques well known to those skilled in the art, such as but not limited to PCR cloning; TA cloning; ligation-independent cloning (LIC); seamless ligation cloning extract (SLICE); mating-assisted genetically integrated cloning (MAGIC); sequence and ligation independent cloning (SLIC); In-Fusion; Gibson assembly; polymerase incomplete primer extension (PIPE); circular polymerase extension cloning (CPEC); site-directed mutagenesis; CrispR; riboswitch; recombineering; ssDNA mutagenesis; transposon mutagenesis.

[0141] In another and / or additional preferred embodiment of the present invention, the sugar importer described herein is derived from the major facilitator superfamily (MFS) of transporters and comprises a polypeptide sequence comprising the IPR001927 domain as defined by InterPro90.0 released on August 4, 2022, and a polypeptide sequence comprising 12 transmembrane (TM) domains having a conserved domain shown as [AGSV][HNQ][ACDEGNQSTV]XX[FWY]XXXXX (non-L) present in the first TM domain, where X can be any amino acid residue, and the second amino acid residue of SEQ ID NO:17 is aligned with Lys18 of the polypeptide having SEQ ID NO:22.

[0142] In a more preferred embodiment, the sugar importer described herein is derived from the major facilitator superfamily (MFS) of transporters and comprises a polypeptide sequence that includes the IPR001927 domain as defined by InterPro 90.0 released on August 4, 2022, and a polypeptide sequence that includes 12 transmembrane (TM) domains having a conserved domain [AGS]Q[ACGNQSTV]XX[FWY] shown in SEQ ID NO:18 present in the first TM domain, where X can be any amino acid residue, and where the second amino acid residue of SEQ ID NO:18 aligns with Lys18 of the polypeptide having SEQ ID NO:22.

[0143] In another and / or additional preferred embodiment of the present invention, the sugar importer described herein further has uptake activity with respect to one or more other sugars different from LN3, where the one or more other sugars different from LN3 are selected from the list including monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

[0144] In another and / or additional preferred embodiment of the present invention, the sugar importer described herein has uptake activity with respect to LN3, but does not have uptake activity with respect to LNT (lacto-N-tetraose, Galβ1-3GlcNAcβ1-3Galβ1-4Glc).

[0145] In another and / or additional preferred embodiment of the present invention, the sugar importer described herein has uptake activity with respect to LN3, but does not have uptake activity with respect to LNnT (lacto-N-neotetraose, Galβ1-4GlcNAcβ1-3Galβ1-4Glc). In a more preferred embodiment of the present invention, the sugar importer described herein has uptake activity with respect to LN3, but does not have uptake activity with respect to LNT and with respect to LNnT.

[0146] Within the scope of the present invention, the cell is genetically engineered to express, preferably overexpress, a polynucleotide sequence encoding the sugar importer described herein.

[0147] In a preferred embodiment of the cell and / or method, the cell is modified with one or more expression modules. The expression module, also referred to as a transcription unit, comprises a polynucleotide for expressing a recombinant gene, which includes a coding gene sequence and suitable transcriptional and / or translational control signals operably linked to the coding sequence. The control signals include a promoter sequence, untranslated regions, ribosome binding sites, terminator sequences. The expression module may contain elements for expressing a single recombinant gene but may also contain elements for expressing multiple recombinant genes, or may be organized into an operon structure for the integrated expression of two or more recombinant genes. The polynucleotide can be produced by recombinant DNA techniques using well-known techniques in the art. Methods well known to those skilled in the art for constructing expression modules include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al., (2001) Molecular Cloning: a laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press, CSH, New York or Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989 and annual updates).

[0148] Expression of each of the expression modules can be constitutive or induced by natural or chemical inducers. As used herein, constitutive expression should be understood as the expression of a gene that is continuously transcribed in an organism. Expression induced by a natural inducer should be understood as facultative or regulated expression of a gene that is expressed only under certain natural conditions of the host (e.g., the organism is in labor, or during lactation), as a response to environmental changes (e.g., including but not limited to, hormones, heat, cold, pH shift, light, oxidative or osmotic stress / signal transduction), or depending on the developmental stage or position in the cell cycle of the host cell (including but not limited to, apoptosis and autophagy). Expression induced by a chemical inducer should be understood as facultative or regulated expression of a gene that is expressed only upon sensing an external chemical (e.g., IPTG, arabinose, lactose, allolactose, rhamnose or fucose) via an inducible promoter or via a genetic circuit that induces or represses transcription or translation of the polynucleotide into a polypeptide.

[0149] The expression module can be integrated into the genome of the cell or can be presented to the cell on a vector. The vector can be in the form of a plasmid, cosmid, phage, liposome, or virus to be stably transformed / transfected into the genetically modified cell. Such vectors include, in particular, chromosomal, episomal, and virus-derived vectors, such as vectors derived from bacterial plasmids, phages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses, and vectors derived from combinations thereof, such as those derived from plasmid and phage genetic elements (e.g., cosmids and phagemids). These vectors can contain selectable markers, such as, but not limited to, antibiotic markers, auxotrophic markers, toxin-antitoxin markers, RNA sense / antisense markers. The expression system construct can contain regulatory and control regions that cause expression. Generally, any system or vector suitable for maintaining, propagating, or expressing polynucleotides and / or expressing polypeptides in a host can be used for expression in this regard. Suitable DNA sequences can be inserted into the expression system by any of a variety of well-known and conventional techniques (e.g., those set forth in Sambrook et al. (see above)). For recombinant production, cells can be genetically modified to incorporate the expression system of the invention or portions or polynucleotides thereof. Introduction of polynucleotides into cells can be effected by methods described in many standard laboratory manuals (e.g., Davis et al., Basic Methods in Molecular Biology, (1986); and Sambrook et al., 1989 (see above)).

[0150] As used herein, an expression module comprises a polynucleotide for expressing at least one recombinant gene. The recombinant gene is involved in the expression of a polypeptide that plays a role in the synthesis of one or more biological products of the invention; or the recombinant gene is associated with other pathways in the cell that are not involved in the synthesis of the one or more biological products. The recombinant gene encodes an endogenous protein with modified expression or activity, preferably the endogenous protein is overexpressed; or the recombinant gene encodes a heterologous protein that is heterologously introduced and expressed (preferably, overexpressed) in the modified cell. The endogenous protein can have modified expression in a cell that also expresses a heterologous protein.

[0151] In another and / or additional preferred embodiment of the cells and / or methods of the invention, the expression of each of the expression modules present in the genetically modified cell is constitutive or coordinatable, as described herein.

[0152] In another and / or further preferred embodiment of the cell and / or method, the polynucleotide sequence encoding the sugar importer is operably linked to a control sequence recognized by the cell. In another and / or further preferred embodiment of the cell and / or method, the polynucleotide sequence encoding the sugar importer is foreign to the cell. In a further preferred embodiment, the polynucleotide sequence is integrated into the genome of the cell and / or presented to the cell on a vector.

[0153] In another and / or further preferred embodiment of the cell and / or method of the present invention, the cell is capable of being produced from one or more precursors described herein and preferably produces the one or more bioproducts. In a more preferred embodiment, the cell is capable of producing, preferably producing at least one of the one or more precursors for producing the one or more bioproducts. In an even more preferred embodiment, the cell is capable of producing, preferably producing all of the one or more precursors.

[0154] In a further preferred embodiment, the cell is genetically engineered to produce at least one of the one or more precursors for producing the one or more bioproducts. In a further more preferred embodiment, the cell is genetically engineered to produce all of the one or more precursors.

[0155] In another and / or further preferred embodiment, at least one of the one or more precursors produced by the cell of the present invention and used by the cell to produce the one or more bioproducts described herein is internalized into the cell via the sugar importer described herein.

[0156] In another and / or further preferred embodiment of the cell and / or method of the present invention, the cell is further genetically engineered to produce one or more bioproducts described herein.

[0157] In another and / or further preferred embodiment of the cell and / or method of the present invention, the cell comprises one or more pathways for monosaccharide synthesis. The pathways for monosaccharide synthesis include enzymes such as carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, kinases, phosphatases, aldolases, hydrolases, dehydrogenases, enzymes involved in the synthesis of any one or more nucleoside triphosphates (e.g., UTP, GTP, ATP, and CTP), enzymes involved in the synthesis of any one or more nucleoside mono- or diphosphates (e.g., UMP and UDP, respectively), and enzymes involved in the synthesis of phosphoenolpyruvate (PEP). More preferably, the cell is genetically engineered to produce one or more monosaccharides.

[0158] In another and / or additional preferred embodiment of the cells and / or methods of the present invention, the cells comprise one or more pathways for the synthesis of phosphorylated monosaccharides. The pathways for the synthesis of phosphorylated monosaccharides comprise enzymes involved in the synthesis of one or more monosaccharides, one or more nucleoside mono-, di- and / or tri-phosphates, and enzymes involved in the synthesis of phosphoenolpyruvate (PEP), such as but not limited to PEP synthase, carboxylase, decarboxylase, isomerase, epimerase, reductase, enolase, phosphorylase, carboxykinase, kinase, phosphatase, aldolase, hydrolase, and dehydrogenase. Preferably, the cells are genetically engineered to produce one or more phosphorylated monosaccharides.

[0159] In another and / or additional preferred embodiment of the cells and / or methods of the present invention, the cells are capable of producing, preferably producing, one or more nucleotide-activated sugars, which are preferably selected from the list consisting of: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose (GDP-Fuc), UDP-glucuronic acid, UDP-galacturonic acid, UDP-2-acetamido-2,6-dideoxy-α-L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy-α-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), CMP-sialic acid (CMP-Neu5Ac), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), GDP-rhamnose, and UDP-xylose. Preferably, the cells comprise one or more pathways for synthesizing one or more of the nucleotide-activated sugars.The pathway for nucleotide-activated sugar synthesis comprises enzymes such as the following: PEP synthase, carboxylase, decarboxylase, isomerase, epimerase, reductase, enolase, phosphorylase, carboxykinase, kinase, phosphatase, aldolase, hydrolase, dehydrogenase, mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, L-fucose kinase / GDP-fucose pyrophosphorylase, L-glutamine-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylglucosamine-6P2-epimerase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, N-acetylmannosamine-6-phosphate 2-epimerase, N-acetylmannosamine-6-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine-1-phosphate uridylyltransferase, glucosamine-1-phosphate acetyltransferase, sialic acid synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphate phosphatase, CMP-sialic acid synthase, galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridylyltransferase, and / or phosphoglucomutase. In a more preferred embodiment of the cells and / or methods of the present invention, the cells use at least one of the nucleotide-activated sugars so produced for the production of one or more biological products of the present invention. In an even more preferred embodiment of the cells and / or methods of the present invention, the cells are genetically engineered to produce one or more of the nucleotide-activated sugars.

[0160] The cells used herein are optionally genetically engineered to express de novo synthesis of UDP-GlcNAc. UDP-GlcNAc can be provided by enzymes expressed in the cells or by the metabolism of the cells. Such cells that produce UDP-GlcNAc can express enzymes that convert, for example, GlcNAc (which is to be added to the cells) into UDP-GlcNAc. These enzymes can be any one or more of the following list: N-acetyl-D-glucosamine kinase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, and N-acetylglucosamine-1-phosphate uridylyltransferase / glucosamine-1-phosphate acetyltransferase, which are from several species, including Homo sapiens and Escherichia coli. Preferably, the cells are modified to produce UDP-GlcNAc. More preferably, the cells are modified for enhanced UDP-GlcNAc production. The modification can be any one or more selected from the group including the following: knockout of N-acetylglucosamine-6-phosphate deacetylase, overexpression of L-glutamine-D-fructose-6-phosphate aminotransferase, overexpression of phosphoglucosamine mutase, and overexpression of N-acetylglucosamine-1-phosphate uridylyltransferase / glucosamine-1-phosphate acetyltransferase.

[0161] Additionally or alternatively, the cells used herein are optionally genetically engineered to express de novo synthesis of CMP-Neu5Ac. CMP-Neu5Ac can be provided by enzymes expressed in the cells or by the metabolism of the cells. Such cells that produce CMP-Neu5Ac can express enzymes that convert, for example, sialic acid into CMP-Neu5Ac. The enzyme can be CMP-sialic acid synthase, such as N-acylneuraminic acid cytidylyltransferase from several species, including Homo sapiens, Neisseria meningitidis, and Pasteurella multocida. Preferably, the cells are modified to produce CMP-Neu5Ac. More preferably, the cells are modified for enhanced CMP-Neu5Ac production. The modification can be any one or more selected from the group including the following: knockout of N-acetylglucosamine-6-phosphate deacetylase, knockout of glucosamine-6-phosphate deaminase, overexpression of CMP-sialic acid synthase, and overexpression of the N-acetyl-D-glucosamine-2-epimerase-encoding gene.

[0162] Additionally or alternatively, the cells used herein are optionally genetically engineered to express de novo synthesis of GDP-fucose. GDP-fucose can be provided by an enzyme expressed in the cell or by the cell's metabolism. Such cells that produce GDP-fucose can express an enzyme that converts, for example, fucose (which is to be added to the cell) into GDP-fucose. The enzyme can be, for example, a bifunctional fucose kinase / fucose-1-phosphate guanylyltransferase, such as Fkp from Bacteroides fragilis, or a combination of a separate fucose kinase and a separate fucose-1-phosphate guanylyltransferase, as known from several species, including Homo sapiens, Sus scrofa, and Rattus norvegicus. Preferably, the cells are modified to produce GDP-fucose. More preferably, the cells are modified for enhanced GDP-fucose production. The modification can be any one or more selected from the group consisting of: knockout of the UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase encoding gene, overexpression of the GDP-L-fucose synthase encoding gene, overexpression of the GDP-mannose 4,6-dehydratase encoding gene, overexpression of the mannose-1-phosphate guanylyltransferase encoding gene, overexpression of the phosphomannomutase encoding gene, and overexpression of the mannose-6-phosphate isomerase encoding gene.

[0163] Additionally or alternatively, the cells used herein are optionally genetically engineered to express de novo synthesis of UDP-Gal. UDP-Gal can be provided by an enzyme expressed in the cell or by the cell's metabolism. Such cells that produce UDP-Gal can express an enzyme that converts, for example, UDP-glucose into UDP-Gal. The enzyme can be, for example, UDP-glucose-4-epimerase GalE, as known from several species, including Homo sapiens, Escherichia coli, and Rattus norvegicus. Preferably, the cells are modified to produce UDP-Gal. More preferably, the cells are modified for enhanced UDP-Gal production. The modification can be any one or more selected from the group consisting of: knockout of the bifunctional 5'-nucleotidase / UDP-sugar hydrolase encoding gene, knockout of the galactose-1-phosphate uridylyltransferase encoding gene, and overexpression of the UDP-glucose-4-epimerase encoding gene.

[0164] Additionally or alternatively, the cells used herein are optionally genetically engineered to express de novo synthesis of UDP-GalNAc. UDP-GalNAc can be synthesized from UDP-GlcNAc by the action of a one-step reaction using UDP-N-acetylglucosamine 4-epimerase, such as wbgU from Plesiomonas shigelloides, gne from Yersinia enterocolitica, or wbpP from Pseudomonas aeruginosa serotype O6. Preferably, the cells are modified to produce UDP-GalNAc. More preferably, the cells are modified for enhanced UDP-GalNAc production.

[0165] Additionally or alternatively, the cells used herein are optionally genetically engineered to express de novo synthesis of UDP-ManNAc. UDP-ManNAc can be directly synthesized from UDP-GlcNAc via an epimerization reaction by UDP-GlcNAc 2-epimerase (e.g., cap5P from Staphylococcus aureus, RffE from Escherichia coli, Cps19fK from Streptococcus pneumoniae, and RfbC from Salmonella enterica). Preferably, the cells are modified to produce UDP-ManNAc. More preferably, the cells are modified for enhanced UDP-ManNAc production.

[0166] In another preferred embodiment of the cells and / or methods according to the invention, the cells have, preferably express, more preferably overexpress one or more glycosyltransferases selected from the list consisting of: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase, N-acetylmannosaminyltransferase, xylosyltransferase, glucuronosyltransferase, galacturonosyltransferase, glucosaminyltransferase, N-glycolylneuraminyltransferase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine transaminase, UDP-N-acetylglucosamine enolpyruvate transferase, and fucosaminyltransferase.

[0167] In a preferred embodiment of the cells and / or methods of the present invention, the fucosyltransferase is selected from the list consisting of: α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,3 / 4-fucosyltransferase, α-1,4-fucosyltransferase, and α-1,6-fucosyltransferase.

[0168] In an alternative and / or additional embodiment of the methods and / or cells of the present invention, the sialyltransferase is selected from the list consisting of: α-2,3-sialyltransferase, α-2,6-sialyltransferase, and α-2,8-sialyltransferase.

[0169] In an alternative and / or additional embodiment of the cells and / or methods of the present invention, the galactosyltransferase is selected from the list consisting of: β-1,3-galactosyltransferase, N-acetylglucosamine β-1,3-galactosyltransferase, β-1,4-galactosyltransferase, N-acetylglucosamine β-1,4-galactosyltransferase, α-1,3-galactosyltransferase, and α-1,4-galactosyltransferase.

[0170] In an alternative and / or additional embodiment of the cells and / or methods of the present invention, the glucosyltransferase is selected from the list consisting of: α-glucosyltransferase, β-1,2-glucosyltransferase, β-1,3-glucosyltransferase, and β-1,4-glucosyltransferase.

[0171] In an alternative and / or additional embodiment of the cells and / or methods of the present invention, the mannosyltransferase is selected from the list consisting of: α-1,2-mannosyltransferase, α-1,3-mannosyltransferase, and α-1,6-mannosyltransferase.

[0172] In an alternative and / or additional embodiment of the cells and / or methods of the present invention, the N-acetylglucosaminyltransferase is selected from the list consisting of: galactoside β-1,3-N-acetylglucosaminyltransferase and β-1,6-N-acetylglucosaminyltransferase.

[0173] In an alternative and / or additional embodiment of the cells and / or methods of the present invention, the N-acetylgalactosaminyltransferase is selected from the list consisting of: α-1,3-N-acetylgalactosaminyltransferase.

[0174] In a further embodiment of the cells and / or methods of the present invention, the cells are modified with respect to the expression or activity of at least one of the glycosyltransferases. In a preferred embodiment, the glycosyltransferase is an endogenous protein of the cell having modified expression or activity, preferably the endogenous glycosyltransferase is overexpressed; alternatively, the glycosyltransferase is a heterologous protein heterologously introduced and expressed (preferably, overexpressed) in the cell. The endogenous glycosyltransferase may have modified expression in a cell that also expresses a heterologous glycosyltransferase.

[0175] In another and / or further preferred embodiment of the cells and / or methods of the present invention, the cells comprise at least one pathway selected from the list consisting of: a fucosylation pathway, a sialylation pathway, a galactosylation pathway, a N-acetylglucosaminylation pathway, a N-acetylgalactosaminylation pathway, a mannosylation pathway, and a N-acetylmannosaminylation pathway, as described herein. In a more preferred embodiment, the cells are genetically engineered to comprise at least one of the pathways. In an even more preferred embodiment, the cells comprise at least one of the pathways, wherein at least one of the pathways has been genetically engineered.

[0176] In a more preferred embodiment, the cells comprise a sialylation pathway. The sialylation pathway is a biochemical pathway consisting of at least one of the enzymes and their respective genes selected from the list consisting of: L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, UDP-GlcNAc 2-epimerase / kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N-acetylglucosamine-1-phosphate uridylyltransferase, glucosamine-1-phosphate acetyltransferase, Neu5Ac synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphatase, sialic acid transporter, cytidine monophosphate (CMP) kinase, and CMP-sialic acid synthase, combined with a sialyltransferase, resulting in any one or more of α2,3, α2,6, and / or α2,8 sialylated oligosaccharides.

[0177] In a more preferred embodiment, the cell is genetically engineered to contain a sialylation pathway. In another more preferred embodiment, the cell has been genetically engineered to contain a sialylation pathway, wherein any one or more of the genes selected from the list consisting of: L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, UDP-GlcNAc 2-epimerase / kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-1-phosphate acetyltransferase, Neu5Ac synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphate phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, and sialyltransferase have modified and / or enhanced expression.

[0178] In another and / or additional preferred embodiment, the cell contains a fucosylation pathway. The fucosylation pathway is a biochemical pathway composed of at least one of the enzymes and their respective genes selected from the list consisting of: mannose-6-phosphate isomerase, phosphomannose mutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-1-phosphate guanylyltransferase, in combination with a fucosyltransferase, resulting in α1,2, α1,3, α1,4, and / or α1,6 fucosylated oligosaccharides.

[0179] In a more preferred additional and / or alternative embodiment, the cell is genetically engineered to contain a fucosylation pathway. In another more preferred additional and / or alternative embodiment, the cell has been genetically engineered to contain a fucosylation pathway, wherein any one or more of the genes selected from the list consisting of: mannose-6-phosphate isomerase, phosphomannose mutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-1-phosphate guanylyltransferase, and fucosyltransferase have modified and / or enhanced expression.

[0180] In another and / or additional preferred embodiment, the cell comprises a galactosylation pathway. A galactosylation pathway is a biochemical pathway consisting of at least one of the enzymes and their respective genes selected from the list comprising: galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridylyltransferase, phosphoglucomutase, in combination with a galactosyltransferase, resulting in a galactosylated compound comprising a monosaccharide, disaccharide or oligosaccharide having α or β-linked galactose on any one or more of the 2, 3, 4 and 6 hydroxyl groups of the monosaccharide, disaccharide or oligosaccharide.

[0181] In a more preferred additional and / or alternative embodiment, the cell is genetically engineered to comprise a galactosylation pathway. In another more preferred additional and / or alternative embodiment, the cell has been genetically engineered to comprise a galactosylation pathway, wherein any one or more of the genes selected from the list comprising: galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridylyltransferase, phosphoglucomutase and galactosyltransferase have modified and / or enhanced expression.

[0182] In another and / or additional preferred embodiment, the cell comprises an "N-acetylglucosaminylation" pathway. An N-acetylglucosaminylation pathway is a biochemical pathway consisting of at least one of the enzymes and their respective genes selected from the list comprising: L-glutamine-D-fructose-6-phosphate aminotransferase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N-acetylglucosamine-1-phosphate uridylyltransferase, glucosamine-1-phosphate acetyltransferase, in combination with a glycosyltransferase, resulting in a GlcNAc-modified compound comprising a monosaccharide, disaccharide or oligosaccharide having α or β-linked N-acetylglucosamine (GlcNAc) on any one or more of the 3, 4 and 6 hydroxyl groups of the monosaccharide, disaccharide or oligosaccharide.

[0183] In a further and / or alternative more preferred embodiment, the cell is genetically engineered to comprise an N - acetylglucosaminylation pathway. In another further and / or alternative more preferred embodiment, the cell has been genetically engineered to comprise an N - acetylglucosaminylation pathway, wherein any one or more of the genes selected from the list comprising: L - glutamine - D - fructose - 6 - phosphate aminotransferase, N - acetylglucosamine - 6 - phosphate deacetylase, phosphoglucosamine mutase, N - acetylglucosamine - 1 - phosphate uridylyltransferase, glucosamine - 1 - phosphate acetyltransferase, and glycosyltransferases that transfer GlcNAc have modified and / or enhanced expression.

[0184] In another and / or further preferred embodiment, the cell comprises a "galactosaminylation" pathway. The galactosaminylation pathway is a biochemical pathway consisting of at least one of the enzymes and their respective genes selected from the list comprising: L - glutamine - D - fructose - 6 - phosphate aminotransferase, phosphoglucosamine mutase, N - acetylglucosamine 1 - phosphate uridylyltransferase, glucosamine - 1 - phosphate acetyltransferase, UDP - N - acetylglucosamine 4 - epimerase, UDP - glucose 4 - epimerase, N - acetylgalactosamine kinase, and / or UDP - N - acetylgalactosamine pyrophosphorylase, combined with a glycosyltransferase, resulting in GalNAc - modified compounds containing monosaccharides, disaccharides, or oligosaccharides that have α or β - linked N - acetylgalactosamine on the monosaccharide, disaccharide, or oligosaccharide.

[0185] In a further and / or alternative more preferred embodiment, the cell is genetically engineered to comprise a galactosaminylation pathway. In another further and / or alternative more preferred embodiment, the cell has been genetically engineered to comprise a galactosaminylation pathway, wherein any one or more of the genes selected from the list comprising: L - glutamine - D - fructose - 6 - phosphate aminotransferase, phosphoglucosamine mutase, N - acetylglucosamine 1 - phosphate uridylyltransferase, glucosamine - 1 - phosphate acetyltransferase, UDP - N - acetylglucosamine 4 - epimerase, UDP - glucose 4 - epimerase, N - acetylgalactosamine kinase, and / or UDP - N - acetylgalactosamine pyrophosphorylase and glycosyltransferases that transfer GalNAc have modified and / or enhanced expression.

[0186] In another and / or additional preferred embodiment, the cell comprises a "mannosylation" pathway. The mannosylation pathway is a biochemical pathway composed of at least one of the enzymes and their respective genes selected from the list comprising: mannose-6-phosphate isomerase, phosphomannomutase and / or mannosyl-1-phosphate guanylyltransferase, combined with a mannosyltransferase, resulting in a mannosylated compound comprising a monosaccharide, disaccharide or oligosaccharide having an α or β-linked mannose on the monosaccharide, disaccharide or oligosaccharide.

[0187] In a more preferred additional and / or alternative embodiment, the cell is genetically engineered to comprise a mannosylation pathway. In another even more preferred additional and / or alternative embodiment, the cell has been genetically engineered to comprise a mannosylation pathway, wherein any one or more of the genes selected from the list comprising: mannose-6-phosphate isomerase, phosphomannomutase and / or mannosyl-1-phosphate guanylyltransferase and mannosyltransferase have modified and / or enhanced expression.

[0188] In another and / or additional preferred embodiment, the cell comprises an "N-acetylmannosaminylation" pathway. The N-acetylmannosaminylation pathway is a biochemical pathway composed of at least one of the enzymes and their respective genes selected from the list comprising: L-glutamine-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-1-phosphate uridyltransferase, glucosamine-1-phosphate acetyltransferase, glucosamine-1-phosphate acetyltransferase, UDP-GlcNAc 2-epimerase and / or ManNAc kinase, combined with a glycosyltransferase, resulting in a ManNAc-modified compound comprising a monosaccharide, disaccharide or oligosaccharide having an α or β-linked N-acetylmannosamine on the monosaccharide, disaccharide or oligosaccharide.

[0189] In a further and / or alternative more preferred embodiment, the cell is genetically engineered to contain an N-acetylmannosamine pathway. In another further and / or alternative more preferred embodiment, the cell has been genetically engineered to contain an N-acetylmannosamine pathway, wherein any one or more of the genes selected from the list comprising: L-glutamine-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-1-phosphate uridyltransferase, glucosamine-1-phosphate acetyltransferase, UDP-GlcNAc 2-epimerase and / or ManNAc kinase and glycosyltransferases that transfer ManNAc have modified and / or enhanced expression.

[0190] In another and / or further preferred embodiment, the cell is genetically engineered for enhanced production of one or more bioproducts, enhanced uptake of one or more precursors and / or acceptors used in the synthesis of one or more bioproducts, better efflux of one or more bioproducts, reduced production of by-products (e.g., acids), increased availability of cofactors (e.g., ATP, NADP, NADPH), and / or better metabolic flux through any of the sialylation, fucosylation, galactosylation, N-acetylglucosaminylation, N-acetylgalactosaminylation, mannosylation and / or N-acetylmannosaminylation pathways present in the cell.

[0191] In another preferred embodiment of the cell and / or method according to the invention, the cell comprises a catabolic pathway for a selected monosaccharide, disaccharide or oligosaccharide that is at least partially inactivated, the monosaccharide, disaccharide or oligosaccharide being involved in and / or required for the synthesis of one or more bioproducts.

[0192] In another preferred embodiment of the present invention, the cells are bacterial, fungal, yeast, plant, animal or protozoal cells. The latter bacteria preferably belong to the phylum Proteobacteria or Firmicutes or Cyanobacteria or Deinococcus-Thermus or Actinobacteria. The latter bacteria belonging to the phylum Proteobacteria preferably belong to the family Enterobacteriaceae, preferably to the species Escherichia coli. The latter bacteria preferably relate to any strain belonging to the species Escherichia coli, such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More particularly, the latter term relates to a cultured Escherichia coli strain - designated as the Escherichia coli K12 strain - which is well adapted to the laboratory environment and, unlike wild-type strains, has lost its ability to thrive in the gut. Well-known examples of the Escherichia coli K12 strain are K12 wild-type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200. Thus, the present invention particularly relates to mutated and / or transformed Escherichia coli cells or strains as indicated above, wherein the Escherichia coli strain is a K12 strain. More preferably, the Escherichia coli K-12 strain is Escherichia coli MG1655. The latter bacteria belonging to the phylum Firmicutes preferably belong to the genus Bacillus, preferably to the order Lactobacilliales, which has members such as Lactobacillus lactis, Leuconostoc mesenteroides, or to the order Bacillales, which has members such as those from the genus Bacillus, such as Bacillus subtilis or Bacillus amyloliquefaciens. The latter bacteria belonging to the phylum Actinobacteria preferably belong to the family Corynebacteriaceae, which has members Corynebacterium glutamicum or Corynebacterium afermentans, or to the family Streptomycetaceae, which has members Streptomyces griseus or Streptomyces fradiae.The bacterium belonging to the phylum Proteobacteria behind is preferably belonging to the family Vibrionaceae, which has a member Vibrio natriegens. The yeast behind is preferably belonging to the phylum Ascomycota or Basidiomycota or Deuteromycota or Zygomycetes. The yeast behind is preferably belonging to the genus Saccharomyces (having members such as Saccharomyces cerevisiae, S. bayanus, S. boulardii), the genus Zygosaccharomyces, the genus Pichia (having members such as Pichia pastoris, P. anomala, P. kluyveri), the genus Komagataella, the genus Hansenula, the genus Kluyveromyces (having members such as Kluyveromyces lactis, K. marxianus, K. thermotolerans), the genus Debaromyces, the genus Candida, the genus Schizosaccharomyces, the genus Schwanniomyces, the genus Torulaspora, the genus Yarrowia (for example, Yarrowia lipolytica) or the genus Starmerella (for example, Starmerella bombicola).The latter yeast is preferably selected from Pichia pastoris, Yarrowia lipolytica, Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha, Kluyveromyces marxianus, Pichia methanolica, Pichia stipites, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Zygosaccharomyces rouxii, and Zygosaccharomyces bailii. The latter fungus preferably belongs to the genus Rhizopus, Dictyostelium, Penicillium, Mucor, or Aspergillus. Plant cells include cells of flowering and non-flowering plants, as well as algal cells, such as Chlamydomonas, Chlorella, etc. Preferably, the plant is tobacco, alfalfa, rice, tomato, cotton, oilseed rape, soybean, maize or corn plant. The latter animal cell is preferably derived from a non-human mammal (e.g., cow, bison, pig, sheep, mouse, rat, primate (e.g., chimpanzee, orangutan, gorilla, monkey (e.g., Old World monkey, New World monkey), lemur), dog, cat, rabbit, horse, dairy cow, goat, bullock, deer, musk, bovine, whale, dolphin, hippopotamus, elephant, rhinoceros, giraffe, zebra, lion, cheetah, tiger, panda, red panda, otter), bird (e.g., chicken, duck, ostrich, turkey, pheasant), fish (e.g., sailfish, salmon, tuna, sea bass, trout, catfish), invertebrate (e.g., lobster, crab, shrimp, clam, oyster, mussel, sea urchin), reptile (e.g., snake, alligator, sea turtle), amphibian (e.g., frog) or insect (e.g., fly, nematode), or is a genetically modified cell line derived from human cells (excluding embryonic stem cells).Both human and non-human mammalian cells are preferably selected from the list comprising the following: epithelial cells, such as mammary epithelial cells, embryonic kidney cells (e.g., HEK293 or HEK 293T cells), fibroblasts, COS cells, Chinese hamster ovary (CHO) cells, murine myeloma cells, such as N20, SP2 / 0 or YB2 / 0 cells, NIH-3T3 cells, non-mammary adult stem cells or their derivatives, such as those described in WO21067641, preferably mesenchymal stem cells or their derivatives as described in WO21067641, lactocytes derived from mammalian induced pluripotent stem cells (preferably, human induced pluripotent stem cells), lactocytes as part of mammary gland-like organoids, post-partum mammary epithelial cells, polarized mammary cells, preferably polarized mammary cells selected from the group consisting of live primary mammary epithelial cells, live mammary myoepithelial cells, live mammary progenitor cells, live immortalized mammary epithelial cells, live immortalized mammary myoepithelial cells, live immortalized mammary progenitor cells, non-mammary adult stem cells or their derivatives, as is well known to those skilled in the art, from, for example, WO 2021 / 219634, WO 2022 / 054053, WO 2021 / 141762, WO 2021 / 142241, WO 2021 / 067641 and WO 2021 / 242866. The latter insect cells are preferably derived from Spodoptera frugiperda (e.g., Sf9 or Sf21 cells), Bombyx mori, Mamestra brassicae, Trichoplusia ni (e.g., BTI-TN-5B1-4 cells) or Drosophila melanogaster (e.g., Drosophila S2 cells). The latter protozoan cells are preferably Leishmania tarentolae cells.

[0193] More preferably, the cells are selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably selected from the group consisting of yeast cells, bacterial cells, archaeal cells, algal cells and fungal cells as described herein.

[0194] In another and / or additional preferred embodiment, the cells are Escherichia coli or yeast having a lactose permease positive phenotype, preferably wherein the lactose permease is encoded by the gene LacY or LAC12, respectively.

[0195] According to another aspect, the present invention provides a method for producing one or more of the biological products described herein, wherein the method comprises the steps of: culturing and / or incubating the cells described herein, preferably single cells, which is carried out under conditions that permit the expression of the sugar importers described herein and the production of one or more biological products; and preferably, separating the one or more biological products from the culture or incubation. Preferably, purifying the one or more biological products from the culture or incubation described herein.

[0196] Within the scope of the present invention, the permissive conditions are understood to be conditions relating to physical or chemical parameters (including but not limited to, temperature, pH, pressure, osmotic pressure, and product / precursor / acceptor concentration).

[0197] In a particular embodiment, the permissive conditions may include a temperature range of about 30 + / - 20 degrees Celsius, a pH range of 2 - 10, preferably a pH range of 3 - 7.

[0198] In a preferred embodiment of the method and / or cells of the present invention, the one or more biological products described herein are produced by the cells described herein cultured in cell culture or incubated in cell incubation. Within the context of the present invention, the cell culture and / or incubation includes in vitro and / or ex vivo culture and / or incubation of the cells.

[0199] In a preferred embodiment of the method of the present invention, the culture medium contains at least one carbon source selected from the group consisting of glucose, fructose, sucrose, and glycerol.

[0200] In another preferred embodiment of the method of the present invention, the culture or incubation medium contains at least one compound selected from the group consisting of lactose, galactose, glucose, UDP-GlcNAc, GlcNAc, UDP-Gal, UDP-Glc, and LN3. In a preferred embodiment, the culture or incubation medium contains LN3. In a more preferred embodiment, the culture or incubation medium contains LN3 produced by the cells. In an even more preferred embodiment, the culture or incubation medium contains LN3 produced by the cells of the present invention, and the cells are cultured or incubated in the culture or incubation medium for the production of one or more biological products described herein.

[0201] Preferably, the one or more biological products produced are recovered from the culture or incubation medium and / or the cells, or separated from the culture or incubation, as explained herein. More preferably, all of the biological products produced are recovered from the culture or incubation medium and / or the cells, or separated from the culture or incubation, as explained herein.

[0202] According to one embodiment of the method of the present invention, the conditions allowing for the production of said one or more biological products include the use of a culture or incubation medium comprising one or more precursors for the production of said one or more biological products described herein.

[0203] Preferably, the culture or incubation medium comprises at least one precursor selected from the group consisting of: monosaccharides such as galactose, glucose, fucose, sialic acid, GlcNAc, GalNAc; nucleotide-activated sugars such as CMP-sialic acid, UDP-Gal, UDP-GlcNAc, GDP-fucose; disaccharides such as lactose, melibiose, lacto-N-biose and N-acetyl lactosamine; and oligosaccharides such as lacto-N-triose (LN3), lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT). In a more preferred embodiment of the method of the present invention, the precursor is selected from the list consisting of galactose, glucose, UDP-GlcNAc, GlcNAc, UDP-Gal, UDP-Glc and LN3. In an even more preferred embodiment of the method of the present invention, the precursor is LN3.

[0204] According to another and / or additional preferred embodiment of the method of the present invention, one or more precursors present in the culture or incubation medium are produced by the cells of the present invention that are cultured or incubated in the culture or incubation medium for the production of said one or more biological products described herein. More preferably, all precursors present in the culture or incubation medium are produced by the cells of the present invention that are cultured or incubated in the culture or incubation medium.

[0205] According to another and / or additional preferred embodiment of the method of the present invention, one or more precursors present in the culture or incubation medium are taken up by the cells of the present invention that are cultured or incubated in the culture or incubation medium, via the sugar transporters expressed in said cells described herein. More preferably, all precursors present in the culture or incubation medium are taken up by the cells of the present invention that are cultured or incubated in the culture or incubation medium, via the sugar transporters expressed in said cells described herein.

[0206] According to a more preferred embodiment, one or more precursors present in the culture or incubation medium are produced and taken up by the cells of the invention that are cultured or incubated in the culture or incubation medium for the production of one or more biological products, wherein the one or more precursors are taken up by the cells via the sugar transporters described herein. In an even more preferred embodiment, all precursors present in the culture or incubation medium are produced and taken up by the cells of the invention that are cultured or incubated in the culture or incubation medium for the production of one or more biological products, wherein all of the precursors are taken up by the cells via the sugar transporters described herein.

[0207] In another more preferred embodiment, one of the one or more precursors present in the culture or incubation medium is LN3, wherein the LN3 is produced by the cells of the invention that are cultured or incubated in the culture or incubation medium. In another more preferred embodiment, one of the one or more precursors present in the culture or incubation medium is LN3, wherein the LN3 is taken up by the cells of the invention that are cultured or incubated in the culture or incubation medium via the sugar transporters described herein. In an even more preferred embodiment, one of the one or more precursors present in the culture or incubation medium is LN3, wherein the LN3 is produced and taken up (via the sugar transporters described herein) by the cells of the invention that are cultured or incubated in the culture or incubation medium.

[0208] According to another and / or additional preferred embodiment of the method of the invention, the conditions allowing the production of the one or more biological products include using a culture or incubation medium comprising one or more acceptors as defined herein for the production of the one or more biological products described herein.

[0209] According to an alternative and / or additional embodiment of the method of the invention, the conditions allowing the production of the one or more biological products include adding to the culture or incubation medium at least one precursor and / or acceptor feed for the production of the one or more biological products.

[0210] According to an alternative embodiment of the method of the invention, the conditions allowing the production of the one or more biological products include using a culture or incubation medium that does not have any precursors and / or acceptors for the production of the one or more biological products and combining therewith the further addition of at least one precursor and / or acceptor feed for the production of the one or more biological products to the culture or incubation medium.

[0211] According to one embodiment of the method of the present invention, the cultivation or incubation is contained in a reactor or incubator, as defined herein. The volume of the reactor or incubator varies from the microliter (μL) scale to 10,000 m 3 (cubic meters). In a preferred embodiment, the volume of the reactor or incubator varies from 250 mL (milliliters) to 10,000 m 3 (cubic meters).

[0212] According to another and / or further preferred embodiment of the method of the present invention, the cells produce one or more biological products in a higher yield and / or higher purity compared to cells having the same genetic composition but lacking the expression of the sugar importer described herein. In a more preferred embodiment, the one or more biological products are one or more LN3-derived oligosaccharides described herein. According to a more preferred embodiment, the cells produce one or more LN3-derived oligosaccharides in a higher yield and / or higher purity compared to cells having the same genetic composition but lacking the expression of the sugar importer described herein.

[0213] In a preferred embodiment of the present invention, the method results in the production of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L of the one or more biological products in the final volume of the cultivation or incubation.

[0214] In another preferred embodiment, the method for producing one or more biological products described herein includes at least one of the following steps:

[0215] i) Using a cultivation or incubation medium comprising at least one precursor and / or acceptor;

[0216] ii) Adding at least one precursor and / or acceptor feed to the cultivation or incubation medium in a reactor or incubator, wherein the total reactor or incubator volume varies from 250 mL (milliliters) to 10,000 m 3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the cultivation or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the cultivation or incubation medium before the addition of the precursor and / or acceptor feed;

[0217] iii) Adding at least one precursor and / or acceptor feed to the cultivation or incubation medium in a reactor or incubator, wherein the total reactor or incubator volume varies from 250 mL (milliliters) to 10,000 m 3(cubic meters) varies, preferably in a continuous manner, and preferably such that the final volume of the culture or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the culture or incubation medium before addition of the precursor and / or acceptor feed, and wherein preferably, the pH of the precursor and / or acceptor feed is set between 2.0 and 10.0, and wherein preferably, the temperature of the precursor and / or acceptor feed is maintained between 20 °C and 80 °C;

[0218] iv) adding at least one precursor and / or acceptor feed to the culture or incubation medium in a continuous manner by means of a precursor and / or acceptor feed solution over the course of 1 day, 2 days, 3 days, 4 days, 5 days;

[0219] v) adding at least one precursor and / or acceptor feed to the culture or incubation medium in a continuous manner by means of a precursor and / or acceptor feed solution over the course of 1 day, 2 days, 3 days, 4 days, 5 days, wherein preferably, the pH of the precursor and / or acceptor feed solution is set between 2.0 and 10.0; and wherein preferably, the temperature of the precursor and / or acceptor feed solution is maintained between 20 °C and 80 °C;

[0220] The method results in one or more bioproducts, wherein the concentration in the final volume of the culture or incubation is at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L. In a more preferred embodiment of the method of the invention, the precursor is selected from the list comprising galactose, glucose, UDP-GlcNAc, GlcNAc, UDP-Gal, UDP-Glc and LN3. In another more preferred embodiment of the method of the invention, the acceptor is lactose.

[0221] In another and / or additional preferred embodiment, the method for producing one or more bioproducts as described herein comprises at least one of the following steps:

[0222] i) using a culture or incubation medium comprising at least one precursor and / or acceptor;

[0223] ii) adding at least one precursor and / or acceptor to the culture or incubation medium in a reactor or incubator in one pulse or in a discontinuous (pulsating) manner, wherein the total reactor or incubator volume ranges from 250 mL (milliliters) to 10,000 m 3(cubic meters) varies, preferably such that the final volume of the culture or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the culture or incubation medium before addition of the precursor and / or acceptor feed pulse;

[0224] iii) adding at least one precursor and / or acceptor feed to the culture or incubation medium in the reactor or incubator in one pulse or in a discontinuous (pulsed) manner, wherein the total reactor or incubator volume varies from 250 mL (milliliters) to 10,000 m 3 (cubic meters), preferably such that the final volume of the culture or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the culture or incubation medium before addition of the precursor and / or acceptor feed, and wherein preferably, the pH of the precursor and / or acceptor feed pulse is set between 2.0 and 10.0, and wherein preferably, the temperature of the precursor and / or acceptor feed pulse is maintained between 20 °C and 80 °C;

[0225] iv) adding at least one precursor and / or acceptor feed to the culture or incubation medium in a discontinuous (pulsed) manner by means of a precursor and / or acceptor feed solution over a period of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days;

[0226] v) adding at least one precursor and / or acceptor feed to the culture or incubation medium in a discontinuous (pulsed) manner by means of a precursor and / or acceptor feed solution over a period of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, wherein preferably, the pH of the precursor and / or acceptor feed solution is set between 2.0 and 10.0; and wherein preferably, the temperature of the precursor and / or acceptor feed solution is maintained between 20 °C and 80 °C;

[0227] The method results in one or more bioproducts, wherein the concentration in the final volume of the culture or incubation is at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L. In a more preferred embodiment of the method of the invention, the precursor is selected from the list comprising galactose, glucose, UDP-GlcNAc, GlcNAc, UDP-Gal, UDP-Glc and LN3. In another more preferred embodiment of the method of the invention, the acceptor is lactose.

[0228] In another and / or additional preferred embodiment, the method for producing one or more of the biological products described herein includes at least one of the following steps:

[0229] i) using a culture or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per liter of initial reactor or incubator volume, wherein the reactor or incubator volume varies from 250 mL to 10,000 m 3 (cubic meters);

[0230] ii) adding to the culture or incubation medium in the reactor or incubator at least one precursor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per liter of initial reactor or incubator volume, wherein the total reactor or incubator volume varies from 250 mL (milliliters) to 10,000 m 3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the culture or incubation medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the culture or incubation medium before the addition of the precursor feed;

[0231] iii) adding to the culture or incubation medium in the reactor or incubator at least one precursor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per liter of initial reactor or incubator volume, wherein the total reactor or incubator volume varies from 250 mL (milliliters) to 10,000 m 3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the culture or incubation medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the culture or incubation medium before the addition of the precursor feed, and wherein preferably, the pH of the precursor feed is set between 2.0 and 10.0, and wherein preferably, the temperature of the precursor feed is maintained between 20°C and 80°C;

[0232] iv) adding at least one precursor feed to the culture or incubation medium in a continuous manner by means of a precursor feed solution over a period of 1 day, 2 days, 3 days, 4 days, 5 days;

[0233] v) During the course of 1 day, 2 days, 3 days, 4 days, 5 days, at least one precursor feed is added continuously to the culture or incubation medium by means of a precursor feed solution, wherein the concentration of the precursor feed solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably 550 g / L, most preferably 600 g / L; and wherein preferably, the pH of the precursor feed solution is set between 2.0 and 10.0; and wherein preferably, the temperature of the precursor feed solution is maintained between 20 °C and 80 °C;

[0234] The method results in one or more bioproducts, wherein the concentration in the final volume of the culture or incubation is at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L. In a more preferred embodiment of the method of the present invention, the precursor is selected from the list comprising galactose, glucose, UDP-GlcNAc, GlcNAc, UDP-Gal, UDP-Glc and LN3.

[0235] In another and / or additional preferred embodiment, the method for producing one or more bioproducts as described herein comprises at least one of the following steps:

[0236] i) Using a culture or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of acceptor per liter of initial reactor or incubator volume, wherein the reactor or incubator volume varies from 250 mL to 10,000 m 3 (cubic meters);

[0237] ii) Adding to the culture or incubation medium in the reactor or incubator at least one acceptor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of acceptor per liter of initial reactor or incubator volume, wherein the total reactor or incubator volume is from 250 mL (milliliters) to 10,000 m 3(cubic meters) varies, preferably in a continuous manner, and preferably such that the final volume of the culture or incubation medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the culture or incubation medium before addition of the acceptor feed;

[0238] iii) adding to the culture or incubation medium in a reactor or incubator at least one acceptor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of acceptor per liter of the initial reactor or incubator volume, wherein the total reactor or incubator volume varies from 250 mL (milliliters) to 10,000 m 3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the culture or incubation medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the culture or incubation medium before addition of the acceptor feed, and wherein preferably, the pH of the acceptor feed is set between 2.0 and 10.0, and wherein preferably, the temperature of the acceptor feed is maintained between 20°C and 80°C;

[0239] iv) adding at least one acceptor feed to the culture or incubation medium in a continuous manner by means of an acceptor feed solution over the course of 1 day, 2 days, 3 days, 4 days, 5 days;

[0240] v) adding at least one acceptor feed to the culture or incubation medium in a continuous manner by means of an acceptor feed solution over the course of 1 day, 2 days, 3 days, 4 days, 5 days, wherein the concentration of the acceptor feed solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably 550 g / L, most preferably 600 g / L; and wherein preferably, the pH of the acceptor feed solution is set between 2.0 and 10.0; and wherein preferably, the temperature of the acceptor feed solution is maintained between 20°C and 80°C;

[0241] The method results in one or more biological products, with a concentration in the final volume of the culture or incubation of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L. In a more preferred embodiment of the method of the invention, the acceptor is lactose.

[0242] In another and / or additional preferred embodiment, the method for producing one or more biological products described herein includes at least one of the following steps:

[0243] i) Using a culture or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per liter of the initial reactor or incubator volume, wherein the reactor or incubator volume varies from 250 mL to 10,000 m 3 (cubic meters);

[0244] ii) Adding, in a pulse or in a discontinuous (pulsatile) manner, at least one precursor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per liter of the initial reactor or incubator volume to the culture or incubation medium in the reactor or incubator, wherein the total reactor or incubator volume varies from 250 mL (milliliters) to 10,000 m 3 (cubic meters), preferably such that the final volume of the culture or incubation medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the culture or incubation medium before the addition of the precursor feed pulse;

[0245] iii) Adding, in a pulse or in a discontinuous (pulsatile) manner, at least one precursor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per liter of the initial reactor or incubator volume to the culture or incubation medium in the reactor or incubator, wherein the total reactor or incubator volume varies from 250 mL (milliliters) to 10,000 m 3 (cubic meters), preferably such that the final volume of the culture or incubation medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the culture or incubation medium before the addition of the precursor feed, and wherein preferably, the pH of the precursor feed pulse is set between 2.0 and 10.0, and wherein preferably, the temperature of the precursor feed pulse is maintained between 20 °C and 80 °C;

[0246] iv) adding at least one precursor feed discontinuously (pulsed) to the culture or incubation medium by means of a precursor feed solution over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days;

[0247] v) adding at least one precursor feed discontinuously (pulsed) to the culture or incubation medium by means of a precursor feed solution over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, wherein the concentration of the precursor feed solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably 550 g / L, most preferably 600 g / L; and wherein preferably, the pH of the precursor feed solution is set between 2.0 and 10.0; and wherein preferably, the temperature of the precursor feed solution is maintained between 20 °C and 80 °C;

[0248] The method results in one or more bioproducts, wherein the concentration in the final volume of the culture or incubation is at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L. In a more preferred embodiment of the method of the invention, the precursor is selected from the list comprising galactose, glucose, UDP-GlcNAc, GlcNAc, UDP-Gal, UDP-Glc and LN3.

[0249] In another and / or additional preferred embodiment, the method for producing one or more bioproducts as described herein comprises at least one of the following steps:

[0250] i) using a culture or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams acceptor per liter of initial reactor or incubator volume, wherein the reactor or incubator volume varies from 250 mL to 10,000 m 3 (cubic meters);

[0251] ii) adding at least one acceptor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of acceptor per liter of the initial reactor or incubator volume to the culture or incubation medium in the reactor or incubator in one pulse or in a discontinuous (pulsed) manner, wherein the total reactor or incubator volume varies from 250 mL (milliliters) to 10,000 m 3 (cubic meters), preferably such that the final volume of the culture or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the culture or incubation medium before the addition of the acceptor feed pulse;

[0252] iii) adding at least one acceptor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of acceptor per liter of the initial reactor or incubator volume to the culture or incubation medium in the reactor or incubator in one pulse or in a discontinuous (pulsed) manner, wherein the total reactor or incubator volume varies from 250 mL (milliliters) to 10,000 m 3 (cubic meters), preferably such that the final volume of the culture or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the culture or incubation medium before the addition of the acceptor feed, and wherein preferably, the pH of the acceptor feed pulse is set between 2.0 and 10.0, and wherein preferably, the temperature of the acceptor feed pulse is maintained between 20 °C and 80 °C;

[0253] iv) adding at least one acceptor feed to the culture or incubation medium in a discontinuous (pulsed) manner by means of an acceptor feed solution over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days;

[0254] v) During the process of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, at least one acceptor feed is added to the culture or incubation medium in a discontinuous (pulsating) manner by means of an acceptor feed solution, wherein the concentration of the acceptor feed solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably 550 g / L, most preferably 600 g / L; and wherein preferably, the pH of the acceptor feed solution is set between 2.0 and 10.0; and wherein preferably, the temperature of the acceptor feed solution is maintained between 20 °C and 80 °C;

[0255] The method results in one or more biological products, wherein the concentration in the final volume of the culture or incubation is at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L. In a more preferred embodiment of the method of the present invention, the acceptor is lactose.

[0256] In a more preferred embodiment, the method for producing one or more biological products described herein includes at least one of the following steps:

[0257] i) Use a culture or incubation medium containing at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of the initial reactor or incubator volume, wherein the reactor or incubator volume varies from 250 mL to 10,000 m 3 (cubic meters);

[0258] ii) Add a lactose feed containing at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of the initial reactor or incubator volume to the culture or incubation medium in the reactor or incubator, wherein the total reactor or incubator volume ranges from 250 mL (milliliters) to 10,000 m 3(cubic meters) varies, preferably in a continuous manner, and preferably such that the final volume of the culture or incubation medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the culture or incubation medium before addition of the lactose feed;

[0259] iii) adding to the culture or incubation medium in a reactor or incubator a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of the initial reactor or incubator volume, wherein the total reactor or incubator volume varies from 250 mL (milliliters) to 10,000 m 3 (cubic meters) varies, preferably in a continuous manner, and preferably such that the final volume of the culture or incubation medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the culture or incubation medium before addition of the lactose feed, and wherein preferably, the pH of the lactose feed is set between 2.0 and 10.0, preferably between 3.0 and 7.0, and wherein preferably, the temperature of the lactose feed is maintained between 20 °C and 80 °C;

[0260] iv) adding the lactose feed to the culture or incubation medium in a continuous manner by means of a feed solution over the course of 1 day, 2 days, 3 days, 4 days, 5 days;

[0261] v) adding the lactose feed to the culture or incubation medium in a continuous manner by means of a feed solution over the course of 1 day, 2 days, 3 days, 4 days, 5 days, wherein the concentration of the lactose feed solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably 400 g / L, more preferably 450 g / L, more preferably 500 g / L, more preferably 550 g / L, most preferably 600 g / L; and wherein preferably, the pH of the lactose feed is set between 2.0 and 10.0, preferably between 3.0 and 7.0; and wherein preferably, the temperature of the lactose feed is maintained between 20 °C and 80 °C;

[0262] The method results in one or more biological products, with a concentration in the final volume of the culture or incubation of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L.

[0263] Preferably, the lactose feeding is achieved by adding lactose at a concentration of at least 5 mM from the start of the culture or incubation, preferably at a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably at a concentration of >300 mM.

[0264] In another embodiment of the method, the lactose feeding is achieved by adding lactose to the culture or incubation medium at such a concentration that a lactose concentration of at least 5 mM, preferably 10 mM or 30 mM, is obtained throughout the production period of the culture or incubation.

[0265] In a further embodiment of the method described herein, the cells are cultured or incubated for at least about 60, 80, 100 or about 120 hours, or are cultured in a continuous manner.

[0266] In a preferred embodiment, a carbon source, preferably sucrose, is provided in the culture medium for 3 or more days, preferably up to 7 days; and / or at least 100, advantageously at least 105, more advantageously at least 110, even more advantageously at least 120 grams of sucrose per liter of the initial culture volume is provided in the culture medium in a continuous manner, such that the final volume of the culture medium is not more than three times, advantageously not more than twice, more advantageously less than twice the volume of the culture medium before the culture.

[0267] Preferably, when implementing the method described herein, a first stage of exponential cell growth is provided by adding a carbon source, preferably glucose or sucrose, to the culture medium, and then lactose is added to the culture medium in a second stage.

[0268] In an alternative preferred embodiment, in the method described herein, lactose has been added together with a carbon substrate in the first stage of exponential growth.

[0269] Another aspect provides cells to be stably cultured in a medium, where the medium can be any type of growth medium, including a basal medium, a complex medium or a growth medium (which is rich in certain compounds, such as but not limited to vitamins, trace elements, amino acids).

[0270] The microorganisms or cells used herein are capable of growing on monosaccharides, disaccharides, oligosaccharides, polysaccharides, polyols, glycerol, complex media, or mixtures thereof (as the main carbon source). The term "main" means the most important carbon source for the production of one or more target biological products, biomass formation, carbon dioxide and / or by-product formation (e.g., acids and / or alcohols such as acetate, lactate, and / or ethanol) by the microorganisms or cells, i.e., 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99% of all the required carbon is derived from the carbon sources indicated above. In one embodiment of the present invention, the carbon source is the sole carbon source for the organism, i.e., 100% of the required carbon is derived from the carbon sources indicated above. Common main carbon sources include, but are not limited to: glucose, glycerol, fructose, sucrose, maltose, lactose, arabinose, maltooligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemicellulose, molasses, corn steep liquor, high fructose syrup, acetate, citrate, lactate, and pyruvate. As used herein, the precursors defined herein cannot be used as a carbon source for the production of one or more biological products of the present invention.

[0271] According to the present invention, the methods described herein preferably include the following steps: separating one or more biological products of the present invention from the culture or incubation, or recovering one or more biological products from the culture or incubation medium and / or cells.

[0272] The term "separating from the culture or incubation" means harvesting, collecting, or recovering the one or more biological products from the cells and / or the medium in which they are cultured or incubated.

[0273] The one or more biological products can be separated from the aqueous medium in which the cells are cultured or incubated in a conventional manner. In the case where the one or more biological products are still present in the cells producing the one or more biological products, conventional means for releasing or extracting the one or more biological products from the cells can be used, such as cell disruption using high pH, heat shock, sonication, French press, homogenization, enzymatic hydrolysis, chemical hydrolysis, solvent hydrolysis, detergents, hydrolysis, etc. Then, the culture or incubation medium and / or cell extracts can be further used together and separately to separate the one or more biological products.

[0274] This preferably involves clarifying the one or more biological products to remove suspended particles and contaminants, in particular cells, cell components, insoluble metabolites and debris produced by culturing or incubating genetically modified cells. In this step, the one or more biological products can be clarified in a conventional manner. Preferably, the one or more biological products are clarified by centrifugation, flocculation, decantation and / or filtration. Another step of separating the one or more biological products preferably involves removing substantially all of the ultimately remaining proteins, peptides, amino acids, RNA and DNA, as well as any endotoxins and glycolipids, which can interfere with subsequent separation steps, preferably after they have been clarified. In this step, the remaining proteins and related impurities can be removed from the one or more biological products in a conventional manner. Preferably, by ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated carbon or carbon treatment, treatment with non-ionic surfactants, enzymatic digestion, tangential flow high performance filtration, tangential flow ultrafiltration, electrophoresis (e.g., using plate-polyacrylamide or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE)), affinity chromatography (using affinity ligands including, for example, DEAE-Sepharose, poly-L-lysine and polymyxin-B, endotoxin-selective adsorption matrices), ion exchange chromatography (e.g., but not limited to cation exchange, anion exchange, mixed bed ion exchange, inside-out ligand attachment), hydrophobic interaction chromatography and / or gel filtration (i.e., size exclusion chromatography), particularly by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography or electrodialysis, the remaining proteins, salts, by-products, colors, endotoxins and other related impurities are removed from the one or more biological products. Except for size exclusion chromatography, the remaining proteins and related impurities are retained by the chromatography medium or the selected membrane.

[0275] In a further preferred embodiment, the methods described herein also provide for further purification of the one or more biological products of the invention. Further purification of the one or more biological products can be accomplished, for example, by using (activated) carbon or charcoal, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment, ion exchange, temperature adjustment, pH adjustment or pH adjustment with an alkaline or acidic solution to remove any remaining DNA, proteins, LPS, endotoxins or other impurities. Alcohols, such as ethanol, and aqueous alcohol mixtures can also be used. Another purification step is accomplished by crystallization, evaporation or precipitation of the one or more biological products. Another purification step is drying, such as spray drying, freeze drying, spray freeze drying, freeze spray drying, ribbon drying, belt drying, vacuum ribbon drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying or vacuum roller drying of the biological products produced.

[0276] In an exemplary embodiment, the separation and purification of the one or more bioproducts is carried out in a process comprising the following steps in any order:

[0277] a) contacting the culture or incubation or a clarified form thereof with a nanofiltration membrane having a molecular weight cut-off (MWCO) of 600 - 3500 Da, thereby ensuring retention of the produced bioproduct and allowing at least a portion of the protein, salts, by-products, color, and other related impurities to pass through;

[0278] b) using the membrane, subjecting the retentate from step a) to a diafiltration process with an aqueous solution of an inorganic electrolyte, followed optionally by diafiltration with pure water to remove excess electrolyte,

[0279] c) and collecting the retentate rich in the one or more bioproducts in the form of a salt comprising cations from the electrolyte.

[0280] In an alternative exemplary embodiment, the separation and purification of the one or more bioproducts is carried out in a process comprising the following steps in any order: subjecting the culture or incubation or a clarified form thereof to two membrane filtration steps using different membranes, wherein

[0281] - one membrane has a molecular weight cut-off of from about 300 to about 500 daltons, and

[0282] - the other membrane has a molecular weight cut-off of from about 600 to about 800 daltons.

[0283] In an alternative exemplary embodiment, the separation and purification of the one or more bioproducts is carried out by treating the culture or incubation or a clarified form thereof in one step with a strong cation exchange resin in the H+-form and in another step with a weak anion exchange resin in the free base form, wherein the steps may be carried out in any order.

[0284] In an alternative exemplary embodiment, the separation and purification of the one or more bioproducts is carried out as follows. The culture or incubation containing the produced bioproduct, biomass, media components, and contaminants is subjected to the following purification steps:

[0285] i) separating the biomass from the culture or incubation,

[0286] ii) treating with a cation exchanger to remove positively charged materials,

[0287] iii) treating with an anion exchanger to remove negatively charged materials,

[0288] iv) A nanofiltration step and / or an electrodialysis step,

[0289] wherein a purified solution is provided, which contains the produced bioproduct at a purity of greater than or equal to 80%. Optionally, the purified solution is dried by any one or more drying steps selected from the list consisting of: spray drying, freeze drying, spray freeze drying, freeze spray drying, belt drying, band drying, vacuum belt drying, vacuum band drying, drum drying, roller drying, vacuum drum drying, and vacuum roller drying.

[0290] In an alternative exemplary embodiment, the separation and purification of the one or more bioproducts is carried out in a process comprising the following steps in any order: enzymatic treatment of the culture or incubation; removal of biomass from the culture or incubation; ultrafiltration; nanofiltration; and a column chromatography step. Preferably, such column chromatography is single-column or multi-column. More preferably, the column chromatography step is simulated moving bed chromatography. Such simulated moving bed chromatography preferably includes: i) at least 4 columns, wherein at least one column contains a weak or strong cation exchange resin; and / or ii) four zones I, II, III, and IV with different flow rates; and / or iii) an eluent containing water; and / or iv) an operating temperature of 15 to 60 degrees Celsius.

[0291] In a particular embodiment, the present invention provides the produced bioproduct dried to a powder by any one or more drying steps selected from the list consisting of: spray drying, freeze drying, spray freeze drying, freeze spray drying, belt drying, band drying, vacuum belt drying, vacuum band drying, drum drying, roller drying, vacuum drum drying, and vacuum roller drying, wherein the dried powder contains <15 wt% water, preferably <10 wt% water, more preferably <7 wt% water, and most preferably <5 wt% water.

[0292] A further aspect of the present invention provides an isolated nucleic acid molecule encoding a sugar importer having lacto-N-triose uptake activity as described herein.

[0293] Another aspect of the present invention provides a vector comprising the isolated nucleic acid molecule encoding a sugar importer having lacto-N-triose uptake activity as described herein.

[0294] The present invention further provides a sugar transporter having an uptake activity with respect to LN3 and comprising a polypeptide sequence having a deletion, insertion, and / or mutation of one or more amino acid residues at the following positions: i) the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO: 12, 02, 01, 09, 19, 20, or 21, and / or ii) the C-terminus of the last TM domain of any one of SEQ ID NO: 12, 02, 01, 09, 19, 20, or 21. In a preferred embodiment, the sugar transporter of the present invention has an uptake activity with respect to LN3 and comprises a polypeptide sequence having a deletion, insertion, and / or mutation of one or more amino acid residues at the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO: 12, 02, 01, 09, 19, 20, or 21, wherein the deletion, insertion, and / or mutation of the one or more amino acid residues is in a non-TM helix or at the N-terminus of a non-TM helix.

[0295] In another and / or additional preferred embodiment, the sugar transporter of the present invention has an uptake activity with respect to LN3 and comprises a polypeptide sequence having a deletion, insertion, and / or mutation of one or more amino acid residues at the C-terminus of the last TM domain of any one of SEQ ID NO: 12, 02, 01, 09, 19, 20, or 21, wherein the deletion, insertion, and / or mutation of the one or more amino acid residues is in a non-TM helix or at the C-terminus of a non-TM helix.

[0296] In another and / or additional preferred embodiment, i) a variant sugar transporter having a deletion, insertion, and / or mutation of one or more amino acid residues at the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO: 12, 02, 01, 09, 19, 20, or 21 and / or ii) a variant sugar transporter having a deletion, insertion, and / or mutation of one or more amino acid residues at the C-terminus of the last TM domain of any one of SEQ ID NO: 12, 02, 01, 09, 19, 20, or 21 has a higher, faster, and / or more efficient (i.e., lower energy-consuming) uptake ratio of the sugar compared to the native sugar transporter lacking the deletion, insertion, and / or mutation.

[0297] Preferably, the sugar importer comprises, consists of, or consists essentially of a polypeptide sequence having SEQ ID NO:03 and has uptake activity with respect to LN3; SEQ ID NO:03 is a mutant polypeptide of the sugar importer having SEQ ID NO:02, wherein SEQ ID NO:03 has a deletion of 39 amino acid residues at the N-terminus of the first TM domain of SEQ ID NO:02 and an insertion of 19 amino acid residues at the N-terminus of the first TM domain of SEQ ID NO:02. The first TM domain of SEQ ID NO:02 starts at Ser at position 40. Both polypeptides having SEQ ID NO:02 and 03 have uptake activity with respect to LN3. In a more preferred embodiment, the sugar importer described herein is a polypeptide having SEQ ID NO:03 and has uptake activity with respect to LN3.

[0298] Alternatively, the sugar importer described herein comprises, consists of, or consists essentially of a polypeptide sequence having SEQ ID NO:04 and has uptake activity with respect to LN3; SEQ ID NO:04 is a mutant polypeptide of the sugar importer having SEQ ID NO:02, wherein SEQ ID NO:04 has a deletion of 39 amino acid residues at the N-terminus of the first TM domain of SEQ ID NO:02 and an insertion of 19 amino acid residues at the N-terminus of the first TM domain of SEQ ID NO:02. The first TM domain of SEQ ID NO:02 starts at Ser at position 40. Both polypeptides having SEQID NO:02 and 04 have uptake activity with respect to LN3. In a more preferred embodiment, the sugar importer described herein is a polypeptide having SEQ ID NO:04 and has uptake activity with respect to LN3.

[0299] Alternatively, the sugar importer described herein comprises, consists of, or consists essentially of a polypeptide sequence having SEQ ID NO:16 and has uptake activity with respect to LN3; SEQ ID NO:16 is a mutant polypeptide of the sugar importer having SEQ ID NO:09, wherein said SEQ ID NO:16 has a deletion of 23 amino acid residues at the N-terminus of the first TM domain of SEQ ID NO:09 and an insertion of 20 amino acid residues at the N-terminus of the first TM domain of SEQ ID NO:09. The first TM domain of SEQ ID NO:09 starts at Val at position 24. In a more preferred embodiment, the sugar importer described herein is a polypeptide having SEQ ID NO:16 and has uptake activity with respect to LN3.

[0300] Another aspect of the present invention provides a sugar importer having an uptake activity with respect to LN3 as described herein for use in producing one or more bioproducts selected from the list consisting of: sugars; monosaccharides; activated monosaccharides; phosphorylated monosaccharides; disaccharides; oligosaccharides; polysaccharides; sugars in milk; sugars in mammalian milk; mammalian milk oligosaccharides (MMO); sugars in human milk; human milk oligosaccharides (HMO); neutral (uncharged) sugars; negatively charged sugars; fucosylated sugars; sialylated sugars; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; O-antigen; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; LN3; lacto-N-tetraose (LNT); lacto-N-neotetraose (LNnT); oligosaccharides derived from LN3; 3'-sialyllactose (3'SL); 6'-sialyllactose (6'SL); sialyllacto-N-tetraose a (LSTa); sialyllacto-N-tetraose b (LSTb); sialyllacto-N-tetraose c (LSTc); sialyllacto-N-tetraose d (LSTd); lacto-N-fucopentaose I; lacto-N-neofucopentaose; lacto-N-fucopentaose II; lacto-N-fucopentaose III; lacto-N-fucopentaose V; lacto-N-neofucopentaose V; lacto-N-difucosylhexose I; lacto-N-neodifucosylhexose; lacto-N-difucosylhexose II; monofucosyllacto-N-hexose III; difucosyllacto-N-hexose a; 6'-galactosyllactose; 3'-galactosyllactose; lacto-N-hexose; lacto-N-neohexose; 2'-fucosyllactose (2'FL); 3-fucosyllactose (3-FL); difucosyllactose (DiFL); chitosan; oligosaccharides containing chitosan; heparosan; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate; acetylhyaluronic acid; hyaluronic acid; and keratan sulfate. In a preferred embodiment, the one or more bioproducts are one or more LN3-derived oligosaccharides. In another preferred embodiment, the sugar importer having an uptake activity with respect to LN3 for use in producing one or more bioproducts further comprises an uptake activity with respect to one or more other sugars different from LN3, wherein the one or more other sugars different from LN3 are selected from the list consisting of monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

[0301] Other further aspects of the present invention provide for the use of i) the cells described herein, ii) the methods described herein, iii) the isolated nucleic acid molecules described herein, iv) the vectors described herein, or v) the sugar importers having LN3 uptake activity described herein for producing one or more bioproducts selected from the list consisting of: sugars; monosaccharides; activated monosaccharides; phosphorylated monosaccharides; disaccharides; oligosaccharides; polysaccharides; sugars in milk; sugars in mammalian milk; mammalian milk oligosaccharides (MMO); sugars in human milk; human milk oligosaccharides (HMO); neutral (uncharged) sugars; negatively charged sugars; fucosylated sugars; sialylated sugars; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; O-antigens; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; LN3; lacto-N-tetraose (LNT); lacto-N-neotetraose (LNnT); oligosaccharides derived from LN3; 3'-sialyllactose (3'SL); 6'-sialyllactose (6'SL); sialyllacto-N-tetraose a (LSTa); sialyllacto-N-tetraose b (LSTb); sialyllacto-N-tetraose c (LSTc); sialyllacto-N-tetraose d (LSTd); lacto-N-fucopentaose I; lacto-N-neofucopentaose; lacto-N-fucopentaose II; lacto-N-fucopentaose III; lacto-N-fucopentaose V; lacto-N-neofucopentaose V; lacto-N-difucosylhexose I; lacto-N-neodifucosylhexose; lacto-N-difucosylhexose II; monofucosyllacto-n-hexose III; difucosyllacto-N-hexose a; 6'-galactosyllactose; 3'-galactosyllactose; lacto-N-hexose; lacto-N-neohexose; 2'-fucosyllactose (2'FL); 3-fucosyllactose (3-FL); difucosyllactose (DiFL); chitosan; oligosaccharides containing chitosan; heparosan; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate; acetylhyaluronic acid; hyaluronic acid; and keratan sulfate. In a preferred embodiment, the one or more bioproducts are one or more LN3-derived oligosaccharides.

[0302] Furthermore, the present invention also relates to one or more biological products obtained by the method according to the present invention. The one or more biological products can be used for manufacturing preparations, used as food additives, prebiotics, symbiotics, for supplementing infant food, adult food, feed for young animals, feed for adult animals, or used as therapeutically or pharmaceutically active compounds, or used in cosmetic applications. In a preferred embodiment, the preparation comprises at least one biological product which is obtainable, preferably obtained, by the method described herein. In another preferred embodiment, a preparation further comprising at least one probiotic microorganism is provided. In another preferred embodiment of the present invention, the preparation is a nutritional composition. In a more preferred embodiment, the preparation is a medical preparation, a dietary supplement, a dairy drink or an infant formula.

[0303] With these new methods, the one or more biological products can be readily and effectively provided without complex, time-consuming and costly synthetic processes.

[0304] In order to identify the biological products of the invention produced as described herein, monosaccharides or monomer building blocks (e.g., monosaccharide or glycan unit composition), anomeric configuration of side chains, presence and position of substituent groups, degree of polymerization / molecular weight, and linkage pattern can be identified by standard methods known in the art such as those listed below: methylation analysis, reductive cleavage, hydrolysis, GC-MS (gas chromatography - mass spectrometry), MALDI-MS (matrix-assisted laser desorption / ionization - mass spectrometry), ESI-MS (electrospray ionization - mass spectrometry), HPLC (high-performance liquid chromatography with ultraviolet or refractive index detection), HPAEC-PAD (high-performance anion-exchange chromatography with pulsed amperometric detection), CE (capillary electrophoresis), IR (infrared) / Raman spectroscopy, and NMR (nuclear magnetic resonance) spectroscopy techniques. Crystal structures can be resolved by using, for example, solid-state NMR, FT-IR (Fourier transform infrared spectroscopy), and WAXS (wide-angle X-ray scattering). Degree of polymerization (DP), DP distribution, and polydispersity can be determined by, for example, viscometry and SEC (SEC-HPLC, high-performance size-exclusion chromatography). To identify the monomeric components of the biological product, methods such as acid-catalyzed hydrolysis, HPLC (high-performance liquid chromatography), or GLC (gas-liquid chromatography) (after conversion to alditol acetates) can be used. To determine glycosidic linkages, the biological product is methylated with methyl iodide and a strong base in DMSO, hydrolyzed, reduced to partially methylated alditols, acetylated to methylated alditol acetates, and analyzed by GLC / MS (gas-liquid chromatography - mass spectrometry). To determine glycan sequences, partial depolymerization is carried out using acid or enzymes to determine the structure. To identify anomeric configurations, the biological product is subjected to enzymatic analysis, e.g., by contacting it with an enzyme specific for a particular type of linkage (e.g., β-galactosidase or α-glucosidase, etc.), and NMR can be used to analyze the product.

[0305] The separate and preferably further purified biological products described herein are incorporated into foods (e.g., human food or feed), dietary supplements, pharmaceutical ingredients, cosmeceutical ingredients, or drugs. In some embodiments, the biological products are mixed with one or more ingredients suitable for foods, feeds, dietary supplements, pharmaceutical ingredients, cosmeceutical ingredients, or drugs.

[0306] In some embodiments, the dietary supplement comprises at least one prebiotic ingredient and / or at least one probiotic ingredient.

[0307] "Prebiotic" refers to substances that promote the growth of microorganisms beneficial to the host, particularly those in the gastrointestinal tract. In some embodiments, the dietary supplement provides a variety of prebiotics, including the bioproducts, which are prebiotic molecules produced and / or purified by the processes disclosed in this specification, to promote the growth of one or more beneficial microorganisms. Examples of prebiotic components for dietary supplements include other prebiotic molecules (such as HMO) and plant polysaccharides (such as inulin, pectin, β-glucan, and xylo-oligosaccharides). "Probiotic" products typically contain live microorganisms that replace or are added to the gastrointestinal microbiota for the benefit of the recipient. Examples of such microorganisms include species of Lactobacillus (such as Lactobacillus acidophilus and Lactobacillus bulgaricus), Bifidobacterium (such as Bifidobacterium animalis, Bifidobacterium longum, and Bifidobacterium infantis (such as Bi-26)), and Saccharomyces boulardii. In some embodiments, one or more bioproducts produced and / or purified by the processes of this specification are orally administered in combination with such microorganisms.

[0308] Examples of further components for dietary supplements include oligosaccharides (such as 2'-fucosyllactose, 3-fucosyllactose, 6'-sialyllactose), disaccharides (such as lactose), monosaccharides (such as glucose, galactose, L-fucose, sialic acid, glucosamine, and N-acetylglucosamine), thickeners (such as gum arabic), acidity regulators (such as trisodium citrate), water, skim milk, and flavoring agents.

[0309] In some embodiments, the biological product is incorporated into human infant food (e.g., infant formula). Infant formula is typically an artificial food used to feed infants as a complete or partial substitute for human breast milk. In some embodiments, the infant formula is sold as a powder and is prepared for bottle or cup feeding to an infant by mixing with water. The composition of infant formula is typically designed to generally mimic human breast milk. In some embodiments, one or more biological products produced and / or purified by the processes in this specification are included in the infant formula to provide nutritional benefits similar to those provided by oligosaccharides in human breast milk. In some embodiments, the one or more biological products are mixed with one or more ingredients of the infant formula. Examples of infant formula ingredients include fat-free milk, a carbohydrate source (e.g., lactose), a protein source (e.g., whey protein concentrate and casein), a fat source (e.g., vegetable oils such as palm oil, high oleic safflower oil, canola oil, coconut oil, and / or sunflower oil; and fish oil), vitamins (e.g., vitamins A, B6, B12, C, and D), minerals (e.g., potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate, and calcium phosphate), and possibly human milk oligosaccharides (HMO). Such HMOs can include, for example, 2’FL, 3-FL, DiFL, 3’SL, 6’SL, lacto-N-triose II, LNT, LNnT, lacto-N-fucopentaose I, lacto-N-neofucopentaose, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucosylhexose I, lacto-N-difucosylhexose II, 6’-galactosyllactose, 3’-galactosyllactose, lacto-N-hexose, and lacto-N-neohexose.

[0310] In some embodiments, the one or more infant formula ingredients include fat-free milk, a carbohydrate source, a protein source, a fat source, and / or vitamins and minerals.

[0311] In some embodiments, the one or more infant formula ingredients include lactose, whey protein concentrate, and / or high oleic safflower oil.

[0312] In some embodiments, the concentration of the biological product in the infant formula is a concentration that is substantially the same as the concentration of the biological product that is typically present in human breast milk.

[0313] In some embodiments, the biological product is incorporated into a feed preparation, wherein the feed is selected from the list consisting of: pet food, animal milk replacers, veterinary products, veterinary feed supplements, nutritional supplements, post-weaning feeds, or creep feeds.

[0314] As will be shown in the examples herein, it has been demonstrated that the sugar importer having uptake activity with respect to LN3 is useful in the cell-based production of one or more bioproducts, preferably LN3-derived oligosaccharides, more preferably LNT and / or LNnT. When using the sugar importer described herein, the methods and cells of the present invention preferably provide at least one of the following further surprising advantages:

[0315] - Higher titre (g / L) of bioproduct,

[0316] - Higher purity of bioproduct,

[0317] - Higher production rate r (g bioproduct / L / h),

[0318] - Higher cell performance index CPI (g bioproduct / g X),

[0319] - Higher specific productivity Qp (g bioproduct / g X / h),

[0320] - Higher yield Y (g bioproduct / g carbon source used) on the carbon source used,

[0321] - Higher yield Ys (g bioproduct / g sucrose) on sucrose,

[0322] - Higher uptake / transformation rate Q (g carbon source / g X / h) of the carbon source used,

[0323] - Higher sucrose uptake / transformation rate Qs (g sucrose / g X / h),

[0324] - Higher lactose conversion / consumption rate rs (g lactose / h),

[0325] - Higher secretion of bioproduct, and / or

[0326] - Higher growth rate of the production host,

[0327] When compared to methods or cells using the same setup or genetic background but not using the sugar importer having uptake activity with respect to LN3 described herein.

[0328] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization described above and below are those well known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. In general, purification steps are carried out according to the manufacturer's instructions.

[0329] Further advantages can be derived from the specific embodiments and examples. It goes without saying that the features mentioned above and those yet to be explained below can be used not only in the combinations specifically specified, but also in other combinations or independently, without departing from the scope of the invention.

[0330] Furthermore, the present invention relates to the following specific embodiments:

[0331] 1. A cell for producing one or more biological products, wherein the cell:

[0332] - is capable of producing, preferably producing, one or more precursors used in the production of at least one of the one or more biological products, preferably the cell is genetically modified to produce at least one of the one or more precursors used in the production of at least one of the one or more biological products, more preferably the cell is genetically modified to produce all of the one or more precursors used in the production of at least one of the one or more biological products, and

[0333] - is genetically modified to express, preferably overexpress, a polynucleotide sequence encoding a sugar importer that internalizes at least one of the one or more precursors into the cell.

[0334] 2. The cell according to embodiment 1, wherein the sugar importer has uptake activity with respect to lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc).

[0335] 3. The cell according to any one of embodiments 1 or 2, wherein one of the one or more precursors is LN3.

[0336] 4. The cell according to any one of embodiments 1 to 3, wherein one of the one or more precursors is LN3, and wherein the sugar importer has uptake activity with respect to the LN3 produced by the cell.

[0337] 5. A cell for producing one or more biological products, wherein the cell is genetically modified to express, preferably overexpress, a polynucleotide sequence encoding a sugar importer having uptake activity with respect to lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc).

[0338] 6. The cell according to any one of the foregoing embodiments, wherein the sugar importer:

[0339] - is derived from the major facilitator superfamily (MFS) of transporters,

[0340] - comprising a polypeptide sequence that contains the IPR001927 domain as defined by InterPro 90.0 released on August 4, 2022,

[0341] - comprising a polypeptide sequence that is at least 25% identical to any one of the polypeptide sequences shown in SEQ ID NO:04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13 over a segment of at least 150 amino acid residues, preferably at least 200 amino acid residues,

[0342] - comprising a polypeptide sequence that is at least 25% identical to any one of the full-length polypeptide sequences shown in SEQ ID NO:04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13,

[0343] - comprising a polypeptide sequence shown by any one of SEQ ID NO:04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13,

[0344] - comprising a polypeptide sequence that has a deletion, insertion, and / or mutation of one or more amino acid residues at the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO:04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13, preferably the deletion, insertion, and / or mutation is in a non-TM helix or at the N-terminus of a non-TM helix, and / or

[0345] - comprising a polypeptide sequence that has a deletion, insertion, and / or mutation of one or more amino acid residues at the C-terminus of the last transmembrane (TM) domain of any one of SEQ ID NO:04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13, preferably the deletion, insertion, and / or mutation is in a non-TM helix or at the C-terminus of a non-TM helix.

[0346] 7. The cell according to any one of embodiments 2 to 6, wherein the sugar importer further comprises uptake activity with respect to one or more other sugars different from LN3, and the one or more other sugars different from LN3 are selected from the list comprising monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

[0347] 8. A cell according to any one of the foregoing embodiments, wherein the one or more biological products are selected from the list comprising: sugars; monosaccharides; activated monosaccharides; phosphorylated monosaccharides; disaccharides; oligosaccharides; polysaccharides; sugars in milk; sugars in mammalian milk; mammalian milk oligosaccharides (MMO); sugars in human milk; human milk oligosaccharides (HMO); neutral (uncharged) sugars; negatively charged sugars; fucosylated sugars; sialylated sugars; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; O-antigen; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; LN3; lacto-N-tetraose (LNT); lacto-N-neotetraose (LNnT); oligosaccharides derived from LN3; 3'-sialyllactose (3'SL); 6'-sialyllactose (6'SL); sialyllacto-N-tetraose a (LSTa); sialyllacto-N-tetraose b (LSTb); sialyllacto-N-tetraose c (LSTc); sialyllacto-N-tetraose d (LSTd); lacto-N-fucopentaose I; lacto-N-neofucopentaose; lacto-N-fucopentaose II; lacto-N-fucopentaose III; lacto-N-fucopentaose V; lacto-N-neofucopentaose V; lacto-N-difucosylhexose I; lacto-N-neodifucosylhexose; lacto-N-difucosylhexose II; monofucosyllacto-N-hexose III; difucosyllacto-N-hexose a; 6'-galactosyllactose; 3'-galactosyllactose; lacto-N-hexose; lacto-N-neohexose; 2'-fucosyllactose (2'FL); 3-fucosyllactose (3-FL); difucosyllactose (DiFL); chitosan; oligosaccharides containing chitosan; heparosan; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate; acetylhyaluronic acid; hyaluronic acid; and keratan sulfate, preferably the one or more biological products are one or more LN3-derived oligosaccharides.

[0348] 9. A cell according to any one of the foregoing embodiments, wherein the polynucleotide sequence is operably linked to a control sequence recognized by the cell, and / or wherein the polynucleotide sequence is foreign to the cell, the polynucleotide sequence further i) integrated into the genome of the cell and / or ii) presented to the cell on a vector.

[0349] 10. A cell according to any one of the foregoing embodiments, wherein the cell is further genetically engineered to produce the one or more biological products.

[0350] 11. A cell according to any one of embodiments 5 to 10, wherein the cell is capable of producing the one or more biological products from one or more precursors, preferably producing the one or more biological products from one or more precursors.

[0351] 12. A cell according to embodiment 11, wherein the cell is capable of producing, preferably producing at least one of the one or more precursors, preferably the cell is capable of producing, preferably producing all of the one or more precursors.

[0352] 13. A cell according to embodiment 12, wherein the cell is genetically engineered to produce at least one of the one or more precursors, preferably the cell is genetically engineered to produce all of the one or more precursors.

[0353] 14. A cell according to any one of embodiments 11 to 13, wherein at least one of the one or more precursors is internalized into the cell via the sugar importer.

[0354] 15. A cell according to any one of embodiments 1 to 4, 11 to 14, wherein the one or more precursors are selected from the list comprising: sugars; monosaccharides; activated monosaccharides; phosphorylated monosaccharides; disaccharides; oligosaccharides; polysaccharides; sugars in milk; sugars in mammalian milk; mammalian milk oligosaccharides (MMO); sugars in human milk; human milk oligosaccharides (HMO); neutral (uncharged) sugars; negatively charged sugars; fucosylated sugars; sialylated sugars; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; O-antigens; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; LN3; lacto-N-tetraose (LNT); lacto-N-neotetraose (LNnT); oligosaccharides derived from LN3; 3'-sialyllactose (3'SL); 6'-sialyllactose (6'SL); sialyllacto-N-tetraose a (LSTa); sialyllacto-N-tetraose b (LSTb); sialyllacto-N-tetraose c (LSTc); sialyllacto-N-tetraose d (LSTd); lacto-N-fucopentaose I; lacto-N-neofucopentaose; lacto-N-fucopentaose II; lacto-N-fucopentaose III; lacto-N-fucopentaose V; lacto-N-neofucopentaose V; lacto-N-difucosylhexose I; lacto-N-neodifucosylhexose; lacto-N-difucosylhexose II; monofucosyllacto-n-hexose III; difucosyllacto-N-hexose a; 6'-galactosyllactose; 3'-galactosyllactose; lacto-N-hexose; lacto-N-neohexose; 2'-fucosyllactose (2'FL); 3-fucosyllactose (3-FL); difucosyllactose (DiFL); chitosan; oligosaccharides containing chitosan; heparosan; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate; acetylhyaluronic acid; hyaluronic acid; and keratan sulfate; preferably the one or more biological products are one or more LN3-derived oligosaccharides.

[0355] 16. A cell according to any of the foregoing embodiments, wherein the cell has, preferably expresses, more preferably overexpresses one or more glycosyltransferases selected from the list consisting of: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase, N-acetylmannosaminyltransferase, xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosaminyltransferase, N-glycolylneuraminyltransferase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosaminyl transferase, UDP-N-acetylglucosamine enolpyruvate transferase, and fucosaminyltransferase,

[0356] - Preferably, the fucosyltransferase is selected from the list consisting of: α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,3 / 4-fucosyltransferase, α-1,4-fucosyltransferase, and α-1,6-fucosyltransferase,

[0357] - Preferably, the sialyltransferase is selected from the list consisting of: α-2,3-sialyltransferase, α-2,6-sialyltransferase, and α-2,8-sialyltransferase,

[0358] - Preferably, the galactosyltransferase is selected from the list consisting of: β-1,3-galactosyltransferase, N-acetylglucosamine β-1,3-galactosyltransferase, β-1,4-galactosyltransferase, N-acetylglucosamine β-1,4-galactosyltransferase, α-1,3-galactosyltransferase, and α-1,4-galactosyltransferase,

[0359] - Preferably, the glucosyltransferase is selected from the list consisting of: α-glucosyltransferase, β-1,2-glucosyltransferase, β-1,3-glucosyltransferase, and β-1,4-glucosyltransferase,

[0360] - Preferably, the mannosyltransferase is selected from the list consisting of: α-1,2-mannosyltransferase, α-1,3-mannosyltransferase, and α-1,6-mannosyltransferase,

[0361] - Preferably, the N-acetylglucosaminyltransferase is selected from the list consisting of: galactoside β-1,3-N-acetylglucosaminyltransferase and β-1,6-N-acetylglucosaminyltransferase,

[0362] - Preferably, the N-acetylgalactosaminyltransferase is α-1,3-N-acetylgalactosaminyltransferase.

[0363] 17. A cell according to any one of the foregoing embodiments, wherein the cell is capable of producing, preferably producing, one or more nucleotide-activated sugars, which are preferably selected from the list consisting of: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose (GDP-Fuc), UDP-glucuronic acid, UDP-galacturonic acid, UDP-2-acetamido-2,6-dideoxy-α-L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy-α-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), CMP-sialic acid (CMP-Neu5Ac), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), GDP-rhamnose, and UDP-xylose, preferably the cell is genetically engineered to produce one or more of the nucleotide-activated sugars.

[0364] 18. A cell according to any one of the foregoing embodiments, wherein the cell comprises at least one pathway selected from the list consisting of: fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway, and N-acetylmannosaminylation pathway, preferably the cell is genetically engineered to comprise at least one of the pathways, more preferably the cell comprises at least one of the pathways, wherein at least one of the pathways has been genetically engineered.

[0365] 19. A cell according to any of the foregoing embodiments, wherein the cell comprises a catabolic pathway for a selected monosaccharide, disaccharide or oligosaccharide that is at least partially inactivated, the monosaccharide, disaccharide or oligosaccharide being involved in and / or required for the production of the one or more biological products.

[0366] 20. A cell according to any of the foregoing embodiments, wherein the cell is a bacterial, fungal, yeast, plant, animal or protozoal cell,

[0367] - Preferably, the bacterium belongs to a phylum selected from the group consisting of Proteobacteria, Firmicutes, Cyanobacteria, Deinococcus-Thermus and Actinobacteria; more preferably, the bacterium belongs to a family selected from the group consisting of Enterobacteriaceae, Bacillaceae, Lactobacillaceae, Corynebacteriaceae and Vibrionaceae; even more preferably, the bacterium is selected from the list consisting of Escherichia coli strains, Bacillus subtilis strains, Vibrio natriegens strains; even more preferably, the Escherichia coli strain is a K12 strain; most preferably, the Escherichia coli K12 strain is Escherichia coli MG1655,

[0368] - Preferably, the fungus belongs to a genus selected from the group consisting of Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus,

[0369] - Preferably, the yeast belongs to a genus selected from the group consisting of Saccharomycodes, Zygosaccharomyces, Pichia, Komagataella, Hansenula, Yarrowia, Starmerella, Kluyveromyces, Debaryomyces, Candida, Schizosaccharomyces, Schwanniomyces or Torulaspora; more preferably, the yeast is selected from the group consisting of Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Pichia methanolica, Pichia stipitis, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Yarrowia lipolytica, Zygosaccharomyces rouxii and Zygosaccharomyces bailii,

[0370] - Preferably, the plant cell is an algal cell or is derived from a tobacco, alfalfa, rice, tomato, cotton, oilseed rape, soybean, maize or corn plant,

[0371] - Preferably, the animal cells are derived from insect, amphibian, reptilian, invertebrate, fish, avian or mammalian cells (excluding human embryonic stem cells); more preferably, the mammalian cells are selected from the list consisting of: epithelial cells, embryonic kidney cells, fibroblasts, COS cells, Chinese hamster ovary (CHO) cells, murine myeloma cells, NIH-3T3 cells, milk cells derived from mammalian induced pluripotent stem cells, more preferably the mammalian induced pluripotent stem cells are human induced pluripotent stem cells, mammary epithelial cells after parturition, polarized mammary cells; more preferably, the polarized mammary cells are selected from the group consisting of live primary mammary epithelial cells, live mammary myoepithelial cells, live mammary progenitor cells, live immortalized mammary epithelial cells, live immortalized mammary myoepithelial cells, live immortalized mammary progenitor cells, non-mammary adult stem cells or derivatives thereof; more preferably, the insect cells are derived from Spodoptera frugiperda, Bombyx mori, Mamestra brassicae, Trichoplusia ni or Drosophila melanogaster,

[0372] - Preferably, the protozoan cells are Leishmania agamae cells.

[0373] 21. A cell according to any of the foregoing embodiments, wherein the cell is selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably selected from the group consisting of yeast cells, bacterial cells, archaeal cells, algal cells and fungal cells.

[0374] 22. A cell according to any of the foregoing embodiments, wherein the cell is Escherichia coli or yeast having a lactose permease positive phenotype, preferably wherein the lactose permease is encoded by the gene LacY or LAC12, respectively.

[0375] 23. A method for producing one or more biological products, the method comprising the steps of:

[0376] a) culturing and / or incubating a cell according to any of embodiments 1 to 22, preferably a single cell, under conditions that permit expression of the sugar importer and production of the one or more biological products,

[0377] b) preferably, separating the one or more biological products from the culture or incubation, preferably purifying the one or more biological products from the culture or incubation.

[0378] 24. The method according to embodiment 23, wherein the one or more biological products are selected from the list comprising: sugars; monosaccharides; activated monosaccharides; phosphorylated monosaccharides; disaccharides; oligosaccharides; polysaccharides; sugars in milk; sugars in mammalian milk; mammalian milk oligosaccharides (MMO); sugars in human milk; human milk oligosaccharides (HMO); neutral (uncharged) sugars; negatively charged sugars; fucosylated sugars; sialylated sugars; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; O-antigen; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; LN3; lacto-N-tetraose (LNT); lacto-N-neotetraose (LNnT); oligosaccharides derived from LN3; 3'-sialyllactose (3'SL); 6'-sialyllactose (6'SL); sialyllacto-N-tetraose a (LSTa); sialyllacto-N-tetraose b (LSTb); sialyllacto-N-tetraose c (LSTc); sialyllacto-N-tetraose d (LSTd); lacto-N-fucopentaose I; lacto-N-neofucopentaose; lacto-N-fucopentaose II; lacto-N-fucopentaose III; lacto-N-fucopentaose V; lacto-N-neofucopentaose V; lacto-N-difucosylhexose I; lacto-N-neodifucosylhexose; lacto-N-difucosylhexose II; monofucosyllacto-N-hexose III; difucosyllacto-N-hexose a; 6'-galactosyllactose; 3'-galactosyllactose; lacto-N-hexose; lacto-N-neohexose; 2'-fucosyllactose (2'FL); 3-fucosyllactose (3-FL); difucosyllactose (DiFL); chitosan; oligosaccharides containing chitosan; heparosan; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate; acetylhyaluronic acid; hyaluronic acid; and keratan sulfate; preferably the one or more biological products are one or more LN3-derived oligosaccharides.

[0379] 25. The method according to any one of embodiments 23 or 24, wherein the cells produce the one or more biological products in a higher yield and / or higher purity compared to cells having the same genetic composition but lacking the expression of the sugar importer.

[0380] 26. The method according to any one of embodiments 23 to 25, wherein the one or more biological products are one or more LN3-derived oligosaccharides, and wherein the cells produce the one or more LN3-derived oligosaccharides in a higher yield and / or higher purity compared to cells having the same genetic composition but lacking the expression of the sugar importer.

[0381] 27. The method according to any one of embodiments 23 to 26, wherein the culture medium comprises at least one carbon source selected from the group consisting of glucose, fructose, sucrose, and glycerol. <{

[0382] 28. The method according to any one of embodiments 23 to 27, wherein the culture or incubation medium comprises at least one compound selected from the group consisting of lactose, galactose, glucose, UDP-GlcNAc, GlcNAc, UDP-Gal, UDP-Glc, and LN3.

[0383] 29. The method according to any one of embodiments 23 to 28, wherein the culture or incubation medium comprises one or more precursors for producing the one or more biological products.

[0384] 30. The method according to embodiment 29, wherein the one or more precursors in the culture or incubation medium are produced by the cells, and / or wherein the one or more precursors are taken up by the cells via the sugar importer.

[0385] 31. The method according to any one of embodiments 23 to 30, wherein the one or more biological products, preferably all of the biological products, are recovered from the culture or incubation medium and / or the cells.

[0386] 32. The method according to any one of embodiments 23 to 31, wherein the method results in the production of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L of the one or more biological products in the final volume of the culture or incubation.

[0387] 33. Use of the cells according to any one of embodiments 1 to 22 for producing one or more biological products, preferably the one or more biological products are one or more LN3-derived oligosaccharides.

[0388] 34. Use of the method according to any one of embodiments 23 to 32 for producing one or more biological products, preferably the one or more biological products are one or more LN3-derived oligosaccharides.

[0389] 35. A sugar importer having uptake activity with respect to lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc) and comprising a polypeptide sequence having a deletion, insertion, and / or mutation of one or more amino acid residues at the following positions:

[0390] - the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO:04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11, or 13, preferably the deletion, insertion, and / or mutation is in a non-TM helix or at the N-terminus of a non-TM helix, and / or

[0391] - the C-terminus of the last TM domain of any one of SEQ ID NO:04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11, or 13, preferably the deletion, insertion, and / or mutation is in a non-TM helix or at the C-terminus of a non-TM helix.

[0392] 36. The sugar importer according to embodiment 35, wherein the sugar importer further has uptake activity with respect to one or more other sugars different from LN3, and the one or more other sugars different from LN3 are selected from the list including monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

[0393] 37. A sugar importer having uptake activity with respect to lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc) for use in the production of one or more biological products, wherein the sugar importer:

[0394] - is derived from the major facilitator superfamily (MFS) of transporters,

[0395] - comprises a polypeptide sequence comprising the IPR001927 domain as defined by InterPro 90.0 released on August 4, 2022,

[0396] - comprises a polypeptide sequence that is at least 25% identical to any one of the polypeptide sequences shown by SEQ ID NO:04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11, or 13 over a segment of at least 150 amino acid residues, preferably at least 200 amino acid residues,

[0397] - comprises a polypeptide sequence that is at least 25% identical to any one of the full-length polypeptide sequences shown by SEQ ID NO:04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11, or 13,

[0398] - Comprising a polypeptide sequence shown by any one of SEQ ID NO:04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13,

[0399] - Comprising a polypeptide sequence having a deletion, insertion and / or mutation of one or more amino acid residues at the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO:04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13, preferably the deletion, insertion and / or mutation is in a non-TM helix or at the N-terminus of a non-TM helix, and / or

[0400] - Comprising a polypeptide sequence having a deletion, insertion and / or mutation of one or more amino acid residues at the C-terminus of the last transmembrane (TM) domain of any one of SEQ ID NO:04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13, preferably the deletion, insertion and / or mutation is in a non-TM helix or at the C-terminus of a non-TM helix,

[0401] and wherein the biological product is selected from the list comprising: sugars; monosaccharides; activated monosaccharides; phosphorylated monosaccharides; disaccharides; oligosaccharides; polysaccharides; sugars in milk; sugars in mammalian milk; mammalian milk oligosaccharides (MMO); sugars in human milk; human milk oligosaccharides (HMO); neutral (uncharged) sugars; negatively charged sugars; fucosylated sugars; sialylated sugars; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; O-antigens; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; LN3; lacto-N-tetraose (LNT); lacto-N-neotetraose (LNnT); oligosaccharides derived from LN3; 3'-sialyllactose (3'SL); 6'-sialyllactose (6'SL); sialyllacto-N-tetraose a (LSTa); sialyllacto-N-tetraose b (LSTb); sialyllacto-N-tetraose c (LSTc); sialyllacto-N-tetraose d (LSTd); lacto-N-fucopentaose I; lacto-N-neofucopentaose; lacto-N-fucopentaose II; lacto-N-fucopentaose III; lacto-N-fucopentaose V; lacto-N-neofucopentaose V; lacto-N-difucosylhexose I; lacto-N-neodifucosylhexose; lacto-N-difucosylhexose II; monofucosyllacto-N-hexose III; difucosyllacto-N-hexose a; 6'-galactosyllactose; 3'-galactosyllactose; lacto-N-hexose; lacto-N-neohexose; 2'-fucosyllactose (2'FL); 3-fucosyllactose (3-FL); difucosyllactose (DiFL); chitosan; oligosaccharides containing chitosan; heparosan; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate; acetylhyaluronic acid; hyaluronic acid; and keratan sulfate; preferably the one or more biological products are one or more LN3-derived oligosaccharides.

[0402] 38. A sugar importer having uptake activity with respect to LN3 for use in producing one or more biological products according to embodiment 37, wherein the sugar importer further comprises uptake activity with respect to one or more other sugars different from LN3, and wherein the one or more other sugars different from LN3 are selected from the list comprising monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

[0403] 39. An isolated nucleic acid molecule encoding a sugar importer having uptake activity with respect to lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc), wherein the sugar importer:

[0404] - is derived from the major facilitator superfamily (MFS) of transporters,

[0405] - comprises a polypeptide sequence comprising the IPR001927 domain as defined by InterPro 90.0 released on August 4, 2022,

[0406] - comprises a polypeptide sequence that is at least 25% identical to any one of the polypeptide sequences shown by SEQ ID NO:04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13 over a segment of at least 150 amino acid residues, preferably at least 200 amino acid residues,

[0407] - comprises a polypeptide sequence that is at least 25% identical to any one of the full-length polypeptide sequences shown by SEQ ID NO:04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13,

[0408] - comprises a polypeptide sequence shown by any one of SEQ ID NO:04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13,

[0409] - comprises a polypeptide sequence having a deletion, insertion and / or mutation of one or more amino acid residues at the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO:04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13, preferably the deletion, insertion and / or mutation is in a non-TM helix or at the N-terminus of a non-TM helix, and / or

[0410] - comprises a polypeptide sequence having a deletion, insertion and / or mutation of one or more amino acid residues at the C-terminus of the last transmembrane (TM) domain of any one of SEQ ID NO:04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13, preferably the deletion, insertion and / or mutation is in a non-TM helix or at the C-terminus of a non-TM helix.

[0411] 40. A vector comprising the isolated nucleic acid molecule according to embodiment 39.

[0412] Use of the isolated nucleic acid molecule according to embodiment 39 for producing one or more biological products selected from the list consisting of: sugars; monosaccharides; activated monosaccharides; phosphorylated monosaccharides; disaccharides; oligosaccharides; polysaccharides; sugars in milk; sugars in mammalian milk; mammalian milk oligosaccharides (MMO); sugars in human milk; human milk oligosaccharides (HMO); neutral (uncharged) sugars; negatively charged sugars; fucosylated sugars; sialylated sugars; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; O-antigens; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; LN3; lacto-N-tetraose (LNT); lacto-N-neotetraose (LNnT); oligosaccharides derived from LN3; 3'-sialyllactose (3'SL); 6'-sialyllactose (6'SL); sialyllacto-N-tetraose a (LSTa); sialyllacto-N-tetraose b (LSTb); sialyllacto-N-tetraose c (LSTc); sialyllacto-N-tetraose d (LSTd); lacto-N-fucopentaose I; lacto-N-neofucopentaose; lacto-N-fucopentaose II; lacto-N-fucopentaose III; lacto-N-fucopentaose V; lacto-N-neofucopentaose V; lacto-N-difucosylhexose I; lacto-N-neodifucosylhexose; lacto-N-difucosylhexose II; monofucosyllacto-N-hexose III; difucosyllacto-N-hexose a; 6'-galactosyllactose; 3'-galactosyllactose; lacto-N-hexose; lacto-N-neohexose; 2'-fucosyllactose (2'FL); 3-fucosyllactose (3-FL); difucosyllactose (DiFL); chitosan; oligosaccharides containing chitosan; heparosan; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate; acetylhyaluronic acid; hyaluronic acid; and keratan sulfate; preferably the one or more biological products are one or more LN3-derived oligosaccharides.

[0413] Use of the vector according to embodiment 40 for producing one or more biological products selected from the list comprising: sugars; monosaccharides; activated monosaccharides; phosphorylated monosaccharides; disaccharides; oligosaccharides; polysaccharides; sugars in milk; sugars in mammalian milk; mammalian milk oligosaccharides (MMO); sugars in human milk; human milk oligosaccharides (HMO); neutral (uncharged) sugars; negatively charged sugars; fucosylated sugars; sialylated sugars; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; O-antigen; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; LN3; lacto-N-tetraose (LNT); lacto-N-neotetraose (LNnT); oligosaccharides derived from LN3; 3'-sialyllactose (3'SL); 6'-sialyllactose (6'SL); sialyllacto-N-tetraose a (LSTa); sialyllacto-N-tetraose b (LSTb); sialyllacto-N-tetraose c (LSTc); sialyllacto-N-tetraose d (LSTd); lacto-N-fucopentaose I; lacto-N-neofucopentaose; lacto-N-fucopentaose II; lacto-N-fucopentaose III; lacto-N-fucopentaose V; lacto-N-neofucopentaose V; lacto-N-difucosylhexose I; lacto-N-neodifucosylhexose; lacto-N-difucosylhexose II; monofucosyllacto-n-hexose III; difucosyllacto-N-hexose a; 6'-galactosyllactose; 3'-galactosyllactose; lacto-N-hexose; lacto-N-neohexose; 2'-fucosyllactose (2'FL); 3-fucosyllactose (3-FL); difucosyllactose (DiFL); chitosan; oligosaccharides containing chitosan; heparosan; chondroitin sulfate; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate; acetylhyaluronic acid; hyaluronic acid; and keratan sulfate; preferably the one or more biological products are one or more LN3-derived oligosaccharides.

[0414] 43. Use of a sugar importer having uptake activity for lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc) for producing one or more biological products selected from the list consisting of: sugars; monosaccharides; activated monosaccharides; phosphorylated monosaccharides; disaccharides; oligosaccharides; polysaccharides; sugars in milk; sugars in mammalian milk; mammalian milk oligosaccharides (MMO); sugars in human milk; human milk oligosaccharides (HMO); neutral (uncharged) sugars; negatively charged sugars; fucosylated sugars; sialylated sugars; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-disaccharide; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; O-antigen; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; LN3; lacto-N-tetraose (LNT); lacto-N-neotetraose (LNnT); oligosaccharides derived from LN3; 3'-sialyllactose (3'SL); 6'-sialyllactose (6'SL); sialyllacto-N-tetraose a (LSTa); sialyllacto-N-tetraose b (LSTb); sialyllacto-N-tetraose c (LSTc); sialyllacto-N-tetraose d (LSTd); lacto-N-fucopentaose I; lacto-N-neofucopentaose; lacto-N-fucopentaose II; lacto-N-fucopentaose III; lacto-N-fucopentaose V; lacto-N-neofucopentaose V; lacto-N-difucosylhexose I; lacto-N-neodifucosylhexose; lacto-N-difucosylhexose II; monofucosyllacto-n-hexose III; difucosyllacto-N-hexose a; 6'-galactosyllactose; 3'-galactosyllactose; lacto-N-hexose; lacto-N-neohexose; 2'-fucosyllactose (2'FL); 3-fucosyllactose (3-FL); difucosyllactose (DiFL); chitosan; oligosaccharides containing chitosan; heparosan; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate; acetylhyaluronic acid; hyaluronic acid; and keratan sulfate; preferably the one or more biological products are one or more LN3-derived oligosaccharides,

[0415] wherein the sugar importer:

[0416] - is derived from the major facilitator superfamily (MFS) of transporters,

[0417] - comprising a polypeptide sequence that comprises the IPR001927 domain as defined by InterPro 90.0 released on August 4, 2022,

[0418] - comprising a polypeptide sequence that is at least 25% identical to any one of the polypeptide sequences shown in SEQ ID NO: 04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13 over a segment of at least 150 amino acid residues, preferably at least 200 amino acid residues,

[0419] - comprising a polypeptide sequence that is at least 25% identical to any one of the full-length polypeptide sequences shown in SEQ ID NO: 04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13,

[0420] - comprising a polypeptide sequence as shown by any one of SEQ ID NO: 04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13,

[0421] - comprising a polypeptide sequence having a deletion, insertion and / or mutation of one or more amino acid residues at the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO: 04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13, preferably said deletion, insertion and / or mutation being in a non-TM helix or at the N-terminus of a non-TM helix, and / or

[0422] - comprising a polypeptide sequence having a deletion, insertion and / or mutation of one or more amino acid residues at the C-terminus of the last transmembrane (TM) domain of any one of SEQ ID NO: 04, 12, 02, 01, 03, 05, 06, 07, 08, 09, 10, 11 or 13, preferably said deletion, insertion and / or mutation being in a non-TM helix or at the C-terminus of a non-TM helix.

[0423] More particularly, the present invention relates to the following preferred specific embodiments:

[0424] 1. A cell for producing one or more biological products, wherein the cell:

[0425] - capable of producing, preferably producing, one or more precursors used in the production of at least one of the one or more biological products, preferably the cell is genetically engineered to produce at least one of the one or more precursors used in the production of at least one of the one or more biological products, more preferably the cell is genetically engineered to produce all of the one or more precursors used in the production of at least one of the one or more biological products, and

[0426] - genetically engineered to express, preferably overexpress, a polynucleotide sequence encoding a sugar importer that internalizes at least one of the one or more precursors into the cell.

[0427] 2. The cell according to particular embodiment 1, wherein the sugar importer has uptake activity with respect to lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc).

[0428] 3. The cell according to any one of particular embodiments 1 or 2, wherein:

[0429] - one of the one or more precursors is LN3, or

[0430] - one of the one or more precursors is LN3 and the sugar importer has uptake activity with respect to the LN3 produced by the cell.

[0431] 4. A cell for producing one or more biological products, wherein the cell is genetically engineered to express, preferably overexpress, a polynucleotide sequence encoding a sugar importer having uptake activity with respect to lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc).

[0432] 5. The cell according to any one of the foregoing particular embodiments, wherein the sugar importer comprises a polypeptide sequence that:

[0433] - is derived from the major facilitator superfamily (MFS) of transporters,

[0434] - comprises an IPR domain selected from the list consisting of: IPR001927, IPR002178, IPR016152, IPR018043, IPR020846, IPR036259, and IPR039672 as defined by InterPro 90.0 released on August 4, 2022,

[0435] - comprises the PF13347 domain and / or the PF00359 domain as defined by PFAM 32.0 released in September 2018,

[0436] - Comprising a PANTHER domain selected from the list consisting of: PTHR11328, PTHR11328:SF24, PTHR11328:SF36, and PTHR11328:SF39 as defined by PANTHER 18.0 released on September 17, 2023,

[0437] - Comprising the cd17332 domain and / or the cd00211 domain as defined by the Conserved Domain Database CDD 3.20 released in September 2022,

[0438] - Being at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% identical to any one of the polypeptide sequences shown by SEQ ID NO:04, 12, 02, 01, 03, 09, 16, 19, 20, or 21 on a segment of at least 50 amino acid residues, at least 100 amino acid residues, at least 150 amino acid residues, at least 200 amino acid residues, at least 250 amino acid residues,

[0439] - Being at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% identical to any one of the full-length polypeptide sequences shown by SEQ ID NO:04, 12, 02, 01, 03, 09, 16, 19, 20, or 21,

[0440] - Being shown by any one of SEQ ID NO:04, 12, 02, 01, 03, 09, 16, 19, 20, or 21,

[0441] - Having a deletion, insertion, and / or mutation of one or more amino acid residues at the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO:12, 02, 01, 09, 19, 20, or 21, preferably the deletion, insertion, and / or mutation is in a non-TM helix or at the N-terminus of a non-TM helix, and / or

[0442] - Having a deletion, insertion, and / or mutation of one or more amino acid...

Claims

1. A cell for producing one or more biological products, wherein the cell: - is capable of producing, preferably producing, one or more precursors used in the production of at least one of the one or more biological products, preferably the cell is genetically modified to produce at least one of the one or more precursors used in the production of at least one of the one or more biological products, more preferably the cell is genetically modified to produce all of the one or more precursors used in the production of at least one of the one or more biological products, and - is genetically modified to express, preferably overexpress, a polynucleotide sequence encoding a sugar importer that internalizes at least one of the one or more precursors into the cell.

2. The cell according to claim 1, wherein the sugar importer has uptake activity with respect to lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc).

3. The cell according to any one of claims 1 or 2, wherein: - one of the one or more precursors is LN3, or - one of the one or more precursors is LN3 and the sugar importer has uptake activity with respect to the LN3 produced by the cell.

4. A cell for producing one or more biological products, wherein the cell is genetically modified to express, preferably overexpress, a polynucleotide sequence encoding a sugar importer having uptake activity with respect to lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc).

5. The cell according to any one of the preceding claims, wherein the sugar importer comprises a polypeptide sequence that: - is derived from the major facilitator superfamily (MFS) of transporters, - comprises an IPR domain selected from the list consisting of: IPR001927, IPR002178, IPR016152, IPR018043, IPR020846, IPR036259, and IPR039672 as defined by InterPro90.0 released on August 4, 2022, - comprises a PF13347 domain and / or a PF00359 domain as defined by PFAM 32.0 released in September 2018, - comprises a PANTHER domain selected from the list consisting of: PTHR11328, PTHR11328:SF24, PTHR11328:SF36, and PTHR11328:SF39 as defined by PANTHER 18.0 released on September 17, 2023, - comprises a cd17332 domain and / or a cd00211 domain as defined by the Conserved Domain Database CDD 3.20 released in September 2022, -Identical by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% with any one of the polypeptide sequences shown by SEQ ID NO:04, 12, 02, 01, 03, 09, 16, 19, 20 or 21 on a segment of at least 50 amino acid residues, at least 100 amino acid residues, at least 150 amino acid residues, at least 200 amino acid residues, at least 250 amino acid residues, -Identical by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% with any one of the full-length polypeptide sequences shown by SEQ ID NO:04, 12, 02, 01, 03, 09, 16, 19, 20 or 21, -Shown by any one of SEQ ID NO:04, 12, 02, 01, 03, 09, 16, 19, 20 or 21, -Having a deletion, insertion and / or mutation of one or more amino acid residues at the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO:12, 02, 01, 09, 19, 20 or 21, preferably the deletion, insertion and / or mutation is in a non-TM helix or at the N-terminus of a non-TM helix, and / or -Having a deletion, insertion and / or mutation of one or more amino acid residues at the C-terminus of the last transmembrane (TM) domain of any one of SEQ ID NO:12, 02, 01, 09, 19, 20 or 21, preferably the deletion, insertion and / or mutation is in a non-TM helix or at the C-terminus of a non-TM helix.

6. The cell according to any one of the preceding claims, wherein the sugar importer is derived from the major facilitator superfamily (MFS) of transporters, and -Comprising a polypeptide sequence that comprises the IPR001927 domain defined by InterPro 90.0 released on August 4, 2022, and -Comprising a polypeptide sequence that comprises 12 transmembrane (TM) domains, which have the conserved domain [AGSV][HNQ][ACDEGNQSTV]XX[FWY]XXXXX (not L) shown by SEQ ID NO:17 present in the first TM domain, where X can be any amino acid residue, preferably having the conserved domain [AGS]Q[ACGNQSTV]XX[FWY] shown by SEQ ID NO:18, where X can be any amino acid residue, wherein the second amino acid residue of SEQ ID NO:17, preferably the second amino acid residue of SEQ ID NO:18 is aligned with Lys18 of the polypeptide having SEQ ID NO:

22.

7. The cell according to any one of claims 2 to 6, wherein the sugar importer further comprises an uptake activity with respect to one or more other sugars different from LN3, and the one or more other sugars different from LN3 are selected from the list consisting of monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

8. The cell according to any one of claims 2 to 7, wherein the sugar importer has an uptake activity with respect to LN3, but does not have an uptake activity with respect to a) LNT (lacto-N-tetraose, Galβ1-3GlcNAcβ1-3Galβ1-4Glc) and / or b) LNnT (lacto-N-neotetraose, Galβ1-4GlcNAcβ1-3Galβ1-4Glc).

9. The cell according to any one of the preceding claims, wherein the one or more biological products are selected from the list comprising: sugars; monosaccharides; activated monosaccharides; phosphorylated monosaccharides; disaccharides; oligosaccharides; polysaccharides; sugars in milk; sugars in mammalian milk; mammalian milk oligosaccharides (MMO); sugars in human milk; human milk oligosaccharides (HMO); neutral (uncharged) sugars; negatively charged sugars; fucosylated sugars; sialylated sugars; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; O-antigen; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; LN3; lacto-N-tetraose (LNT); lacto-N-neotetraose (LNnT); oligosaccharides derived from LN3; 3'-sialyllactose (3'SL); 6'-sialyllactose (6'SL); sialyllacto-N-tetraose a (LSTa); sialyllacto-N-tetraose b (LSTb); sialyllacto-N-tetraose c (LSTc); sialyllacto-N-tetraose d (LSTd); lacto-N-fucopentaose I; lacto-N-neofucopentaose; lacto-N-fucopentaose II; lacto-N-fucopentaose III; lacto-N-fucopentaose V; lacto-N-neofucopentaose V; lacto-N-difucosylhexose I; lacto-N-neodifucosylhexose; lacto-N-difucosylhexose II; monofucosyllacto-N-hexose III; difucosyllacto-N-hexose a; 6'-galactosyllactose; 3'-galactosyllactose; lacto-N-hexose; lacto-N-neohexose; 2'-fucosyllactose (2'FL); 3-fucosyllactose (3-FL); difucosyllactose (DiFL); chitosan; oligosaccharides containing chitosan; heparosan; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate; acetylhyaluronic acid; hyaluronic acid; and keratan sulfate, preferably the one or more biological products are one or more LN3-derived oligosaccharides.

10. The cell according to any one of the preceding claims, wherein the cell is further genetically modified to produce the one or more biological products.

11. The cell according to any one of claims 4 to 10, wherein the cell is capable of producing the one or more biological products from one or more precursors, preferably producing the one or more biological products from one or more precursors.

12. The cell according to claim 11, wherein the cell is capable of producing, preferably producing, at least one of the one or more precursors, preferably the cell is genetically engineered to produce at least one of the one or more precursors; more preferably the cell is capable of producing, preferably producing, all of the one or more precursors, even more preferably the cell is genetically engineered to produce all of the one or more precursors.

13. The cell according to any one of claims 11 or 12, wherein at least one of the one or more precursors is internalized into the cell via the sugar importer.

14. The cell according to any one of claims 1 to 3, 11 to 13, wherein the one or more precursors are selected from the list comprising: sugars; monosaccharides; activated monosaccharides; phosphorylated monosaccharides; disaccharides; oligosaccharides; polysaccharides; sugars in milk; sugars in mammalian milk; mammalian milk oligosaccharides (MMO); sugars in human milk; human milk oligosaccharides (HMO); neutral (uncharged) sugars; negatively charged sugars; fucosylated sugars; sialylated sugars; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; O-antigen; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; LN3; lacto-N-tetraose (LNT); lacto-N-neotetraose (LNnT); oligosaccharides derived from LN3; 3'-sialyllactose (3'SL); 6'-sialyllactose (6'SL); sialyllacto-N-tetraose a (LSTa); sialyllacto-N-tetraose b (LSTb); sialyllacto-N-tetraose c (LSTc); sialyllacto-N-tetraose d (LSTd); lacto-N-fucopentaose I; lacto-N-neofucopentaose; lacto-N-fucopentaose II; lacto-N-fucopentaose III; lacto-N-fucopentaose V; lacto-N-neofucopentaose V; lacto-N-difucosylhexose I; lacto-N-neodifucosylhexose; lacto-N-difucosylhexose II; monofucosyllacto-N-hexose III; difucosyllacto-N-hexose a; 6'-galactosyllactose; 3'-galactosyllactose; lacto-N-hexose; lacto-N-neohexose; 2'-fucosyllactose (2'FL); 3-fucosyllactose (3-FL); difucosyllactose (DiFL); chitosan; oligosaccharides containing chitosan; heparosan; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate; acetylhyaluronic acid; hyaluronic acid; and keratan sulfate; preferably the one or more biological products are one or more LN3-derived oligosaccharides.

15. The cell according to any one of the preceding claims, wherein the cell: i) having, preferably expressing, more preferably overexpressing one or more glycosyltransferases selected from the list consisting of: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase, N-acetylmannosaminyltransferase, xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosaminyltransferase, N-glycolylneuraminyltransferase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosaminyltransferase, UDP-N-acetylglucosamine enolpyruvate transferase, and fucosaminyltransferase, - preferably, the fucosyltransferase is selected from the list consisting of: α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,3 / 4-fucosyltransferase, α-1,4-fucosyltransferase, and α-1,6-fucosyltransferase, - preferably, the sialyltransferase is selected from the list consisting of: α-2,3-sialyltransferase, α-2,6-sialyltransferase, and α-2,8-sialyltransferase, - preferably, the galactosyltransferase is selected from the list consisting of: β-1,3-galactosyltransferase, N-acetylglucosamine β-1,3-galactosyltransferase, β-1,4-galactosyltransferase, N-acetylglucosamine β-1,4-galactosyltransferase, α-1,3-galactosyltransferase, and α-1,4-galactosyltransferase, - preferably, the glucosyltransferase is selected from the list consisting of: α-glucosyltransferase, β-1,2-glucosyltransferase, β-1,3-glucosyltransferase, and β-1,4-glucosyltransferase, - preferably, the mannosyltransferase is selected from the list consisting of: α-1,2-mannosyltransferase, α-1,3-mannosyltransferase, and α-1,6-mannosyltransferase, - preferably, the N-acetylglucosaminyltransferase is selected from the list consisting of: galactoside β-1,3-N-acetylglucosaminyltransferase and β-1,6-N-acetylglucosaminyltransferase, - preferably, the N-acetylgalactosaminyltransferase is α-1,3-N-acetylgalactosaminyltransferase, ii) capable of producing, preferably producing, one or more nucleotide-activated sugars, preferably selected from the list comprising: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose (GDP-Fuc), UDP-glucuronic acid, UDP-galacturonic acid, UDP-2-acetamido-2,6-dideoxy-α-L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy-α-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), CMP-sialic acid (CMP-Neu5Ac), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), GDP-rhamnose and UDP-xylose, preferably the cell is genetically engineered to produce one or more of the nucleotide-activated sugars, iii) comprising at least one pathway selected from the list comprising: fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway, preferably the cell is genetically engineered to comprise at least one of the pathways, more preferably the cell comprises at least one of the pathways, wherein at least one of the pathways has been genetically engineered, and / or iv) comprising a catabolic pathway for the selected monosaccharide, disaccharide or oligosaccharide that is at least partially inactivated, the monosaccharide, disaccharide or oligosaccharide being involved in and / or required for the production of the one or more biological products.

16. A cell according to any one of the preceding claims, wherein the cell is selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably selected from the group consisting of yeast cells, bacterial cells, archaeal cells, algal cells, and fungal cells.

17. A cell according to any one of the preceding claims, wherein the cell is Escherichia coli (E. coli) or yeast having a lactose permease-positive phenotype, preferably wherein the lactose permease is encoded by the gene LacY or LAC12, respectively.

18. A method for producing one or more biological products, the method comprising the following steps: c) culturing and / or incubating a cell according to any one of claims 1 to 17, preferably a single cell, under conditions that permit expression of the sugar importer and production of the one or more biological products, d) preferably, separating the one or more biological products from the culture or incubation, preferably purifying the one or more biological products from the culture or incubation.

19. The method according to claim 18, wherein the one or more biological products are selected from the list comprising: sugars; monosaccharides; activated monosaccharides; phosphorylated monosaccharides; disaccharides; oligosaccharides; polysaccharides; sugars in milk; sugars in mammalian milk; mammalian milk oligosaccharides (MMO); sugars in human milk; human milk oligosaccharides (HMO); neutral (uncharged) sugars; negatively charged sugars; fucosylated sugars; sialylated sugars; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; O-antigens; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; LN3; lacto-N-tetraose (LNT); lacto-N-neotetraose (LNnT); oligosaccharides derived from LN3; 3'-sialyllactose (3'SL); 6'-sialyllactose (6'SL); sialyllacto-N-tetraose a (LSTa); sialyllacto-N-tetraose b (LSTb); sialyllacto-N-tetraose c (LSTc); sialyllacto-N-tetraose d (LSTd); lacto-N-fucopentaose I; lacto-N-neofucopentaose; lacto-N-fucopentaose II; lacto-N-fucopentaose III; lacto-N-fucopentaose V; lacto-N-neofucopentaose V; lacto-N-difucosylhexose I; lacto-N-neodifucosylhexose; lacto-N-difucosylhexose II; monofucosyllacto-N-hexose III; difucosyllacto-N-hexose a; 6'-galactosyllactose; 3'-galactosyllactose; lacto-N-hexose; lacto-N-neohexose; 2'-fucosyllactose (2'FL); 3-fucosyllactose (3-FL); difucosyllactose (DiFL); chitosan; oligosaccharides containing chitosan; heparosan; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate; acetylhyaluronic acid; hyaluronic acid; and keratan sulfate; preferably the one or more biological products are one or more LN3-derived oligosaccharides.

20. The method according to any one of claims 18 or 19, wherein the cells produce the one or more biological products in a higher yield and / or higher purity compared to cells having the same genetic composition but lacking the expression of the sugar importer.

21. The method according to any one of claims 18 to 20, wherein the one or more biological products are one or more LN3-derived oligosaccharides, and wherein the cells produce the one or more LN3-derived oligosaccharides in a higher yield and / or higher purity compared to cells having the same genetic composition but lacking the expression of the sugar importer.

22. The method according to any one of claims 18 to 21, wherein - the culture medium comprises at least one carbon source selected from the group consisting of glucose, fructose, sucrose, and glycerol, - the culture or incubation medium comprises at least one compound selected from the group consisting of lactose, galactose, glucose, UDP-GlcNAc, GlcNAc, UDP-Gal, UDP-Glc, and LN3, and / or - the culture or incubation medium comprises one or more precursors for producing the one or more biological products.

23. The method according to claim 22, wherein the one or more precursors in the culture or incubation medium are produced by the cells, and / or wherein the one or more precursors are taken up by the cells via the sugar importer.

24. The method according to any one of claims 18 to 23, wherein the one or more biological products, preferably all of the biological products, are recovered from the culture or incubation medium and / or the cells.

25. The method according to any one of claims 18 to 24, wherein the method results in the production of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L of the one or more biological products in the final volume of the culture or incubation.

26. Use of the cell according to any one of claims 1 to 17 for producing one or more biological products, preferably the one or more biological products are one or more LN3-derived oligosaccharides.

27. Use of the method according to any one of claims 18 to 25 for producing one or more biological products, preferably the one or more biological products are one or more LN3-derived oligosaccharides.

28. A sugar importer having uptake activity with respect to lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc) and comprising a polypeptide sequence having a deletion, insertion, and / or mutation of one or more amino acid residues at the following positions: - the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO:12, 02, 01, 09, 19, 20, or 21, preferably the deletion, insertion, and / or mutation is in a non- - TM helix or at the N-terminus of a non-TM helix, and / or - the C-terminus of the last TM domain of any one of SEQ ID NO:12, 02, 01, 09, 19, 20, or 21, preferably the deletion, insertion, and / or mutation is in a non-TM helix or at the C-terminus of a non-TM helix.

29. The sugar importer according to claim 28, wherein the sugar importer further comprises an uptake activity with respect to one or more other sugars different from LN3, and the one or more other sugars different from LN3 are selected from the list including monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

30. The sugar importer according to any one of claims 28 or 29, wherein the sugar importer has an uptake activity with respect to LN3, but does not have an uptake activity with respect to a) LNT and / or b) LNnT.

31. Use of a sugar importer having an uptake activity with respect to lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc) for producing one or more biological products, wherein the sugar importer comprises a polypeptide sequence that: - is derived from the major facilitator superfamily (MFS) of transporters, - contains an IPR domain selected from the list including: IPR001927, IPR002178, IPR016152, IPR018043, IPR020846, IPR036259, and IPR039672 defined by InterPro90.0 released on August 4, 2022, - contains a PF13347 domain and / or a PF00359 domain defined by PFAM 32.0 released in September 2018, - contains a PANTHER domain selected from the list including: PTHR11328, PTHR11328:SF24, PTHR11328:SF36, and PTHR11328:SF39 defined by PANTHER 18.0 released on September 17, 2023, - contains a cd17332 domain and / or a cd00211 domain defined by the Conserved Domain Database CDD 3.20 released in September 2022, - is at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% identical to any one of the polypeptide sequences shown by SEQ ID NO:04, 12, 02, 01, 03, 09, 16, 19, 20, or 21 on a segment of at least 50 amino acid residues, at least 100 amino acid residues, at least 150 amino acid residues, at least 200 amino acid residues, at least 250 amino acid residues, - is at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% identical to any one of the full-length polypeptide sequences shown by SEQ ID NO:04, 12, 02, 01, 03, 09, 16, 19, 20, or 21, - is shown by any one of SEQ ID NO:04, 12, 02, 01, 03, 09, 16, 19, 20, or 21, - having a deletion, insertion, and / or mutation of one or more amino acid residues at the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO: 12, 02, 01, 09, 19, 20, or 21, preferably said deletion, insertion, and / or mutation being in a non-TM helix or at the N-terminus of a non-TM helix, and / or - having a deletion, insertion, and / or mutation of one or more amino acid residues at the C-terminus of the last transmembrane (TM) domain of any one of SEQ ID NO: 12, 02, 01, 09, 19, 20, or 21, preferably said deletion, insertion, and / or mutation being in a non-TM helix or at the C-terminus of a non-TM helix And wherein said biological product is selected from the list comprising: sugars; monosaccharides; activated monosaccharides; phosphorylated monosaccharides; disaccharides; oligosaccharides; polysaccharides; sugars in milk; sugars in mammalian milk; mammalian milk oligosaccharides (MMO); sugars in human milk; human milk oligosaccharides (HMO); neutral (uncharged) sugars; negatively charged sugars; fucosylated sugars; sialylated sugars; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; O-antigens; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, which are preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, which are preferably selected from the group consisting of N-glycans and O-glycans; LN3; lacto-N-tetraose (LNT); lacto-N-neotetraose (LNnT); oligosaccharides derived from LN3; 3'-sialyllactose (3'SL); 6'-sialyllactose (6'SL); sialyllacto-N-tetraose a (LSTa); sialyllacto-N-tetraose b (LSTb); sialyllacto-N-tetraose c (LSTc); sialyllacto-N-tetraose d (LSTd); lacto-N-fucopentaose I; lacto-N-neofucopentaose; lacto-N-fucopentaose II; lacto-N-fucopentaose III; lacto-N-fucopentaose V; lacto-N-neofucopentaose V; lacto-N-difucosylhexose I; lacto-N-neodifucosylhexose; lacto-N-difucosylhexose II; monofucosyllacto-n-hexose III; difucosyllacto-N-hexose a; 6'-galactosyllactose; 3'-galactosyllactose; lacto-N-hexose; lacto-N-neohexose; 2'-fucosyllactose (2'FL); 3-fucosyllactose (3-FL); difucosyllactose (DiFL); chitosan; oligosaccharides containing chitosan; heparosan; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate; acetylhyaluronic acid; hyaluronic acid; and keratan sulfate; preferably said one or more biological products are one or more LN3-derived oligosaccharides.

32. The use according to claim 31, wherein said sugar importer is derived from the major facilitator superfamily (MFS) of transporters, and - comprises such a polypeptide sequence, said polypeptide sequence comprising the IPR001927 domain as defined by InterPro 90.0 released on August 4, 2022, and - Comprising such a polypeptide sequence, said polypeptide sequence comprising 12 transmembrane (TM) domains, which have a conserved domain shown by SEQ ID NO:17 [AGSV][HNQ][ACDEGNQSTV]XX[FWY]XXXXX (not L) present in the first TM domain, where X can be any amino acid residue, preferably having a conserved domain shown by SEQ ID NO:18 [AGS]Q[ACGNQSTV]XX[FWY], where X can be any amino acid residue, wherein the second amino acid residue of SEQ ID NO:17, preferably the second amino acid residue of SEQ ID NO:18 is aligned with Lys18 of the polypeptide having SEQ ID NO:

22.

33. The use according to any one of claims 31 or 32, wherein the sugar importer further comprises uptake activity with respect to one or more other sugars different from LN3, wherein the one or more other sugars different from LN3 are selected from the list comprising monosaccharides, disaccharides, oligosaccharides and polysaccharides.

34. The use according to any one of claims 31 to 33, wherein the sugar importer has uptake activity with respect to LN3, but does not have uptake activity with respect to a) LNT and / or b) LNnT.

35. An isolated nucleic acid molecule encoding a sugar importer having uptake activity with respect to lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc), wherein the sugar importer comprises such a polypeptide sequence, said polypeptide sequence: - Derived from the major facilitator superfamily (MFS) of transporters, - Comprising an IPR domain selected from the list comprising: IPR001927, IPR002178, IPR016152, IPR018043, IPR020846, IPR036259 and IPR039672 defined by InterPro90.0 released on August 4, 2022, - Comprising the PF13347 domain and / or the PF00359 domain defined by PFAM 32.0 released in September 2018, - Comprising a PANTHER domain selected from the list comprising: PTHR11328, PTHR11328:SF24, PTHR11328:SF36 and PTHR11328:SF39 defined by PANTHER 18.0 released on September 17, 2023, - Comprising the cd17332 domain and / or the cd00211 domain defined by the Conserved Domain Database CDD 3.20 released in September 2022, -Identical to any one of the polypeptide sequences shown in SEQ ID NO:04, 12, 02, 01, 03, 09, 16, 19, 20 or 21 by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% over a segment of at least 50 amino acid residues, at least 100 amino acid residues, at least 150 amino acid residues, at least 200 amino acid residues, at least 250 amino acid residues, -Identical to any one of the full-length polypeptide sequences shown in SEQ ID NO:04, 12, 02, 01, 03, 09, 16, 19, 20 or 21 by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, -Shown by any one of SEQ ID NO:04, 12, 02, 01, 03, 09, 16, 19, 20 or 21, -Having a deletion, insertion and / or mutation of one or more amino acid residues at the N-terminus of the first transmembrane (TM) domain of any one of SEQ ID NO:12, 02, 01, 09, 19, 20 or 21, preferably said deletion, insertion and / or mutation is in a non-TM helix or at the N-terminus of a non-TM helix, and / or -Having a deletion, insertion and / or mutation of one or more amino acid residues at the C-terminus of the last transmembrane (TM) domain of any one of SEQ ID NO:12, 02, 01, 09, 19, 20 or 21, preferably said deletion, insertion and / or mutation is in a non-TM helix or at the C-terminus of a non-TM helix.

36. The isolated nucleic acid molecule according to claim 35, wherein the sugar importer is derived from the major facilitator superfamily (MFS) of transporters, and -Comprising a polypeptide sequence that comprises the IPR001927 domain as defined by InterPro 90.0 released on August 4, 2022, and -Comprising a polypeptide sequence that comprises 12 transmembrane (TM) domains, which have a conserved domain [AGSV][HNQ][ACDEGNQSTV]XX[FWY]XXXXX (non-L) as shown in SEQ ID NO:17 present in the first TM domain, where X can be any amino acid residue, preferably having a conserved domain [AGS]Q[ACGNQSTV]XX[FWY] as shown in SEQ ID NO:18, where X can be any amino acid residue, wherein the second amino acid residue of SEQ ID NO:17, preferably the second amino acid residue of SEQ ID NO:18 aligns with Lys18 of the polypeptide having SEQ ID NO:

22.

37. A vector comprising the isolated nucleic acid molecule according to any one of claims 35 or 36.

38. Use of the isolated nucleic acid molecule according to any one of claims 35 or 36 for producing one or more biological products selected from the list consisting of: sugars; monosaccharides; activated monosaccharides; phosphorylated monosaccharides; disaccharides; oligosaccharides; polysaccharides; sugars in milk; sugars in mammalian milk; mammalian milk oligosaccharides (MMO); sugars in human milk; human milk oligosaccharides (HMO); neutral (uncharged) sugars; negatively charged sugars; fucosylated sugars; sialylated sugars; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; O-antigen; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; LN3; lacto-N-tetraose (LNT); lacto-N-neotetraose (LNnT); oligosaccharides derived from LN3; 3'-sialyllactose (3'SL); 6'-sialyllactose (6'SL); sialyllacto-N-tetraose a (LSTa); sialyllacto-N-tetraose b (LSTb); sialyllacto-N-tetraose c (LSTc); sialyllacto-N-tetraose d (LSTd); lacto-N-fucopentaose I; lacto-N-neofucopentaose; lacto-N-fucopentaose II; lacto-N-fucopentaose III; lacto-N-fucopentaose V; lacto-N-neofucopentaose V; lacto-N-difucosylhexose I; lacto-N-neodifucosylhexose; lacto-N-difucosylhexose II; monofucosyllacto-N-hexose III; difucosyllacto-N-hexose a; 6'-galactosyllactose; 3'-galactosyllactose; lacto-N-hexose; lacto-N-neohexose; 2'-fucosyllactose (2'FL); 3-fucosyllactose (3-FL); difucosyllactose (DiFL); chitosan; oligosaccharides containing chitosan; heparosan; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate; acetylhyaluronic acid; hyaluronic acid; and keratan sulfate; preferably the one or more biological products are one or more LN3-derived oligosaccharides.

39. Use of the carrier according to claim 37 for producing one or more bioproducts selected from the list comprising: sugars; monosaccharides; activated monosaccharides; phosphorylated monosaccharides; disaccharides; oligosaccharides; polysaccharides; sugars in milk; sugars in mammalian milk; mammalian milk oligosaccharides (MMO); sugars in human milk; human milk oligosaccharides (HMO); neutral (uncharged) sugars; negatively charged sugars; fucosylated sugars; sialylated sugars; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; oligosaccharides containing lactose; non-fucosylated neutral (uncharged) oligosaccharides; O-antigens; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigen oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; LN3; lacto-N-tetraose (LNT); lacto-N-neotetraose (LNnT); oligosaccharides derived from LN3; 3'-sialyllactose (3'SL); 6'-sialyllactose (6'SL); sialyllacto-N-tetraose a (LSTa); sialyllacto-N-tetraose b (LSTb); sialyllacto-N-tetraose c (LSTc); sialyllacto-N-tetraose d (LSTd); lacto-N-fucopentaose I; lacto-N-neofucopentaose; lacto-N-fucopentaose II; lacto-N-fucopentaose III; lacto-N-fucopentaose V; lacto-N-neofucopentaose V; lacto-N-difucosylhexose I; lacto-N-neodifucosylhexose; lacto-N-difucosylhexose II; monofucosyllacto-N-hexose III; difucosyllacto-N-hexose a; 6'-galactosyllactose; 3'-galactosyllactose; lacto-N-hexose; lacto-N-neohexose; 2'-fucosyllactose (2'FL); 3-fucosyllactose (3-FL); difucosyllactose (DiFL); chitosan; oligosaccharides containing chitosan; heparosan; chondroitin sulfate; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate; acetylhyaluronic acid; hyaluronic acid; and keratan sulfate; preferably the one or more bioproducts are one or more LN3-derived oligosaccharides.

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