Production of sugars from cells with reduced lactobionic acid synthesis

Abstract

The invention is in the technical field of synthetic biology, metabolic engineering and cell culture. The present invention provides a cell for producing sugars, wherein the synthesis of lactobionic acid in the cell is deteriorated in function or knocked out. The invention further provides the use of said cells in culture or incubation. Methods for producing sugars by using the cells and purifying the sugars are also described.

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 sugars, wherein lactobionic acid synthesis in the cells is functionally impaired or knocked out. The present invention further provides the use of the cells in culture or incubation. The present invention also describes methods for producing sugars using the cells and purifying the sugars. Background Art

[0002] Sugars (e.g., disaccharides and oligosaccharides) are extremely diverse in their chemical structure and are composed of a wide variety of monosaccharides (e.g., glucose, galactose, N-acetylglucosamine, xylose, rhamnose, fucose, mannose, N-acetylneuraminic acid, N-acetylgalactosamine, galactosamine, glucosamine, glucuronic acid, and galacturonic acid). Sugars are widely distributed in all living organisms and play important roles in a variety of physiological and pathological processes (e.g., cell migration, signal transduction, cell-cell adhesion, inflammation, and immune responses). Economical production of these sugars is crucial to fully benefit from their biological advantages. An important group of sugars includes mammalian milk oligosaccharides (MMOs) and human milk oligosaccharides (HMOs), found in mammalian and human milk, respectively. A wide variety of synthetic methods have been developed, ranging from extraction to chemical synthesis to enzymatic synthesis. These methods are currently the least utilized, while biotechnological fermentation production is currently being pursued and commercialized. Methods for producing sugars, in particular oligosaccharides, are well known to those skilled in the art (e.g., as described in Faijes et al. (2019), US2010120096A, JP2013201913, WO2022 / 034067).

[0003] Lactobionic acid (LBA) can be produced by oxidation of lactose to a lactone by the dehydrogenase (lactose oxidase) system, which is further hydrolyzed to LBA. LBA can also be produced after oxidation of maltose. LBA can also be produced by lactose dehydrogenase and lactonase. LBA can also be formed from sugars other than lactose (such as, but not limited to, D-glucose, D-galactose, D-mannose, D-talose, D-xylose, D-ribose, L-arabinose, cellobiose, and D-fructose). LBA is classified as a bioacid, chemically consisting of gluconic acid bonded to galactose. LBA is known for its antioxidant, antimicrobial, chelating, stabilizer, acidulant, and wetting properties. Although LBA is of great interest to industry, its applications are rare. Due to a lack of testing and knowledge about its health effects, ingestion of LBA is still considered unsafe, although in vivo testing is beginning. Summary of the Invention SUMMARY OF THE INVENTION

[0005] It is an object of the present invention to provide tools and methods with the help of which sugars can be produced, preferably in an efficient, timely and cost-effective manner, and which produce large amounts of desired sugars with small to zero amounts of LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA.

[0006] According to the present invention, this and other objectives are achieved by providing methods and cells for producing sugars. The present invention also provides methods for purifying the sugars. Further, the present invention provides cells that have been genetically engineered as described herein and wherein the synthesis of LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA is functionally impaired or knocked out. The present invention also provides purified sugars produced by the above-mentioned processes. Upon reading the present invention, the further benefits of the teachings of the present invention will be apparent to those skilled in the art.

[0007] definition

[0008] The words used in this specification to describe the present invention and its various embodiments should be understood not only in their generally defined meanings, but also to include structures, materials, or actions that extend beyond the generally defined meanings through specific definitions in this specification. Therefore, if an element in the context of this specification can be understood to include more than one meaning, its use in the claims must be understood to be general to all possible meanings supported by this specification and by the word itself.

[0009] The various aspects and embodiments of the present invention disclosed herein should be understood not only in the order and scenario specifically described in this specification, but also in any order and any combination thereof. Each embodiment identified in this article 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 that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference in its entirety. Unless otherwise specifically stated, all words used in the singular should be considered to include the plural, and vice versa. Unless otherwise defined, all technical and scientific terms used in this article generally have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used in this article and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described in this article are those well-known and commonly used 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.

[0010] In this specification, embodiments of the present invention have been disclosed, and although specific terms are employed, these terms are used in a descriptive sense only and not for limiting purposes, and the scope of the invention is set forth in the claims below. It must be understood that the illustrative embodiment is set forth for illustrative purposes only, and it should not be considered as limiting the invention. It will be apparent to those skilled in the art that changes, other embodiments, improvements, details and uses may be made consistent with the letter and spirit of the disclosure herein and within the scope of the disclosure, and the scope of the disclosure is limited only by the claims and interpreted in accordance with patent law, including the doctrine of equivalents. In the claims below, the reference characters used to indicate claim steps are provided for convenience of description only and are not intended to imply any particular order for performing the steps, unless otherwise specifically stated.

[0011] Throughout the application, unless otherwise expressly stated, the feature "synthesize" is used interchangeably with the feature "produce". Throughout the application, unless otherwise expressly stated, the expression "can ..." is preferably replaced by the active voice of the verb, and vice versa. For example, the expression "can express" is preferably replaced by "express", and vice versa, i.e., "express" is preferably replaced by "can express". In this document and in its claims, the verbs "comprise", "have" and "comprises" and their variations are used in their non-restrictive sense to mean that the items following the word are included, but items not specifically mentioned are not excluded. Throughout the application, the verb "comprise (comprising)" can be replaced by "consisting of" or "consisting essentially of", and vice versa. In addition, the verb "consisting of" can be replaced by "consisting essentially of", which means that the composition defined in this document may contain additional components other than those specifically identified, and the additional components do not change the unique characteristics of the invention. In this document and in its claims, unless otherwise specifically stated, the verbs "comprise," "have," and "include" and their conjugations may be replaced with "consisting of" (and their conjugations) or "consisting essentially of" (and their conjugations), and vice versa. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that only one of the element be present. Thus, the indefinite article "a" or "an" often means "at least one."

[0012] Throughout this document and in the claims, unless expressly stated otherwise, the articles "a" and "an" are preferably replaced by "at least one," more preferably by "at least two," more preferably by "at least three," more preferably by "at least four," more preferably by "at least five," more preferably by "at least six," and most preferably by "at least two." When used in connection with a numerical value (e.g., "about 10") or in connection 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 margin of error is specifically specified, the expression "about" or "approximately" when used in connection with a numerical value is to be interpreted as having the same rounding as the given value. Throughout this document and in the claims thereof, unless expressly stated otherwise, the expression "x to y" (where x and y represent numerical values) refers to a numerical range with x being the lower limit of the range and y being the upper limit of the range. Herein, x and y are also included in the range.

[0013] According to the present invention, the term "polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. It should be understood that DNA or RNA containing rare bases (e.g., inosine) or modified bases (e.g., tritylated bases) are encompassed by the term "polynucleotide". It will be appreciated that DNA and RNA have been subjected to a very wide variety of modifications that 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 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" refers to any peptide or protein comprising two or more amino acids linked 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 may contain other amino acids in addition to the 20 genetically encoded amino acids. "Polypeptides" include those modified by natural processes (e.g., processing and other post-translational modifications), but also include those modified by chemical modification techniques well known to technicians. The same type of modification can be present at several sites in a given polypeptide to the same or varying extent. 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. Polypeptides can be branched or cyclic, with or without branches. Cyclic, branched, or branched annular polypeptides can be produced by natural post-translational processes and can also be prepared by completely synthetic methods.

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

[0016] "Isolated" means altered "by the hand of man" from its natural state, 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 naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated," as the term is employed herein. Similarly, a "synthetic" sequence, as the term is used herein, means any sequence that is produced synthetically rather than directly isolated from a natural source. "Synthetic," as the term is used herein, means any sequence that is produced synthetically rather than directly isolated from a natural source.

[0017] "Recombinant" means genetically modified DNA that is produced by transplanting or splicing genes 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 with respect to cells or host cells 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 being transformed with at least one heterologous or exogenous gene, or as being transformed by the introduction of at least one heterologous or exogenous gene. Recombinant or metabolically engineered or genetically engineered or transgenic cells may contain genes that are not found in the native (non-recombinant) form of the cell. Recombinant cells may also contain genes found in the native form of the cell, wherein the genes have been modified by artificial means and reintroduced into the cell. The term also encompasses cells comprising a 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 mutation, CrispR, riboswitch, recombineering, ssDNA mutagenesis, transposon mutagenesis, and related techniques known to those skilled 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 a nucleic acid endogenous to the cell (e.g., knocking out a gene) using well-known techniques common to those skilled in the art.

[0019] As used herein, a "heterologous sequence" or "heterologous nucleic acid" is a sequence or nucleic acid that is derived from a source foreign to a particular cell (e.g., from a different species) or, if derived from the same source, has been modified from its original form or position in the genome. Thus, a heterologous nucleic acid operably linked to a promoter is derived from a source different from the source from which the promoter is derived or, if derived from the same source, has been modified from its original form or position in the genome. A heterologous sequence can be stably introduced into the genome of a host cell, for example, by transfection, transformation, conjugation, or transduction, where techniques that will depend on the cell and the sequence to be introduced can be applied. 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, NY (1989). The terms "mutant" or "modified" cell, as used within the context of the present invention, refers to a genetically modified cell.

[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's chromosomes, and whose expression control is unchanged compared to the natural control mechanisms that act on its expression. The term "exogenous" refers to any polynucleotide, polypeptide, or protein sequence that originates from outside the cell in question and is not a natural part of the cell, or that does not occur at its natural location in the cell's chromosomes or on a plasmid.

[0021] When used in relation to polynucleotides, genes, nucleic acids, polypeptides or enzymes, the term "heterologous" refers to polynucleotides, genes, nucleic acids, polypeptides or enzymes from or derived from other sources except the host organism. In contrast, "homologous" polynucleotides, genes, nucleic acids, polypeptides or enzymes are used herein to represent polynucleotides, genes, nucleic acids, polypeptides or enzymes derived from the host organism. When referring to gene regulatory sequences or auxiliary nucleic acid sequences (e.g., promoters, 5' untranslated regions, 3' untranslated regions, poly A addition sequences, intron sequences, splice sites, ribosome bind sites, internal ribosome entry sequences, genome homology regions, recombination sites etc.) for maintaining or manipulating gene sequences, "heterologous" means that the regulatory sequences or auxiliary sequences are not naturally associated with the genes to which the regulatory sequences or auxiliary nucleic acid sequences are juxtaposed in constructs, genomes, chromosomes or episomes. Thus, a promoter that is operably linked to a gene to which it is not operably linked in its native state (i.e., in the genome of a non-genetically modified organism) is referred to herein as a "heterologous promoter," even though the promoter may be derived from the same species (or, in some cases, the same organism) as the gene to which it is linked.

[0022] " modified expression " of term gene relates to the change in expression compared to the wild-type expression of described gene in any stage of the production process of desired sugar.Described modified expression is expression lower or higher compared to wild type, wherein term " higher expression " is also defined as " overexpression " of described gene in the case of endogenous gene, or is defined as " expression " in the case of heterologous gene not present in wild-type strain.Lower expression is obtained by means of common well-known technology (such as using siRNA, CrispR, CrispRi, riboswitch, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutant gene, knockout gene, transposon mutagenesis etc.) for those skilled in the art, these technologies are used to change gene in this way, so that described gene is " less able " (that is, statistically significantly " less able " compared to functional wild-type gene) or is not able to produce functional final product completely (such as, the gene of knockout).Term " riboswitch " used in this article is defined as the part of messenger RNA that folds into the complex structure that blocks expression by interfering translation. The binding of effector molecules induces conformational changes, thereby allowing post-transcriptional regulation of expression. In addition to changing the target gene as described above to obtain lower expression, lower expression can also be obtained by changing 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 completely changing the promoter sequence to a constitutive promoter with lower expression intensity than the wild type, or an inducible promoter that causes regulated expression, or a repressible promoter that causes regulated expression. Overexpression or expression is obtained by means of well-known techniques common to those skilled in the art (e.g., use of artificial transcription factors, de novo design of promoter sequences, ribosome engineering, introduction or reintroduction of expression modules at euchromatin, use of high copy number plasmids), wherein the gene is part of an "expression cassette", which refers to any sequence in which a promoter sequence, an untranslated region sequence (including a ribosome binding sequence, a Shine Dalgarno sequence or a Kozak sequence), a coding sequence and optionally a transcription terminator are present and results in the expression of a functionally active protein. The expression is constitutive or conditional or regulated or tunable.

[0023] The term "constitutive expression" is defined as the absence of transcription factors other than subunits of RNA polymerase (e.g., bacterial sigma factors such as sigma) under certain growth conditions. 70 , σ 54Or the related σ-factor, and yeast mitochondrial RNA polymerase specific factor MTF1, which is co-associated with the RNA polymerase core enzyme) to regulate 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 a catalytic machine for synthesizing RNA from a DNA template. RNA polymerase binds to a specific DNA sequence to initiate transcription, for example, via the σ factor in a prokaryotic host or via MTF1 in yeast. Constitutive expression provides a constant expression level without the need for induction or repression.

[0024] The term "regulated expression" is defined as expression that is regulated under certain growth conditions by transcription factors other than subunits of RNA polymerase (e.g., bacterial sigma factors). Examples of such transcription factors are described above. Typically, expression regulation is achieved with the aid of inducers such as, but not limited to, IPTG, arabinose, rhamnose, fucose, allolactose, or pH shifts or temperature shifts or carbon depletion or substrate or generated products.

[0025] The term "control sequence" refers to a sequence recognized by the cellular transcription and translation machinery to allow transcription and translation of a polynucleotide sequence into a polypeptide. Thus, such DNA sequences are necessary for expression of an operably linked coding sequence in a particular host cell, cell, or organism. Such control sequences may include, but are not limited to, promoter sequences, ribosome binding sequences, Shine Dalgarno sequences, Kozak sequences, and transcription terminator sequences. Suitable control sequences for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. DNA for a presequence or secretory leader can be operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer can be operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site can be operably linked to a coding sequence if it is positioned so as to facilitate translation. Furthermore, the control sequences can be controlled by external chemicals (such as, but not limited to, IPTG, arabinose, lactose, allolactose, rhamnose, or fucose) via inducible promoters or via genetic circuits that induce or repress transcription or translation of the polynucleotide into a polypeptide.

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

[0027] The term "wild type" refers to the generally known genetic or phenotypic condition 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 than 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 than the wild-type (i.e., native in the expression host) protein.

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

[0030] As used herein with respect to cells that produce a sugar, the term "non-naturally occurring" indicates that the sugar i) is not produced in nature, or ii) when produced in nature, is not produced by the cell in the same amount; and that the cell has been genetically engineered to produce the sugar or to have increased production of the sugar.

[0031] As the term is used herein, a "variant" is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties. A typical variant of a polynucleotide differs from another reference polynucleotide in nucleotide sequence. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs from another reference polypeptide in amino acid sequence. Typically, the differences are limited, so that the sequences of the reference polypeptide and the variant are generally very similar and, in many regions, are identical. A variant and a reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination. The substituted or inserted amino acid residues may or may not be amino acid residues encoded by the genetic code. A variant of a polynucleotide or polypeptide may be naturally occurring, such as an allelic variant, or it may be an unknown naturally occurring variant. Non-naturally occurring variants of polynucleotides and polypeptides can be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art.

[0032] In some embodiments, the present invention contemplates preparing variants by modifying the structure of the enzymes used in the present invention. Variants can be generated by amino acid substitutions, deletions, additions, or a combination thereof.

[0033] With respect to polynucleotides, a "fragment" refers to a clone or any portion of a polynucleotide molecule, particularly a portion of a polynucleotide.

[0034] Throughout this application, the sequence of a polynucleotide may be represented by a SEQ ID NO or, alternatively, by a Gene ID (Maglott et al. (2011) Nucl. Acids Res. 39, Issue suppl_1, D52-D57) or a GenBank NO (https: / / www.ncbi.nlm.nih.gov / genbank / ). Therefore, the terms "polynucleotide SEQ ID NO," "polynucleotide Gene ID," and "polynucleotide GenBank NO" are used interchangeably unless otherwise expressly stated. Throughout this application, the sequence of a polypeptide may be represented by a SEQ ID NO or, alternatively, by a UniProt ID. Therefore, the terms "polypeptide SEQ ID NO" and "polypeptide UniProt ID" are used interchangeably unless otherwise expressly stated.

[0035] 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), Conserved Domain Database (CDD) nomenclature (https: / / www.ncbi.nlm.nih.gov / cdd) (Lu et al., Nucleic Acids Res. 48 (2020) D265-D268) or PTHR domains (http: / / www.pantherdb.org) (Mi et al., Nucleic Acids. Res. 41 (2013) D377-D386; Thomas et al., Genome Research 13 (2003) 2129-2141). Protein or polypeptide sequence information and functional information can be provided by a wide range of resources about 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 an expert and richly managed protein database called UniProt Knowledgebase (UniProtKB), together with UniProt Reference Clusters (UniRef) and UniProt Archive (UniParc). The UniProt identifier (UniProt ID) is unique for each protein present in the database. Throughout the application, the sequence of the polypeptide is represented by SEQ ID NO or UniProt ID. Unless otherwise stated, the UniProt ID of the protein described corresponds to its sequence version 01 present in the UniProt database (www.uniprot.org) version 2021_03 and accessed on June 9, 2021.

[0036] InterPro provides functional analysis of proteins by classifying them into families and predicting structural domains and important sites. To classify proteins in this way, InterPro uses predictive models, called signatures, provided by several different databases (called member databases) that make up the InterPro federation. The protein signatures from these member databases are combined into a single searchable resource, where their individual strengths are harnessed to create a powerful integrated database and diagnostic tool.

[0037] Those skilled in the art will understand that for the databases used herein, including InterPro 90.0 (released on August 4, 2022) and eggNOG 5.0 (released in 2019), the content of each database is fixed at each release and will not change. When the content of a particular database is changed, a new release version with a new release date is accepted for that particular database. 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.

[0038] The terms "lactobionic acid", "LBA", "4-O-beta-D-galactopyranosyl-D-gluconic acid", "4-O-β-galactopyranosyl-D-gluconic acid", "4-(β-D-galactosyl)-D-gluconic acid", "lactobionate", "maltobionic acid" and "(2R,3R,4R,5R)-2,3,5,6-tetrahydroxy-4-[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyhexanoic acid" are used interchangeably and refer to C 12 H 22 O 12 , which is a disaccharide formed between β-D-galactose and D-gluconic acid.

[0039] As used herein, the term "modified form of LBA" refers to an LBA molecule that is further modified with one or more other molecules (e.g., with one or more monosaccharides, disaccharides, oligosaccharides, chitosan, chemical groups and / or amino groups), or refers to an LBA molecule that is present in a complex with one or more minerals (e.g., calcium, magnesium, potassium, sodium, iron, zinc, copper, chromium, selenium, manganese). An example of a modified form of LBA is a glycosylated form of LBA described herein.

[0040] As used herein, the terms "glucosylated form of LBA" and "glycosylated form of LBA" are used interchangeably and refer to LBA molecules that are further modified with one or more monosaccharides. The monosaccharides are further defined herein. Examples of glycosylated forms of LBA include fucosylated LBA and sialylated LBA. As used herein, the term "fucosylated LBA" refers to LBA molecules that are further modified with one or more fucose residues. Examples of fucosylated LBA include O-6-deoxy-α-L-galactopyranosyl-(1-2)-O-β-D-galactopyranosyl-(1-4)-D-gluconic acid (2'FLBA, 2'-fucosylated LBA) and O-6-deoxy-α-L-galactopyranosyl-(1-3)-O-[β-D-galactopyranosyl-(1-4)-]-D-gluconic acid (3FLBA, 3-fucosylated LBA). As used herein, the term "sialylated LBA" refers to an LBA molecule that is further modified with one or more sialic acid residues. Examples of sialylated LBAs include O-(N-acetyl-α-neuraminic acid)-(2-3)-O-β-D-galactopyranosyl-(1-4)-D-gluconic acid (3'SLBA, 3'-sialylated LBA) and O-(N-acetyl-α-neuraminic acid)-(2-6)-O-β-D-galactopyranosyl-(1-4)-D-gluconic acid (6'S-LBA, 6'-sialylated LBA).

[0041] As used herein, the term "monosaccharide" refers to a sugar that cannot be broken down into simpler sugars by hydrolysis, is classified as an aldose, ketose, deoxysugar, deoxyaminosugar, uronic acid, aldonic acid, ketoaldonic acid, aldaric acid, or sugar alcohol, and contains one or more hydroxyl groups per molecule. A monosaccharide is a sugar that contains only one simple sugar.

[0042] As used herein, the term "phosphorylated monosaccharide" refers to a phosphorylated monosaccharide. 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.

[0043] The terms "activated monosaccharide," "nucleotide-activating sugar," "nucleotide-sugar," "activated sugar," "nucleoside," or "nucleotide donor" are used interchangeably herein and refer to an 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-L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L- Nucleotide sugars include UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetylamino-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-phospho-L-glucose (UDP-L-PneNAC or UDP-2-acetylamino-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-isorhamnose (UDP-L-QuiNAc or UDP-2-acetylamino-2,6-dideoxy-L-glucose), GDP-L-isorhamnose, CMP-sialic acid (CMP-Neu5Ac or CMP-N-acetylneuraminic acid), GDP-fucose (GDP-Fuc), GDP-rhamnose, and UDP-xylose. Nucleotide sugars serve as glycosyl donors in glycosylation reactions. Glycosylation reactions are reactions catalyzed by glycosyltransferases.

[0044] As used herein, the term "glycosyltransferase" refers to an enzyme that catalyzes the transfer of a sugar moiety from a donor to a specific acceptor, thereby forming a glycosidic bond. The donor can be a precursor as defined herein. Glycosyltransferases and related proteins that use nucleotide diphosphate-sugar, nucleotide monophosphate-sugar, and sugar phosphate have been described and classified into 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). As used herein, the glycosyltransferase can be selected from the list comprising, consisting of, or consisting essentially of a fucosyltransferase, a sialyltransferase, a galactosyltransferase, a glucosyltransferase, a mannosyltransferase, an N-acetylglucosamine transferase, an N-acetylgalactosamine transferase, an N-acetylmannosamine transferase, a xylosyltransferase, a glucuronyltransferase, a galacturonyltransferase, a glucosamine transferase, an N-glycolylneuraminic acid transferase, a rhamnosyltransferase, an N-acetylrhamnosyltransferase, a UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine transaminase, a UDP-N-acetylglucosamine enolpyruvyltransferase, and a fucosamine transferase.

[0045] As used herein, the term "disaccharide" refers to a sugar polymer comprising two simple sugars (i.e., monosaccharides). 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, and 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).

[0046] As used herein and as generally understood in the prior art, the term "oligosaccharide" refers to a sugar polymer comprising a small number, typically three to twenty, preferably three to ten simple sugars, i.e., monosaccharides. The oligosaccharides used in the present invention can be linear structures or can contain branches. The bond between two sugar units (e.g., glycosidic bond, galactosidic bond, glucosidic bond, etc.) can be expressed as, for example, 1,4, 1->4, or (1-4), and are used interchangeably herein. For example, the terms "Gal-b1,4-Glc", "Gal-β1,4-Glc", "b-Gal-(1->4)-Glc", "β-Gal-(1->4)-Glc", "Galβ1-4-Glc", "Gal-b(1-4)-Glc" and "Gal-β(1-4)-Glc" have the same meaning, i.e., a β-glycosidic bond connecting carbon-1 of galactose (Gal) to carbon-4 of glucose (Glc). Each monosaccharide can be in a cyclic form (e.g., a 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, of monosaccharides connected by glycosidic bonds.Examples of oligosaccharides include, but are not limited to, Lewis-type antigenic oligosaccharides, milk oligosaccharides, mammalian (including human) milk oligosaccharides, O-antigens, enterobacterial common antigens (ECA), glycan chains present in lipopolysaccharides (LPS), oligosaccharide repeats present in capsular polysaccharides, peptidoglycans (PG), amino sugars, antigens of the human ABO blood group system, animal oligosaccharides (preferably selected from the list consisting of N-glycans and O-glycans), plant oligosaccharides (preferably selected from the list consisting of N-glycans and O-glycans), sialylated oligosaccharides, neutral ( uncharged) oligosaccharides, negatively charged oligosaccharides, fucosylated oligosaccharides, oligosaccharides containing N-acetylglucosamine, oligosaccharides containing lacto-N-biose, oligosaccharides containing N-acetyllactosamine, sialylated oligosaccharides containing N-acetylglucosamine, neutral (uncharged) oligosaccharides containing N-acetylglucosamine, negatively charged oligosaccharides containing N-acetylglucosamine, fucosylated oligosaccharides containing N-acetylglucosamine, non-fucosylated oligosaccharides containing N-acetylglucosamine, sialylated oligosaccharides containing lacto-N-biose, Sugars, neutral (uncharged) oligosaccharides comprising lacto-N-biose, negatively charged oligosaccharides comprising lacto-N-biose, fucosylated oligosaccharides comprising lacto-N-biose, non-fucosylated oligosaccharides comprising lacto-N-biose, sialylated oligosaccharides comprising N-acetyllactosamine, neutral (uncharged) oligosaccharides comprising N-acetyllactosamine, negatively charged oligosaccharides comprising N-acetyllactosamine, fucosylated oligosaccharides comprising N-acetyllactosamine, non-fucosylated oligosaccharides comprising N-acetyllactosamine, chitosan , oligosaccharides including chitosan, heparosan, chondroitin sulfate, glycosaminoglycan oligosaccharides, heparin, heparan sulfate, dermatan sulfate, acetylated hyaluronan, hyaluronic acid, keratan sulfate, erlose (Glc-α1,4-Glc-α1,2-Fru), lactococcal-N-triose II (GlcNAc-β1,3-Gal-β1,4-Fru), lactococcal-N-tetraose, lactococcal-N-neotetraose and globotriose.

[0047] The terms "negatively charged oligosaccharide" or "acidic oligosaccharide" are used interchangeably and refer to oligosaccharides having a negative charge. In a preferred embodiment, the negatively charged oligosaccharide is a sialylated oligosaccharide. As used herein, "sialylated oligosaccharide" is understood to be an oligosaccharide comprising negatively charged sialic acid, i.e., an oligosaccharide having one or more sialic acid residues. It has an acidic nature. Some examples are 3'SL (3'-sialyllactose, Neu5Ac-α2,3-Gal-β1,4-Glc), 3'-sialyllactosamine, 6'SL (6' sialyllactose, Neu5Ac-α2,6-Gal-β1,4-Glc), 8'SL (8' sialyllactose, Neu5Ac-α2,8-Gal-β1,4-Glc), 3,6-disialyllactose (Neu5Ac-α2,3-(Neu5Ac-α2,6)-Gal-β1,4-Glc), 6, 6'-disialyl lactose (Neu5Ac-α2,6-Gal-β1,4-(Neu5Ac-α2,6)-Glc), 8,3-disialyl lactose (Neu5Ac-α2,8-Neu5Ac-α2,3-Gal-β1,4-Glc), 6'-sialyl lactosamine, 3'-sialyl lactosamine, oligosaccharides containing 6'sialyl lactose, SGG hexose (Neu5Acα-2,3Galβ-1,3GalNacβ-1,3Galα-1,4Galβ-1,4Gal ), sialylated tetraose, sialylated pentose, sialylated lacto-N-triose, sialylated lacto-N-tetraose, sialylated lacto-N-neotetraose, LSTc(Neu5Ac-α2,6-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc), LSTd(Neu5Ac-α2,3-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc), monosialyl lacto-N-hexose, disialyl lacto-N-hexose I, monosialyl lacto-N-neohexose I, monosialoyl lacto-N-neohexose II, disialoyl lacto-N-neohexose, disialoyl lacto-N-tetraose, disialoyl lacto-N-hexose II, sialyl lacto-N-tetraose a (LSTa, Neu5Ac-α2,3-Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc), disialoyl lacto-N-hexose I, sialyl lacto-N-tetraose b (LSTb, Gal-β1,3-(Neu5Ac-α2,6)-GlcNAc-β1,3-Gal-β1,4-Glc4-Glc), 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, monofucosylmonosialyllacto-N-octose (sialyl Lea), sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, monofucosyldisialyllacto-N-tetraose, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal, Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal, 3'-KDO-lactose, 3'-KDO-lactosamine, 3'-KDO-6'sialyllactose, 3'-KDO-8'sialyllactosamine, 3'-KDO-10'sialyllactosamine, 3'-KDO-20'sialyllactosamine, 3'-KDO-30'sialyllactosamine, 3'-KDO-40'sialyllactosamine, 3'-KDO-5'sialyllactosamine, 3'-KDO-6'sialyllactosamine, 3'-KDO-8'sialyllactosamine, 3'-KDO-10 ... Acyl lactose, KDO-2,3Galβ-1,3GalNacβ-1,3Galα-1,4Galβ-1,4Gal, KDO-2,3Galβ-1,3GlcNacβ-1,3Galβ-1,4Glc, KDO-2,3Galβ-1,4GlcNacβ-1,3Galβ-1,4Glc, 3'-KDO-3-fucosyllactose, Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal, 3'-sialyl-2'-fucosyllactose, 6'-sialyl-2'-fucosyllactose, 6'-sialyl-3-fucosyllactose, 3'-sialyl Acyl-3-fucosyllactose, Neu5Ac-a2,6-(Neu5Ac-a2,3-)Gal-b1,4-Glc, 3'-sialyl-3-fucosyllactosamine, Fuc-a1,4-(Neu5Ac-a2,3-Gal-b1,3-)GlcNAc, 6'-sialyllacto-N-biose, 3'-sialyllacto-N-biose, Neu5Ac-a2,6-(GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,3-Gal- b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-( Gal-b1,4-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,3-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1, 3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4- Glc, Fuc-a1,4-(Neu5Ac-a2,3-Gal-b1,3-)GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-(Fuc-a1, 3-)Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc , Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc and oligosaccharides carrying one or more sialic acid residues, including but not limited to oligosaccharide moieties of gangliosides selected from the following: GM3 (3' sialyllactose, Neu5Acα-2,3Galβ-4Glc) and oligosaccharides containing the GM3 motif, 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), G T1a(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,

[0048] (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

[0049] (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-2,8Neu5Acα 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 moieties with ceramide or synthesizing the above oligosaccharides on ceramide.

[0050] "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, galacturonic acid, and 2-keto-3-deoxymannooctonic acid (KDO). Charged oligosaccharides are also known as acidic oligosaccharides. In contrast, neutral (uncharged) oligosaccharides are non-sialylated oligosaccharides and, therefore, 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.

[0051] As used herein, the terms "neutral oligosaccharide" and "uncharged oligosaccharide" are used interchangeably and, as generally 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), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-triose II (LN3, GlcNAcβ1-3Galβ1-4Glc), lacto-N-tetraose (LNT, Galβ1-3GlcNAcβ1-3Galβ1-4Glc), lacto-N-neotetraose (LNnT, Galβ1-4GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentose I, lacto-N-neotetraose (LNnT, Galβ1-4GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentose I, lacto-N-neotetraose (LNnT, Galβ1-4GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentose II ... I, lacto-N-neotetraose (LNnT, Galβ1-4GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentose II, lacto-N-neotetraose (LNnT, Fucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Fuc-a1,2-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Fuc-a1,2-Gal-b1,4-(Fuc-a1,3 -)GlcNAc-b1,3-Gal-b1,4-Glc,Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc,Fuc-a1,2-Gal-b1,4-(Fuc-a1, 3-)GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Fuc-a1,4-(Fuc-a1,2-Gal-b1,3-)GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, monofucosyl emulsion- N-hexose-III, difucosyllactose-N-hexose (a), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose, difucosyllacto-N-hexose, difucosyllacto-N-neohexose, trifucosyllacto-N-hexose, a1,3-galactosyl-3-fucosyllactose, Gal-a1,3-(Fuc-a1,2-)Gal-b1,4-(Fuc-a1,3-)Glc, GalNAc-a1,3-(Fuc-a1,2-)Gal-b1,4-(Fuc-a1,3-)Glc3-)Glc, 2-fucosyllactulose, 3-fucosyl-N-acetyllactosamine, 2'-fucosyl-N-acetyllactosamine, difucosyl-N-acetyllactosamine, 4-fucosyllacto-N-biose, 2'-fucosyllacto-N-biose, difucosyllacto-N-biose and GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Glc NAc-b1,6-(GlcNAc-b1,3-)Gal-b1,4-Glc, lacto-N-pentose (LN5), lacto-N-neopentose, p-lacto-N-pentose, p-lacto-N-neopentose, lacto-N-neopentose I, lacto-N-heptose (LN7), lacto-N-neoheptose, p-lacto-N-neoheptose, p-lacto-N-heptose, lacto-N-octose (LNO), lacto-N-neooctose, isolactose N-octose, lacto-N-octose, allolacto-N-neooctose, neo-lacto-N-neooctose, lacto-N-neooctose (pLNnO), allolacto-N-nonose, neo-lacto-N-nonose, lacto-N-nonose (LN9), lacto-N-decose, allolacto-N-decose, neo-lacto-N-decose, lacto-N-neodecose, lacto-N-neodecose, lacto-N-neodecose (pLNnD), a1,3-galactosyl lacto-N-neotetraose Sugar, GlcNAc-b1,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, GlcNAc-b1,6-(Gal-b1,4-Gl cNAc-b1,3-)Gal-b1,4-Glc and GlcNAc-b1,6-(Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc. ,

[0052] As used herein and as generally understood in the prior art, "fucosylated oligosaccharides" are oligosaccharides carrying a fucose residue. Such fucosylated oligosaccharides are sugar structures comprising at least three monosaccharide subunits interconnected via glycosidic bonds, wherein at least one of the monosaccharide subunits is fucose. Fucosylated oligosaccharides may comprise more than one fucose residue, for example two, three or more. Fucosylated oligosaccharides may be neutral oligosaccharides or charged oligosaccharides, which, for example, also comprise a sialic acid structure. 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-fucopentose I (LNFP I), Gal-a1,3-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (Gal-LNFP I), GalNAc-a1,3-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (GalNAc-LNFP I), lacto-N-fucopentose II (LNFP II), lacto-N-fucopentose III (LNFP III), lacto-N-fucopentose V (LNFP V), lacto-N-fucopentose VI (LNFP VI), lacto-N-neofucopentose I, lacto-N-difucohexose I (LDFHI), lacto-N-difucohexose II (LDFH II), monofucosyl lacto-N-hexose III (MFLNH III), difucosyllactose-N-hexose (a), difucosyllactose-N-hexose, difucosyl-lacto-N-neohexose, trifucosyllactose-N-hexose, a1,3-galactosyl-3-fucosyllactose, Gal-a1,3-(Fuc-a1,2-)Gal-b1,4-(Fuc-a1,3-)Glc, GalNAc-a1,3-(Fuc-a1,2-)Gal-b1,4-(Fuc-a1,3-)Glc, 2-fucosyllactulose, 3-fucosyl-N-acetyllactosamine, 2'-fucosyl-N-acetyllactosamine, difucosyl-N-acetyllactosamine, 4-fucose sialyllacto-N-biose, 2'-fucosyllacto-N-biose, difucosyllacto-N-biose and GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, 3'-sialyl-2'-fucosyllactose, 6'-sialyl-2'-fucosyllactose, 6'-sialyl-3-fucosyllactose, 3'-sialyl-3-fucosyllactose, disialylmonofucosyllacto-N-neohexose, monofucosylmonosialyllacto-N-octose (sialyl Lea), sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, monofucosyldisialyllacto-N-tetraose.

[0053] Mammalian milk oligosaccharides include oligosaccharides present in milk from humans and mammals found at any stage during lactation, including colostrum. As used herein, "mammalian milk oligosaccharides" or "MMOs" refer to oligosaccharides such as, but not limited to, those: 3-fucosyllactose, 2'-fucosyllactose, 6-fucosyllactose, 2',3-difucosyllactose, 2',2-difucosyllactose, 3,4-difucosyllactose, 6'-sialylactose, 3'-sialylactose, 3,6-disialylactose, 6,6'-disialylactose, 8,3-disialylactose, 3,6-disialyllacto-N-tetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentose II, lacto-N-fucopentose I, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, sialyllacto-N-tetraose c. , sialyl lacto-N-tetraose b, sialyl lacto-N-tetraose a, lacto-N-difucohexose I, lacto-N-difucohexose II, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, monofucosylmonosialyl lacto-N-tetraose c, monofucosyl p-lacto-N-hexose, monofucosyl lacto-N-hexose III, isomeric fucosylated lacto-N- Hexose III, isomeric fucosylated lacto-N-hexose I, sialyl lacto-N-hexose, sialyl lacto-N-neohexose II, difucosyl-p-lacto-N-hexose, difucosyl lacto-N-hexose, difucosyl lacto-N-hexose a, difucosyl lacto-N-hexose c, galactosylated chitosan, fucosylated oligosaccharides, neutral oligosaccharides and / or sialylated oligosaccharides.

[0054] The term "human milk oligosaccharides" or "HMOs" refers to oligosaccharides found in human breast milk (including premature human milk, colostrum and term human milk). HMOs include fucosylated oligosaccharides, non-fucosylated neutral oligosaccharides and sialylated oligosaccharides. Examples of HMOs include 3-fucosyllactose, 2'-fucosyllactose, 2',3-difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, LN3, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentose II, lacto-N-fucopentose I, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, sialyllactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentose II, lacto-N-fucopentose I, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, sialyllactose, lacto-N-fucopentose VI ... Sialyllacto-N-tetraose c, sialyllacto-N-tetraose b, sialyllacto-N-tetraose a, difucosyllacto-N-tetraose, lacto-N-hexose, lacto-N-difucohexose I, lacto-N-difucohexose II, disialyllacto-N-tetraose, fucosyllacto-N-hexose, difucosyllacto-N-hexose, fucodisialyllacto-N-hexose, disialyllacto-N-hexose.

[0055] As used herein, the term "sugar-producing pathway" refers to a biochemical pathway comprised of enzymes and their respective genes involved in the synthesis of sugars as defined herein. Such sugar-producing pathways may include, but are not limited to, pathways involved in synthesizing nucleotide-activated sugars and transferring the nucleotide-activated sugars to an acceptor to produce the sugars of the present invention. Examples of such pathways include, but are not limited to, the fucosylation pathway, the sialylation pathway, the galactosylation pathway, the N-acetylglucosaminylation pathway, the N-acetylgalactosaminylation pathway, the mannosylation pathway, and the N-acetylmannosaminylation pathway.

[0056] As used herein, the terms "LBA pathway" or "pathway for producing LBA" are used interchangeably and refer to a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of lactobionic acid (LBA). The pathway for producing LBA may include pathways for the synthesis and / or import of cofactors used in the pathway for producing LBA.

[0057] As used herein, the terms "synthesis of a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA" or "a pathway for producing a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA" are used interchangeably and refer to a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA, respectively. The pathway for producing a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA may include pathways for synthesizing and / or importing cofactors used in the pathway for producing a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA, respectively.

[0058] The term "purified" refers to a material that is substantially or essentially free of components that interfere with the activity of a biomolecule. For cells, carbohydrates, nucleic acids, and polypeptides, the term "purified" refers to a material that is substantially or essentially free of components that normally accompany the material (as found in its native state). Typically, the purified carbohydrates, oligosaccharides, proteins, or nucleic acids of the invention are at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% pure, and 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 method for determining purity. Purity or homogeneity can be indicated by a number of 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 means of purification will be employed. For disaccharides and oligosaccharides, purity can be determined using methods such as, but not limited to, thin layer chromatography, gas chromatography, NMR, HPLC, capillary electrophoresis, or mass spectrometry. Further herein, the terms "contaminants" and "impurities" preferably refer to particles, cells, cellular 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).

[0059] As used herein, the term "clarification" refers to the act of treating an aqueous medium (e.g., a culture, incubation medium) to remove suspended particles and contaminants from the production process, such as cells, cellular components, insoluble metabolites, and debris that may interfere with the final purification of the sugars. Such treatment may be performed 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) to carry out.

[0060] The term "culture" refers to the culture medium in which cells are cultured or fermented, the cells themselves, and sugars, LBAs, modified forms of LBAs, glycosylated forms of LBAs, fucosylated LBAs, and / or sialylated LBAs produced by the cells in the whole culture fluid, i.e., inside (intracellularly) and outside (extracellularly) of the cells. As used herein, the terms "culture medium" and "incubation medium" are used interchangeably and refer to the medium in which cells are cultured.

[0061] The term "incubation" refers to a mixture in which i) sugars, or ii) sugars, LBAs and / or modified forms of LBAs, glycosylated forms of LBAs, fucosylated LBAs and / or sialylated LBAs are produced. The mixture may comprise one or more enzymes, one or more precursors and one or more acceptors as defined herein (which are present in a buffer solution) and incubated at a temperature for a certain time so that sugars can be produced in the mixture by catalysis of the one or more enzymes using the one or more precursors and the one or more acceptors. The mixture may also comprise: i) cells obtained after culture or incubation, optionally subjecting the cells to cell lysis; ii) a buffer solution or culture or incubation medium in which the cells are cultured or fermented; and iii) a) sugars, or b) sugars, LBAs and / or modified forms of LBAs, glycosylated forms of LBAs, fucosylated LBAs and / or sialylated LBAs produced by cells in whole culture medium, i.e., inside (intracellularly) and outside (extracellularly) of the cells. Said incubation may also be culturing as defined herein.

[0062] The terms "reactor" and "incubator" refer to containers 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, and CO2 incubators.

[0063] As used herein, the term "cell productivity index (CPI)" refers to the mass of sugars produced by cells divided by the mass of cells produced in culture. The term "CPI" as used herein will also be understood as the mass of LBAs produced by cells divided by the mass of cells produced in culture. Further, the term "CPI" as used herein will also be understood as the mass of sugars and LBAs produced by cells divided by the mass of cells produced in culture. The term "CPI" as used herein will also be understood as the mass of modified forms of LBAs, glycosylated forms of LBAs, fucosylated LBAs and / or sialylated LBAs produced by cells divided by the mass of cells produced in culture. The term "CPI" as used herein will also be understood as the mass of sugars, LBAs and modified forms of LBAs, glycosylated forms of LBAs, fucosylated LBAs and / or sialylated LBAs produced by cells divided by the mass of cells produced in culture.

[0064] As used herein, the term "precursor" refers to a substance that is taken up or synthesized by a cell for the specific production of i) a sugar, ii) an LBA, and / or iii) a sugar, an LBA, a modified form of an LBA, a glycosylated form of an LBA, a fucosylated LBA, and / or a sialylated LBA according to the present invention. In this sense, a precursor can be an acceptor as defined herein, but can also be another substance, a metabolite, a cofactor that is first modified within the cell as part of the biochemical synthesis pathway of i) a sugar, ii) an LBA, and / or iii) a sugar, an LBA, a modified form of an LBA, a glycosylated form of an LBA, a fucosylated LBA, and / or a sialylated LBA. As used herein, the term "precursor" will also be understood as a chemical compound that participates in an incubation or enzymatic reaction to produce another compound (e.g., an intermediate or acceptor, as defined herein) as part of a metabolic pathway for i) a sugar, ii) an LBA, and / or iii) a sugar, an LBA, a modified form of an LBA, a glycosylated form of an LBA, a fucosylated LBA, and / or a sialylated LBA. As used herein, the term "precursor" will also be understood as a donor that is used by a glycosyltransferase to modify an acceptor, as defined herein, with a sugar moiety in a glycosidic bond as part of a metabolic pathway for a sugar. Examples of such precursors include acceptors as defined herein, and / or dihydroxyacetone, glucosamine, N-acetylglucosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, galactosyl lactose, 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 activating 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.

[0065] Optionally, the cells used to produce the sugar are transformed to contain 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 precursors added to the medium for the synthesis of the sugar of the present invention.

[0066] 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, lactulose, lactobionic acid (LBA), lacto-N-triose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-pentose (LNP), lacto-N-neopentose, p-lacto-N-pentose, p-lacto-N-neopentose, Lacto-N-neopentose I, Lacto-N-hexose (LNH), Lacto-N-neohexose (LNnH), p-Lacto-N-neohexose (pLNnH), p-Lacto-N-hexose (pLNH), Lacto-N-heptose, Lacto-N-neoheptose, p-Lacto-N-neoheptose, p-Lacto-N-heptose, Lacto-N-octose (LNO), Lacto-N-neooctose, Allolacto-N-octose, p-Lacto-N-octose saccharides, allolacto-N-neooctose, neolacto-N-neooctose, paralacto-N-neooctose, allolacto-N-nonose, neolacto-N-nonose, lacto-N-nonose, lacto-N-nonose, lacto-N-decose, allolacto-N-decose, neolacto-N-decose, lacto-N-neodecanose, and oligosaccharides comprising one or more N-acetyllactosamine units and / or one or more lacto-N-biose units, or intermediates thereof, fucosylated and sialylated forms thereof, ceramides, N-acylated sphingoids, glucosylceramides, lactosylceramides, lactosylceramides, sphingosine, phytosphingosine, sphingosine synthons, peptide backbones having β-GlcNAc-Asn residues, glycoproteins having terminal GlcNAc and Gal residues, immunoglobulins. Detailed Description of the Invention

[0068] According to a first aspect, the present invention provides a cell which i) is capable of synthesizing and / or synthesizes lactobionic acid (4-O-β-galactopyranosyl-D-gluconic acid, LBA), modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA in the presence of lactose in the culture or incubation medium of the cell, and ii) is genetically modified for the production of sugars, wherein the cell comprises a pathway for the production of the sugars, characterized in that the synthesis of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA is functionally impaired or knocked out in the cell. Herein, the synthesis of said LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA is obtained by expressing at least one gene selected from a list consisting of genes encoding the following: carbohydrate oxidases, dehydrogenases, lactonases, oxygen-dependent FAD-linked oxidoreductases and pyrroloquinoline quinone (PQQ) oxidoreductases, wherein the synthesis of said LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA is functionally impaired or knocked out in said cell by functionally impairing or knocking out said at least one gene, two or more of said genes or all of said genes.

[0069] According to a second aspect, the present invention provides a method for producing sugars, wherein the method comprises culturing and / or incubating the cells described herein in a culture and / or incubation medium under conditions allowing the production of sugars and LBAs.

[0070] In the context of the present invention, permissive conditions are understood to be conditions involving physical or chemical parameters (including but not limited to temperature, pH, pressure, osmotic pressure and product / precursor / acceptor / cofactor concentrations). It is understood herein that said conditions include the presence of lactose in the culture or incubation medium in which the cells are cultured and / or incubated.

[0071] In a particular embodiment, the permissive conditions may include a temperature range of 30 + / - 20 degrees Celsius, and a pH range of 7 + / - 3.

[0072] In a preferred embodiment, the sugar is separated from the culture and / or incubation. In another and / or additional preferred embodiment, the sugar is separated from LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA. In another and / or additional preferred embodiment, the sugar is purified.

[0073] In a particular embodiment of the methods and / or cells of the present invention, the cells are capable of synthesizing LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA, and / or sialylated LBA. LBA can be produced by oxidation of lactose to a lactone by a dehydrogenase (lactose oxidase) system, which is further hydrolyzed to LBA. LBA can also be produced after oxidation of maltose. LBA can also be produced by lactose dehydrogenase and lactonase. LBA can also be formed from sugars other than lactose (for example, but not limited to, D-glucose, D-galactose, D-mannose, D-talose, D-xylose, D-ribose, L-arabinose, cellobiose, and D-fructose).

[0074] In a preferred embodiment of the method and / or cell, the cell synthesizes LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA, and / or a sialylated LBA. In a more preferred embodiment, the cell comprises a pathway for producing LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA, and / or a sialylated LBA. The pathway for producing LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA, and / or a sialylated LBA consists of enzymes involved in the synthesis of the LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA, and / or a sialylated LBA, and their respective genes. The enzymes involved in the synthesis of LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA include, but are not limited to, carbohydrate oxidases, dehydrogenases, lactonases, oxygen-dependent FAD-linked oxidoreductases, pyrroloquinoline quinone (PQQ) oxidoreductases, lactose dehydrogenases, quinone protein glucose dehydrogenases, aldose sugar dehydrogenases, glucose / fructose dehydrogenases, glucose / sorbosone dehydrogenases, cellobiose dehydrogenases, pyrroloquinoline quinone (PQQ) dehydrogenases, malate dehydrogenases and lactose oxidase. The synthesis of LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA may utilize one or more cofactors. Examples of cofactors include, but are not limited to, pyrroloquinoline quinone (PQQ), FAD, divalent metal cations, Ca 2+ 、Cu 2+ Mg 2+ 、Zn 2+, heme, heme C and pyridoxal 5'-phosphate. The pathway for producing LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA may include pathways for synthesizing and / or importing cofactors used in the pathway for producing LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA. In other words, the cells of the present invention may comprise pathways for producing and / or importing PQQ, FAD, divalent metal cations, Ca 2+ 、Cu 2+ Mg 2+ 、Zn 2+ , heme, heme C and pyridoxal 5'-phosphate. Alternatively, the cells of the present invention do not synthesize the cofactors necessary for the synthesis of LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, but have all other enzymes necessary for the synthesis of LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA. The cells can synthesize LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA after being supplemented with one or more cofactors.

[0075] In a further particular embodiment, the synthesis of LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA is functionally impaired or knocked out in the cell. Herein, a cell in which the synthesis of LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA is functionally impaired will be understood as a cell having a lower production of LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA than a cell in which the synthesis of LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA, respectively, is not functionally impaired. According to the present invention, the synthesis of LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA is functionally impaired in a cell by impairing the function of one or more genes involved in the pathway for producing LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA. Alternatively and / or additionally, the synthesis of LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA is functionally impaired in a cell by impairing the function of one or more genes involved in the pathway for producing and / or importing one or more cofactors used in the synthesis of LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA. Preferably, the synthesis of LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA is functionally impaired in the cell by functionally impairing one or more genes involved in a pathway for the production and / or import of one or more cofactors selected from the list comprising, consisting of or essentially consisting of pyrroloquinoline quinone (PQQ), FAD, divalent metal cations, Ca 2 + 、Cu 2+ Mg 2+ 、Zn 2+, heme, heme C and pyridoxal 5'-phosphate.Making a gene functionally deteriorate will be understood as making a gene less able (that is, statistically significantly "less able" or completely unable (for example, the gene of knockout) compared to a functional wild-type gene) to produce a functional final product. Gene functionally deteriorate can be made by means of common well-known techniques for technicians, for example, by any one or more of the insertion, deletion and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from the list comprising the following, consisting of the following or consisting essentially of the following: the promoter sequence, ribosome binding site, non-translated region, coding sequence and transcription terminator sequence of the gene, so that the gene is less able to produce a functional final product. Methods such as siRNA, CrispR, CrispRi, riboswitch, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutant gene and transposon mutagenesis can be used herein. Alternatively and / or additionally, the synthesis of LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA in a cell is functionally impaired by replacing a naturally occurring pathway for producing LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA in the cell with another pathway for producing LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA that gives lower production of LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA than the naturally occurring pathway for producing LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA in the cell. A cell wherein the synthesis of LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA, and / or a sialylated LBA is knocked out is understood to mean that the cell does not produce LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA, and / or a sialylated LBA. Knockout of a pathway in a cell can be accomplished by genomic deletion of one or more genes involved in the pathway or rendering one or more genes involved in the pathway incapable of producing a functional end product. Knockout of a gene from the genome of a cell can be accomplished by methods well known to those skilled in the art.

[0076] In a preferred embodiment of the method and / or cell of the present invention, the synthesis of LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA is obtained by expressing at least one gene selected from the list consisting of genes encoding carbohydrate oxidases, dehydrogenases, lactonases, oxygen-dependent FAD-linked oxidoreductases and pyrroloquinoline quinone (PQQ) oxidoreductases, and said at least one gene is functionally impaired or knocked out as described herein. In another preferred embodiment of the method and / or cell of the present invention, at least one gene involved in the synthesis and / or import of a cofactor involved in the synthesis of LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA is functionally impaired or knocked out, preferably said cofactor is selected from the list comprising, consisting of or essentially consisting of pyrroloquinoline quinone (PQQ), FAD, a divalent metal cation, Ca 2+ 、Cu 2+ Mg 2+ 、Zn 2+, heme, heme C and pyridoxal 5'-phosphate. In a more preferred embodiment, at least one of the genes involved in the synthesis of LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA and / or involved in the synthesis and / or import of a cofactor involved in the synthesis of LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA is functionally impaired by insertion, deletion and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from the list comprising, consisting of or essentially consisting of: a promoter sequence, a ribosome binding site, an untranslated region, a coding sequence and a transcription terminator sequence of the at least one gene. It will be understood herein that by making at least one of the genes deteriorate in function or knocking out at least one of the genes in the cell, the cell is made to have the synthesis of LBA, modified form of LBA, glycosylated form of LBA, fucosylated LBA and / or sialylated LBA that deteriorates in function or knocks out. As a result, the cell produces less LBA, modified form of LBA, glycosylated form of LBA, fucosylated LBA and / or sialylated LBA than a cell in which one of the genes is not deteriorated in function or knocked out. The term "less LBA" can be understood as comprising 0g / L LBA. The term "less LBA" can be understood as comprising the modified form of the LBA of 0g / L. The term "less LBA" can be understood as comprising the glycosylated form of the LBA of 0g / L. The term "less fucosylated LBA" may be understood as comprising 0 g / L of fucosylated LBA. The term "less sialylated LBA" may be understood as comprising 0 g / L of sialylated LBA.

[0077] In a more preferred embodiment, the synthesis of LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA is obtained by expressing at least two genes selected from the list consisting of genes encoding the following: carbohydrate oxidases, dehydrogenases, lactonases, oxygen-dependent FAD-linked oxidoreductases and pyrroloquinoline quinone (PQQ) oxidoreductases, and i) making at least one of the at least two genes functionally impaired, ii) making at least two of the at least two genes functionally impaired, iii) making all of the at least two genes functionally impaired, iv) knocking out at least one of the at least two genes, ii) knocking out at least two of the at least two genes, iii) knocking out all of the at least two genes, as described herein.

[0078] In another more preferred embodiment, the dehydrogenase is selected from the list comprising, consisting of, or consisting essentially of: lactose dehydrogenase, quinoprotein glucose dehydrogenase, aldose sugar dehydrogenase, glucose / fructose dehydrogenase, glucose / sorbosone dehydrogenase, cellobiose dehydrogenase, pyrroloquinoline quinone (PQQ) dehydrogenase, and malate dehydrogenase. In another and / or additional more preferred embodiment, the carbohydrate oxidase is lactose oxidase.

[0079] In another and / or additional more preferred embodiment of the methods and / or cells of the invention, the at least one gene encodes an enzyme, wherein the enzyme is selected from the enzyme class selected from the list comprising, consisting of or essentially consisting of EC:1.1.3.-, EC:1.1.3.5, EC:1.1.3.4, EC:1.1.5.-, EC:1.1.5.2 and EC:1.1.99.18.

[0080] In another and / or additional more preferred embodiment of the method and / or cell of the invention, the at least one gene encodes an enzyme, wherein the enzyme comprises a polypeptide sequence comprising an IPR domain selected from the list comprising, consisting of or essentially consisting of: IPR000172, IPR001479, IPR002372, IPR006094, IPR007867, IPR011041, IPR011042, IPR011047, IPR012132, IPR012938, IPR012951, IPR013154, IPR013428, IPR015402, IPR015920, IPR016166, IPR016169, IPR017511, IPR017512, IPR018391, IPR027424, IPR029056, IPR031640, IPR036188, and IPR036318 as defined in 90.0.

[0081] In another and / or additional more preferred embodiment of the methods and / or cells of the invention, the at least one gene encodes an enzyme, wherein the enzyme comprises a polypeptide sequence comprising a Panther domain selected from the list comprising, consisting of or essentially consisting of PTHR11552, PTHR13460, PTHR32303:SF4, PTHR42973 and PTHR47190 as defined by InterPro 90.0 released on August 4, 2022.

[0082] In another and / or additional more preferred embodiment of the methods and / or cells of the invention, the at least one gene encodes an enzyme, wherein the enzyme comprises a polypeptide sequence comprising a PFAM domain selected from the list comprising, consisting of or essentially consisting of: PF00732, PF01011, PF05199, PF07995, PF08031, PF08240, PF13360, PF13570, PF01565, PF16010 and PF16912 as defined by InterPro 90.0 released on August 4, 2022.

[0083] In another and / or additional more preferred embodiment of the methods and / or cells of the invention, the at least one gene encodes an enzyme, wherein the enzyme comprises a polypeptide sequence comprising the conserved protein domain cd10280 as defined by InterPro 90.0 released on August 4, 2022.

[0084] In another and / or additional more preferred embodiment of the methods and / or cells of the invention, the at least one gene encodes an enzyme, wherein the enzyme is, on August 4, 2022, part of a NOG family selected from the list comprising, consisting of or essentially consisting of: COG0277, COG2133, COG2303 and COG4993 as defined by eggNOG5.0 released in 2019.

[0085] In another and / or additional more preferred embodiment of the method and / or cell of the invention, the at least one gene encodes an enzyme, wherein the enzyme uses a cofactor selected from the list comprising, consisting of or essentially consisting of pyrroloquinoline quinone (PQQ), FAD, a divalent metal cation, Ca 2+ 、Cu 2+ Mg 2+ 、Zn 2+ , heme, heme C and pyridoxal 5'-phosphate.

[0086] In a more preferred embodiment, the at least one gene encodes an enzyme, wherein the enzyme is selected from enzyme class EC: 1.1.5.2; comprises a polypeptide sequence comprising, consisting of, or essentially consisting of: IPR domains IPR001479, IPR002372, IPR011047, IPR017511, and IPR018391 as defined in InterPro 90.0, released on August 4, 2022; comprises a polypeptide sequence comprising, consisting of, or essentially consisting of: Panther domain PTHR32303:SF4 as defined in InterPro 90.0, released on August 4, 2022; comprises a polypeptide sequence comprising, consisting of, or essentially consisting of: 90.0; comprising a polypeptide sequence comprising, consisting of, or essentially consisting of: a conserved protein domain cd10280 as defined by InterPro90.0, released on August 4, 2022; and a part of the NOG family COG4993 as defined by eggNOG5.0, released in 2019.

[0087] In a more preferred embodiment, the at least one gene encodes an enzyme, wherein the enzyme is selected from enzyme class EC: 1.1.5.2; comprising a polypeptide sequence comprising, consisting of, or consisting essentially of: IPR domains IPR001479, IPR002372, IPR011047, IPR017511, IPR017512, and IPR018391 as defined in InterPro 90.0, released on August 4, 2022; comprising a polypeptide sequence comprising, consisting of, or consisting essentially of: Panther domain PTHR32303:SF4 as defined in InterPro 90.0, released on August 4, 2022; comprising a polypeptide sequence comprising, consisting of, or consisting essentially of: 90.0, released on August 4, 2022; and a portion of the NOG family COG4993 defined by eggNOG5.0, released in 2019.

[0088] In another more preferred embodiment, the at least one gene encodes an enzyme, wherein the enzyme is selected from enzyme class EC: 1.1.5.-; comprises a polypeptide sequence comprising, consisting of or essentially consisting of the IPR domains IPR011041, IPR011042 and IPR012938 as defined in InterPro 90.0 released on August 4, 2022; comprises a polypeptide sequence comprising, consisting of or essentially consisting of the PFAM domain PF07995 as defined in InterPro 90.0 released on August 4, 2022; and is a part of the NOG family COG2133 as defined in eggNOG5.0 released in 2019.

[0089] In another more preferred embodiment, the at least one gene encodes an enzyme, wherein the enzyme is selected from enzyme class EC: 1.1.5.2; comprises a polypeptide sequence comprising, consisting of or essentially consisting of the IPR domains IPR011041, IPR011042 and IPR012938 as defined in InterPro 90.0 released on August 4, 2022; and comprises a polypeptide sequence comprising, consisting of or essentially consisting of the PFAM domain PF07995 as defined in InterPro 90.0 released on August 4, 2022.

[0090] In another more preferred embodiment, the at least one gene encodes an enzyme that is a dehydrogenase and uses a cofactor selected from the list comprising, consisting of, or consisting essentially of: pyrroloquinoline quinone (PQQ), FAD, a divalent metal cation, Ca 2+ 、Cu 2+ Mg 2+ 、Zn 2+ , heme, heme C and pyridoxal 5'-phosphate. In another more preferred embodiment, the at least one gene encodes an enzyme that i) is a quinone protein glucose dehydrogenase or an aldose sugar dehydrogenase and ii) uses a cofactor selected from the list consisting of, consisting of or consisting essentially of pyrroloquinoline quinone (PQQ), FAD, a divalent metal cation, Ca 2+ 、Cu 2 + Mg 2+ 、Zn 2+ , heme, heme C and pyridoxal 5'-phosphate. In a most preferred embodiment, the at least one gene encodes a dehydrogenase, such as the gcd gene from Escherichia coli, the gdhB_2 gene from Streptomyces fradiae (UniProt ID A0A1Y2NTL5) or the BA894_20530 gene from Vibrio natriegens (UniProtID A0A1B1EJ52).

[0091] In another more preferred embodiment, the at least one gene encodes an enzyme, wherein the enzyme comprises a polypeptide sequence comprising an IPR domain selected from the list consisting of, consisting of, or essentially consisting of IPR000172, IPR001479, IPR007867, IPR011041, IPR011047, IPR013154, IPR013428, IPR015402, IPR017511, IPR029056, and IPR031640 as defined in InterPro 90.0 released on August 4, 2022.

[0092] In another more preferred embodiment, the at least one gene encodes an enzyme, wherein the enzyme comprises a polypeptide sequence comprising a PFAM domain selected from the list consisting of, consisting of, or essentially consisting of: PF00732, PF05199, PF08240, and PF16912 as defined by InterPro 90.0 released on August 4, 2022.

[0093] In another more preferred embodiment, the at least one gene encodes an enzyme, wherein the enzyme comprises the Panther domain PTHR13460 as defined by InterPro 90.0 released on August 4, 2022.

[0094] In another more preferred embodiment, the at least one gene encodes an enzyme, wherein the enzyme is selected from the class of enzymes selected from the list comprising, consisting of, or consisting essentially of: EC:1.1.3.- and EC:1.1.3.5, EC:1.1.3.4; comprising a polypeptide sequence comprising an IPR domain selected from the list comprising, consisting of, or consisting essentially of: IPR000172, IPR006094, IPR007867, IPR012132, IPR012951, IPR016166, IPR016169, IPR027424, IPR036188 and IPR036318 as defined by InterPro 90.0 released on August 4, 2022; comprising a polypeptide sequence comprising a PFAM domain selected from the list consisting of, consisting of or essentially consisting of: PF00732, PF05199, PF08031 and PF01565 as defined by InterPro 90.0 released on August 4, 2022; and using FAD as a cofactor.

[0095] In another more preferred embodiment, the at least one gene encodes an enzyme, wherein the enzyme is selected from enzyme class EC: 1.1.99.18; comprises a polypeptide sequence comprising an IPR domain selected from the list consisting of, consisting of or essentially consisting of: IPR000172, IPR007867, IPR015920 and IPR036188 as defined in InterPro 90.0 released on August 4, 2022; and comprises a polypeptide sequence comprising a PFAM domain selected from the list consisting of, consisting of or essentially consisting of: PF00732, PF05199 and PF16010 as defined in InterPro 90.0 released on August 4, 2022.

[0096] In another particular embodiment, the cell is genetically modified for the production of sugar, wherein the cell comprises a pathway for the production of the sugar. In a preferred embodiment, the cell is genetically modified for the production of two or more sugars. In another and / or another preferred embodiment, the cell is genetically modified for the production of sugar enhancement, the enhanced uptake of one or more precursors and / or acceptors used in the synthesis of sugar, the better outflow of sugar, the reduced production of by-products (e.g., acid), the increased availability of cofactors (e.g., ATP, NADP, NADPH), and / or the better metabolic flux of any one of sialylation, fucosylation, galactosylation, N-acetylglucosaminylation, N-acetylgalactosaminylation, mannosylation and / or N-acetylmannosaminylation pathways present in the cell.

[0097] In a preferred aspect of the methods and / or cells of the invention, the genetically engineered cells are modified with gene expression modules, wherein expression from any one of the expression modules is constitutive or tunable.

[0098] The expression module is also referred to as a transcription unit and comprises a polynucleotide for expressing a recombinant gene, which comprises a coding gene sequence and suitable transcription and / or translation control signals operably connected to the coding sequence. The control signals include a promoter sequence, an untranslated region, a ribosome binding site, and a terminator sequence. The expression module can include elements for expressing a single recombinant gene but can also include elements for expressing multiple recombinant genes, or can be organized into an operon structure for the integrated expression of two or more recombinant genes. The polynucleotide can be produced by recombinant DNA technology using techniques well known in the art. It is well known to those skilled in the art that methods for constructing expression modules include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination. See, for example, Sambrook et al., (2001) Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press, CSH, New York or the technology described in Current Protocols in Molecular Biology, John Wiley and Sons, NY (1989 and annual updates).

[0099] According to a preferred aspect of the present invention, the cell is modified with one or more expression modules. The expression module can be integrated into the genome of the cell or can be presented to the cell on a carrier. The carrier can exist in the form of plasmid, clay, phage, liposome or virus to be stably transformed / transfected into the cell through metabolic modification. This type of carrier especially includes chromosomal, additional and virus-derived carriers, such as those derived from bacterial plasmids, phages, transposons, yeast episomes, insertion elements, yeast chromosome elements, viruses, and carriers derived from their combinations, such as those derived from plasmid and phage genetic elements (such as clay and phagemids). These carriers can include selective markers, such as but not limited to antibiotic markers, auxotrophic markers, toxin-antitoxin markers, RNA sense / antisense markers. The expression system construct can include regulation and control and cause the control region of expression. Generally, any system or carrier suitable for maintaining, propagating or expressing polynucleotides and / or expressed polypeptides in a host can be used for expression in this respect. The appropriate DNA sequence can be inserted into the expression system by any of a variety of well-known and conventional techniques, for example, those described in Sambrook et al. (see above). For recombinant production, cells can be genetically engineered to incorporate the expression system of the present invention, or portions thereof, or polynucleotides. Introduction of polynucleotides into cells can be accomplished by methods described in many standard laboratory manuals, for example, Sambrook et al., 1989 (see above).

[0100] As used herein, an expression module comprises a polynucleotide for expressing at least one recombinant gene. The recombinant gene is involved in a pathway for producing sugars; or the recombinant gene is associated with other pathways in the cell that are not involved in the synthesis of sugars. The recombinant gene encodes an endogenous protein with modified expression or activity, preferably overexpressed; or the recombinant gene encodes a heterologous protein that is heterogenously introduced and expressed (preferably overexpressed) in the modified cell. The endogenous protein may have modified expression in a cell that also expresses a heterologous protein.

[0101] According to a preferred aspect of the invention, the expression of each of said expression modules is constitutive or tunable, as defined herein.

[0102] In a preferred embodiment of the methods and / or cells of the present invention, the pathway for producing sugars is selected from the list comprising, consisting of, or consisting essentially of a fucosylation pathway, a sialylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N-acetylgalactosaminylation pathway, a mannosylation pathway, and an N-acetylmannosaminylation pathway. In a more preferred embodiment, the cell is genetically engineered to comprise at least one of the pathways. In an even more preferred embodiment, the cell comprises at least one of the pathways, wherein at least one of the pathways has been genetically engineered.

[0103] In another and / or additional preferred embodiment of the method and / or cell, the cell comprises a fucosylation pathway. In a more preferred additional and / or alternative embodiment, the cell is metabolically engineered to comprise a fucosylation pathway. In another even more preferred additional and / or alternative embodiment, the cell has been metabolically engineered to comprise a fucosylation pathway, wherein any one or more of the genes selected from a list comprising, consisting of, or consisting essentially of the following have modified and / or enhanced expression: mannose-6-phosphate isomerase, phosphomannose mutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, fucose-1-phosphate guanylyltransferase, and fucosyltransferase.

[0104] In another and / or additional preferred embodiment of the method and / or cell, the cell comprises a sialylation pathway. In a more preferred embodiment, the cell is metabolically engineered to comprise a sialylation pathway. In another more preferred embodiment, the cell has been metabolically engineered to comprise a sialylation pathway, wherein any one or more of the genes selected from a list comprising, consisting of, or substantially consisting of the following have modified and / or enhanced expression: 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 uridyltransferase, glucosamine-1-phosphate acetyltransferase, Neu5Ac synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, and sialyltransferase.

[0105] In another and / or additional preferred embodiment of the method and / or cell, the cell comprises a galactosylation pathway. In a more preferred additional and / or alternative embodiment, the cell is metabolically engineered to comprise a galactosylation pathway. In another more preferred additional and / or alternative embodiment, the cell is metabolically engineered to comprise a galactosylation pathway, wherein any one or more of the genes selected from a list comprising, consisting of, or substantially consisting of the following have modified and / or enhanced expression: galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridyltransferase, phosphoglucomutase, and galactosyltransferase.

[0106] In another and / or additional preferred embodiment of the method and / or cell, the cell comprises an "N-acetylglucosaminylation" pathway. In a more preferred additional and / or alternative embodiment, the cell is metabolically engineered to comprise an N-acetylglucosaminylation pathway. In another even more preferred additional and / or alternative embodiment, the cell has been metabolically engineered to comprise an N-acetylglucosaminylation pathway in which any one or more of the genes selected from a list comprising, consisting of, or consisting essentially of the following have modified and / or enhanced expression: L-glutamine-D-fructose-6-phosphate aminotransferase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N-acetylglucosamine-1-phosphate uridyltransferase, glucosamine-1-phosphate acetyltransferase, and a glycosyltransferase that transfers GlcNAc.

[0107] In another and / or additional preferred embodiment of the method and / or cell, the cell comprises an "N-acetylgalactosaminylation" pathway. In a more preferred additional and / or alternative embodiment, the cell is metabolically engineered to comprise an N-acetylgalactosaminylation pathway. In another more preferred additional and / or alternative embodiment, the cell has been metabolically engineered to comprise an N-acetylgalactosamination pathway, wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of: L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine 1-phosphate uridyltransferase, glucosamine-1-phosphate acetyltransferase, UDP-N-acetylglucosamine 4-epimerase, UDP-glucose 4-epimerase, N-acetylgalactosamine kinase and / or UDP-N-acetylgalactosamine pyrophosphorylase and GalNAc-transferase has modified and / or enhanced expression.

[0108] In another and / or additional preferred embodiment of the method and / or cell, the cell comprises a "mannosylation" pathway. In a more preferred additional and / or alternative embodiment, the cell is metabolically engineered to comprise a mannosylation pathway. In another even more preferred additional and / or alternative embodiment, the cell has been metabolically engineered to comprise a mannosylation pathway in which any one or more of the genes selected from the list comprising, consisting of, or consisting essentially of mannose-6-phosphate isomerase, phosphomannose mutase, and / or mannose-1-phosphate guanylyltransferase and mannosyltransferase have modified and / or enhanced expression.

[0109] In another and / or additional preferred embodiment of the method and / or cell, the cell comprises an "N-acetylmannosamination" pathway. In a more preferred additional and / or alternative embodiment, the cell is metabolically engineered to comprise an N-acetylmannosamination pathway. In another more preferred additional and / or alternative embodiment, the cell has been metabolically engineered to comprise an N-acetylmannosamination pathway, wherein any one or more of the genes selected from the list comprising, consisting of, or consisting essentially of 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 and ManNAc-transferring glycosyltransferases have modified and / or enhanced expression.

[0110] In another and / or additional preferred embodiment of the methods and / or cells of the invention, the cell comprises one or more pathways for monosaccharide synthesis. The pathways for monosaccharide synthesis comprise, consist of or consist essentially of enzymes such as carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, kinases, phosphatases, aldolases, hydrolases, dehydrogenases, enzymes involved in the synthesis of 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).

[0111] In another and / or additional preferred embodiment of the method and / or cell of the present invention, the cell comprises one or more pathways for the synthesis of phosphorylated monosaccharides. The pathways for the synthesis of phosphorylated monosaccharides comprise, consist of, or consist essentially of enzymes involved in the synthesis of one or more monosaccharides, one or more nucleoside mono-, di-, and / or triphosphates, and enzymes involved in the synthesis of phosphoenolpyruvate (PEP), such as, but not limited to, PEP synthases, carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, kinases, phosphatases, aldolases, hydrolases, and dehydrogenases. In another and / or additional preferred embodiment of the method and / or cell of the present invention, the cell comprises one or more pathways for the synthesis of one or more nucleotide activated sugars. The pathway for nucleotide activated sugar synthesis comprises enzymes such as PEP synthase, carboxylase, decarboxylase, isomerase, epimerase, reductase, enolase, phosphorylase, carboxykinase, kinase, phosphatase, aldolase, hydrolase, dehydrogenase, mannose-6-phosphate isomerase, phosphomannose mutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, L-fucokinase / GDP-fucose pyrophosphorylase, L-glutamine-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acetylglucosamine epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylglucosamine-6P 2-epimerase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, N-acetylmannosamine-6-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine-1-phosphate uridyltransferase, 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 uridyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridyltransferase, phosphoglucomutase and / or N-acetylglucosamine-1-phosphate uridyltransferase.

[0112] In another and / or additional preferred embodiment of the method and / or cell, the cell has, preferably expresses, more preferably overexpresses one or more glycosyltransferases selected from the list comprising, consisting of or essentially consisting of: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosaminetransferase, N-acetylgalactosaminetransferase, N-acetylmannosaminetransferase, xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosaminetransferase, N-glycolylneuraminic acid transferase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine transaminase, UDP-N-acetylglucosamine enolpyruvyltransferase and fucosamine transferase.

[0113] In a more preferred embodiment of the method and / or cell of the invention, the fucosyltransferase is selected from the list comprising, consisting of or essentially consisting of α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,4-fucosyltransferase and α-1,6-fucosyltransferase.

[0114] In an alternative and / or additionally more preferred embodiment of the methods and / or cells of the invention, the sialyltransferase is selected from the list comprising, consisting of or essentially consisting of α-2,3-sialyltransferase, α-2,6-sialyltransferase and α-2,8-sialyltransferase.

[0115] In an alternative and / or additionally more preferred embodiment of the method and / or cell of the invention, the galactosyltransferase is selected from the list comprising, consisting of or essentially consisting of β-1,3-galactosyltransferase, N-acetylglucosamine β-1,3-galactosyltransferase, β-1,4-galactosyltransferase, N-acetylglucosamine β-1,4-galactosyltransferase, alpha-1,3-galactosyltransferase and alpha-1,4-galactosyltransferase.

[0116] In an alternative and / or additionally more preferred embodiment of the methods and / or cells of the invention, the glucosyltransferase is selected from the list comprising, consisting of or essentially consisting of α-glucosyltransferase, β-1,2-glucosyltransferase, β-1,3-glucosyltransferase and β-1,4-glucosyltransferase.

[0117] In an alternative and / or additionally more preferred embodiment of the methods and / or cells of the invention, the mannosyltransferase is selected from the list comprising, consisting of or essentially consisting of α-1,2-mannosyltransferase, α-1,3-mannosyltransferase and α-1,6-mannosyltransferase.

[0118] In an alternative and / or additionally more preferred embodiment of the methods and / or cells of the invention, the N-acetylglucosamine transferase is selected from the list comprising, consisting of or essentially consisting of galactoside β-1,3-N-acetylglucosamine transferase and β-1,6-N-acetylglucosamine transferase.

[0119] In an alternative and / or additionally more preferred embodiment of the methods and / or cells of the invention, the N-acetylgalactosamine transferase is selected from the list comprising, consisting of or essentially consisting of alpha-1,3-N-acetylgalactosamine transferase.

[0120] In an alternative and / or further more preferred embodiment of the methods and / or cells of the invention, the cell is modified in terms of 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 that is 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.

[0121] In another and / or additional preferred embodiment of the method and / or cell, the cell is capable of producing, preferably produces, one or more nucleotide activating sugars, preferably the cell is genetically engineered for the production of one or more of said nucleotide activating sugars. Herein, the one or more nucleotide activating sugars are selected from the list comprising, consisting of, or consisting essentially 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-acetylamino-2,6-dideoxy--L-arabino-4-hexulose, UDP-2-acetylamino-2,6-dideoxy--L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetylamino-2,6-dideoxy-L-mannose), dTDP -N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetylamino-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-l-phospho-succinyl-L-glucosamine (UDP-L-PneNAc or UDP-2-acetylamino-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-isorhamnosamine (UDP-L-QuiNAc or UDP-2 -acetylamino-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-Neu5,7(8,9)Ac2, CMP-Neu5Gc, GDP-rhamnose, and UDP-xylose.

[0122] In another and / or additional preferred embodiment of the method and / or cell, the cell comprises a pathway for synthesizing a nucleotide activated sugar selected from the list comprising, consisting of, or consisting essentially 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-acetylamino-2,6-dideoxy-L-arabino-4-hexulose, UDP-2-acetylamino-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetylamino-2,6 -dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetylamino-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-l-phospho-succinyl-L-olactose (UDP-L-PneNAC or UDP-2-acetylamino-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-isorhamnosamine (UDP-L-QuiN Ac or UDP-2-acetylamino-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-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), GDP-rhamnose, and UDP-xylose.

[0123] Cell employed in this article is optionally genetically modified to express the de novo synthesis of UDP-GlcNAc.UDP-GlcNAc can be provided by the enzyme expressed in cell or by the metabolism of cell.This type of cell that produces UDP-GlcNAc can express the enzyme that for example GlcNAc (it is to be added into cell) is converted into UDP-GlcNAc.These enzymes can be for comprising the following, being made up of the following or being made up of the following list substantially any one or more: N-acetyl-D-glucosamine kinase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase and N-acetylglucosamine-1-phosphate uridyl transferase / glucosamine-1-phosphate acetyltransferase, it is from several species, comprises Homo sapiens (Homo sapiens), Escherichia coli.Preferably, described cell is modified to produce UDP-GlcNAc.More preferably, described cell is modified to produce for the UDP-GlcNAc of enhancing. The modification may be any one or more selected from the list comprising, consisting of, or essentially consisting of: 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 uridyltransferase / glucosamine-1-phosphate acetyltransferase.

[0124] Additionally or alternatively, the cells used in this article are optionally genetically modified to express the de novo synthesis of CMP-Neu5Ac. CMP-Neu5Ac can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cells producing CMP-Neu5Ac can express an enzyme that converts, for example, sialic acid into CMP-Neu5Ac. The enzyme can be a CMP-sialic acid synthetase, 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 to produce CMP-Neu5Ac for enhanced. The modification may be any one or more selected from the group consisting of, comprising, or essentially consisting of: knockout of N-acetylglucosamine-6-phosphate deacetylase, knockout of glucosamine-6-phosphate deaminase, overexpression of CMP-sialic acid synthetase, and overexpression of a gene encoding N-acetyl-D-glucosamine-2-epimerase.

[0125] Additionally or alternatively, the cells used in this article are optionally genetically modified to express the de novo synthesis of GDP-fucose. GDP-fucose can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cells producing GDP-fucose can express an enzyme that, for example, fucose (which is to be added to the cell) is converted into GDP-fucose. The enzyme can be, for example, a bifunctional fucokinase / fucose-1-phosphate guanylyltransferase, such as the Fkp from Bacteroides fragilis, or a separate fucokinase together with a separate fucose-1-phosphate guanylyltransferase, as known from several species, including Homo sapiens, wild boar (Sus scrofa) and Rattus norvegicus (Rattus norvegicus). Preferably, the cell is modified to produce GDP-fucose. More preferably, the cell is modified to produce GDP-fucose for enhanced. The modification can be any one or more selected from the list comprising, consisting of, or essentially consisting of: knockout of a gene encoding UDP-glucose:undecylenyl phosphate glucose-1-phosphotransferase, overexpression of a gene encoding GDP-L-fucose synthase, overexpression of a gene encoding GDP-mannose 4,6-dehydratase, overexpression of a gene encoding mannose-1-phosphate guanylyltransferase, overexpression of a gene encoding phosphomannose mutase, and overexpression of a gene encoding mannose-6-phosphate isomerase.

[0126] Additionally or alternatively, the cells used in this article are optionally genetically modified to express the de novo synthesis of UDP-Gal. UDP-Gal can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cells producing UDP-Gal can express an enzyme that, for example, UDP-glucose is converted into UDP-Gal. This 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 to produce UDP-Gal for enhanced production. The modification can be any one or more selected from a list comprising, consisting of, or essentially consisting of the following: knockout of a bifunctional 5'-nucleotidase / UDP-sugar hydrolase encoding gene, knockout of a galactose-1-phosphate uridyltransferase encoding gene, and overexpression of a UDP-glucose-4-epimerase encoding gene.

[0127] Additionally or alternatively, the cell used in this article is optionally genetically modified to express the de novo synthesis of UDP-GalNAc.UDP-GalNAc can be synthesized from UDP-GlcNAc by the effect of one-step reaction, wherein using UDP-N-acetylglucosamine 4-epimerase, for example from the wbgU of class Shigelloides, from the gne of Yersinia enterocolitica or from the wbpP of Pseudomonas aeruginosa serotype O6.Preferably, the cell is modified to produce UDP-GalNAc.More preferably, the cell is modified to produce for the UDP-GalNAc of enhancing.

[0128] Additionally or alternatively, the cells used in this article are optionally genetically modified to express the de novo synthesis of UDP-ManNAc. UDP-ManNAc can be directly synthesized from UDP-GlcNAc via the epimerization reaction performed by UDP-GlcNAc 2-epimerase (e.g., cap5P from Staphylococcus aureus (Staphylococcus aureus), RWE from Escherichia coli, Cps19fK from Streptococcus pneumoniae (S.pneumoniae) and RfbC from Salmonella enterica (S.enterica). Preferably, the cells are modified to produce UDP-ManNAc. More preferably, the cells are modified to produce UDP-ManNAc for enhanced.

[0129] In another and / or additional preferred embodiment of the method and / or cell, the cell has, preferably expresses, more preferably overexpresses one or more genes selected from the list comprising, consisting of or essentially consisting of: mannose-6-phosphate isomerase, phosphomannose mutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, fucose-1-phosphate guanylyltransferase, 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 uridyltransferase, glucosamine-1-phosphate acetyltransferase, Neu5Ac synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase, and UDP-N-acetylgalactosamine pyrophosphorylase.

[0130] In another and / or additional preferred embodiment of the method and / or cell, the cell is genetically engineered for the production of a saccharide, wherein the saccharide is selected from the list comprising, consisting of or consisting essentially of: a monosaccharide; a phosphorylated monosaccharide; an activated monosaccharide; a disaccharide; an oligosaccharide; a neutral (uncharged) oligosaccharide; a negatively charged, preferably sialylated oligosaccharide; a milk oligosaccharide; a sialylated milk oligosaccharide; a neutral (uncharged) milk oligosaccharide; a fucosylated milk oligosaccharide; a non-fucosylated neutral (uncharged) milk oligosaccharide; a sialylated mammalian milk oligosaccharide; a neutral (uncharged) mammalian milk oligosaccharide; a fucosylated mammalian milk oligosaccharide; a non-fucosylated neutral (uncharged) mammalian milk oligosaccharide; a sialylated human milk oligosaccharide; a neutral (uncharged) human milk oligosaccharide; a Milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; O-antigens; enterobacterial common antigen (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigenic oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides; plant oligosaccharides; fucosylated oligosaccharides; sialylated oligosaccharides; neutral (uncharged) sugars containing N-acetylglucosamine; sugars containing N-acetylglucosamine; sugars containing N-acetyllactosamine; sugars containing lacto-N-biose; non-fucosylated neutral (uncharged) sugars; chitosan; oligosaccharides containing chitosan; heparosan; chondroitin sulfate; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; dermatan sulfate; hyaluronan; hyaluronic acid; and keratan sulfate.

[0131] In a more preferred embodiment, the saccharide is a mammalian milk oligosaccharide (MMO) as described herein. In another more preferred embodiment, the saccharide is a human milk oligosaccharide (HMO) as described herein. In another more preferred embodiment, the saccharide is an animal oligosaccharide selected from the list consisting of N-glycans and O-glycans. In another more preferred embodiment, the saccharide is a plant oligosaccharide selected from the list consisting of N-glycans and O-glycans. In the context of the present invention, N-glycans and O-glycans refer to oligosaccharide structures known to those skilled in the art, wherein the structure is not attached to a protein or peptide. In another more preferred embodiment, the fucosylated oligosaccharide is selected from the list comprising, consisting of or essentially consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lactose-N-hexose and difucosyl-lactose-N-neohexose. In another more preferred embodiment, the sialylated oligosaccharide is selected from the list comprising, consisting of or consisting essentially of 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), disialyllacto-N-tetraose, disialyllacto-N-neotetraose , monosialyllacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II and monofucosyldisialyllacto-N-tetraose.In another more preferred embodiment, the neutral (uncharged) sugar containing N-acetylglucosamine is selected from the list comprising, consisting of or essentially consisting of: lacto-N-biose (LNB), N-acetyllactosamine (LacNAc), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose.

[0132] In a more preferred embodiment, the sugar is selected from the list comprising, consisting of, or consisting essentially of: Fucα1-2Galβ1-3GlcNAc; Galβ1-3[Fucα1-4]GlcNAc; Fucα1-2Galβ1-3[Fucα1-4]GlcNAc; Neu5Acα2-3Galβ1-3[Fucα1-4]GlcNAc; Fucα1-2Galβ1-4GlcNAc; Galβ1-4[Fucα1-3]GlcNAc; Fucα1-2Galβ1-4[Fu cα1-3]GlcNAc; Neu5Acα2-3Galβ1-4[Fucα1-3]GlcNAc; 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose Algal hexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose; 3' sialyl lactose (3'SL), 6' sialyl lactose (6'SL), sialyl lacto-N-tetraose a (LSTa), sialyl lacto-N-tetraose b (LSTb), sialyl lacto-N-tetraose c (LSTc), sialyl lactose -N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialylacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialylacto-N-neohexose I, monosialylacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II and monofucosyldisialyllacto-N-tetraose.

[0133] In the context of the present invention, the sugar is preferably in free form, ie the sugar does not comprise any protecting groups.

[0134] In another and / or additional preferred embodiment of the methods and / or cells of the present invention, the cells are capable of producing, preferably producing, the sugar, LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA, and / or sialylated LBA from one or more precursors as defined herein. In a more preferred embodiment, the precursor is lactose. Preferably, the one or more precursors are fed to the cells from culture medium or incubation. In another more preferred embodiment, the cells are capable of producing, preferably producing, at least one of the one or more precursors. In an even more preferred embodiment, the cells are capable of producing, preferably producing all of the one or more precursors. In another even more preferred embodiment, the cells are genetically engineered to produce at least one of the one or more precursors. In an even more preferred embodiment, the cells are genetically engineered to produce all of the one or more precursors. In another even more preferred embodiment, at least one of the one or more precursors is internalized into the cells via one or more membrane proteins. In another preferred embodiment, the precursor used by the cells to produce the sugar is completely converted into the sugar.

[0135] In a preferred embodiment, the sugars of the present invention are produced by cells cultured in cell culture. Within the context of the present invention, the cell culture includes in vitro and / or in vitro culture of cells. In another and / or further more preferred embodiment, the cell culture is fermentation. In an alternative and / or further more preferred embodiment, the cells are cultured or incubated in a reactor as defined herein. In an alternative and / or further more preferred embodiment, the cells are cultured or incubated in an incubator as defined herein.

[0136] In another and / or additional preferred embodiment, the cells are cultured in a culture medium comprising, consisting of, or essentially consisting of a carbon source comprising, consisting of, or essentially consisting of a monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide, a polyol, glycerol, a complex medium (which comprises molasses, corn steep liquor, peptone, tryptone, or yeast extract). Preferably, the carbon source is selected from a list comprising, consisting of, or essentially consisting of glucose, N-acetylglucosamine (GlcNAc), 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. In a more preferred embodiment, the culture medium or culture medium is a medium of known chemical composition. In a further preferred embodiment, the culture medium or incubation medium is a minimal salt medium comprising, consisting of or consisting essentially of sulfate, phosphate, chloride, ammonium, calcium, magnesium, sodium, potassium, iron, copper, zinc, manganese, cobalt and / or selenium. In another and / or further preferred embodiment, the culture or incubation medium comprises one or more precursors for the production of the sugar and / or the LBA. In a more preferred embodiment, the culture or incubation medium comprises one or more cofactors selected from the list comprising, consisting of or consisting essentially of pyrroloquinoline quinone (PQQ), FAD, divalent metal cations, Ca 2+ 、Cu 2+ Mg 2+ 、Zn 2+ , heme, heme C and pyridoxal 5'-phosphate.

[0137] In another and / or additional preferred embodiment of the method of the present invention, the method for producing a sugar as described herein comprises at least one of the following steps:

[0138] i) adding at least one precursor and / or acceptor feed to a culture medium or a culture medium in a reactor, wherein the total reactor volume is from 250 mL (milliliters) to 10,000 mL 3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the culture medium or culture medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the culture medium or culture medium before the addition of the precursor and / or acceptor feed;

[0139] ii) adding at least one precursor and / or acceptor feed to the culture medium or the culture medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days with the aid of a feed solution;

[0140] iii) adding at least one precursor and / or acceptor feed to the culture medium or the culture medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days with the aid of a feed solution, wherein preferably the pH of the feed solution is set between 3 and 7; and wherein preferably the temperature of the feed solution is maintained between 20° C. and 80° C.;

[0141] The method results in sugars wherein the concentration in the final volume of the culture medium or culturing medium is at least 30 g / L.

[0142] In another and / or additional preferred embodiment of the method of the present invention, the method for producing a sugar as described herein comprises at least one of the following steps:

[0143] i) adding at least one precursor and / or acceptor to the culture medium in one pulse or in a discontinuous (pulsated) manner, wherein the total reactor volume is from 250 mL (milliliters) to 10,000 mL 3 (cubic meters), preferably such that the final volume of the culture medium or culturing medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the culture medium or culturing medium before the addition of the precursor and / or acceptor feed pulse;

[0144] ii) adding at least one precursor and / or acceptor feed to the culture medium or the culture medium in a discontinuous (pulsatile) manner with the aid of a 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;

[0145] iii) adding at least one precursor and / or acceptor feed to the culture medium or the culture medium in a discontinuous (pulsating) manner with the aid of a 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 preferably the pH of the feed solution is set between 3 and 7; and wherein preferably the temperature of the feed solution is maintained between 20° C. and 80° C.;

[0146] The method results in sugars wherein the concentration in the final volume of the culture medium or culturing medium is at least 30 g / L.

[0147] In a further more preferred embodiment, the method for producing a sugar as described herein comprises at least one of the following steps:

[0148] i) adding to the culture medium 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 initial reactor volume, wherein the total reactor volume is from 250 mL (milliliters) to 10,000 mL 3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the culture medium or culture medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the culture medium or culture medium before the addition of the lactose feed;

[0149] ii) adding a lactose feed to the culture medium or the culture medium in a continuous manner with the aid of a feed solution during 1 day, 2 days, 3 days, 4 days, 5 days;

[0150] iii) adding a lactose feed to the culture medium or cultivation medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days with the aid of a feed solution, wherein the concentration of the lactose feed solution is 25 g / L, preferably 50 g / L, more 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, still more preferably 550 g / L, most preferably 600 g / L; and wherein preferably, the pH of the solution is set between 3 and 7; and wherein preferably, the temperature of the feed solution is maintained between 20° C. and 80° C.;

[0151] The method results in sugars wherein the concentration in the final volume of the culture medium or culturing medium is at least 30 g / L.

[0152] Preferably, the lactose feeding is achieved by adding lactose from the start of the culture at a concentration of at least 1 mM, preferably at least 5 mM, preferably at a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably at a concentration >300 mM.

[0153] In another aspect, the lactose feeding is achieved by adding lactose to the culture medium in such a concentration that a lactose concentration of at least 1 mM, preferably 5 mM, 10 mM or 30 mM is obtained throughout the production phase of the culture.

[0154] In a further embodiment of the methods described herein, the cells are cultured for at least about 60, 80, 100, or about 120 hours, or in a continuous manner.

[0155] In a preferred embodiment, the carbon source, preferably sucrose, is provided in the culture medium or 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 g of sucrose per liter of initial culture volume is provided in the culture medium or culture medium in a continuous manner, so that the final volume of the culture medium or culture medium is no more than three times, advantageously no more than two times, more advantageously less than two times the volume of the culture medium or culture medium before culturing.

[0156] Preferably, when performing the methods described herein, a first phase of exponential cell growth is provided by adding a carbon source, preferably glucose or sucrose, to the culture medium or culture medium, followed by addition of lactose to the culture medium or culture medium in a second phase.

[0157] In an alternative preferred embodiment, in the methods described herein, lactose is already added during the first phase of exponential growth together with the carbon substrate.

[0158] In another and / or additional preferred embodiment of the method and / or cell of the present invention, the cell comprises an at least partially inactivated catabolic pathway for a selected mono-, di- or oligosaccharide that is involved in and / or required for the synthesis of said saccharide.

[0159] In another and / or additional preferred embodiment of the methods and / or cells of the present invention, the cell is selected from the list consisting of prokaryotic cells and eukaryotic cells, preferably selected from the list consisting of yeast cells, bacterial cells, archaeal cells, algal cells, plant cells and fungal cells. In another preferred embodiment, the cell is a bacterial, fungal, yeast or plant cell.

[0160] The latter bacterium preferably belongs to the phylum Proteobacteria or Firmicutes or Cyanobacteria or Deinococcus-Thermus or Actinobacteria. The latter bacterium belonging to the phylum Proteobacteria preferably belongs to the family Enterobacteriaceae, preferably to the species Escherichia coli. The latter bacterium preferably relates to any strain belonging to the species Escherichia coli, such as but not limited to E. coli B, E. coli C, E. coli W, E. coli K12, E. coli Nissle. More particularly, the latter term relates to a cultured strain of E. coli - designated as the E. coli K12 strain - which is well adapted to the laboratory environment and, unlike the wild-type strain, has lost its ability to flourish in the intestine. Well-known examples of E. coli K12 strains are K12 wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111, and AA200. Therefore, the present invention particularly relates to mutated and / or transformed E. coli cells or strains as indicated above, wherein the E. coli strain is a K12 strain. More preferably, the E. coli K12 strain is E. coli MG1655. The latter bacterium belonging to the phylum Firmicutes preferably belongs to the Bacillus, preferably the order Lactobacilliales, which has members such as Lactobacillus lactis, Leuconostoc mesenteroides, or the order Bacillales, which has members such as from the genus Bacillus, such as Bacillus subtilis or B. amyloliquefaciens. The latter bacterium belonging to the phylum Actinobacteria preferably belongs to the family Corynebacteriaceae, which has members Corynebacterium glutamicum or C. afermentans, or to the family Streptomycetaceae, which has members Streptomyces griseus or S. fradiae. The latter bacterium belonging to the phylum Proteobacteria preferably belongs to the family Vibrionaceae, which has as its member Vibrio natriegens.The latter yeast preferably belongs to the phylum Ascomycota or Basidiomycota or Deuteromycota or Zygomycetes. The latter yeast preferably belongs to the genus Saccharomyces (with members such as Saccharomyces cerevisiae, S. bayanus, S. boulardii), Zygosaccharomyces, Pichia (with members such as Pichia pastoris, P. anomala, P. kluyveri), Komagataella, Hansenula, Kluyveromyces (with members such as Kluyveromyces lactis lactis, K. marxianus, K. thermotolerans, Debaromyces, Candida, Schizosaccharomyces, Schwanniomyces, Torulaspora, Yarrowia (e.g., Yarrowia lipolytica), or Starmerella (e.g., Starmerella bombicola). The latter yeast is preferably selected from the group consisting of 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. The latter plant cell is preferably an algae cell, or is derived from a rose, tobacco, alfalfa, rice, tomato, cotton, oilseed rape, soybean, maize or corn plant. More preferably, the latter plant cell is selected from the family Rosaceae.

[0161] In another and / or additional preferred embodiment, the cell is E. coli or yeast having a lactose permease positive phenotype, preferably wherein the lactose permease is encoded by the gene LacY or LAC12, respectively.

[0162] In another and / or additional preferred embodiment of the methods and / or cells of the invention, the cells produce 30 g / L or more of said sugar in the whole broth and / or supernatant, and / or wherein said sugar in the whole broth and / or supernatant has a purity of at least 80%, measured based on the total amount of sugar and its precursors produced by said cells in the whole broth and / or supernatant, respectively. In a more preferred embodiment, the cells produce 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, 50 g / L, 51 g / L, 52 g / L, 53 g / L, 54 g / L, 55 g / L, 56 g / L, 57 g / L, 58 g / L, 59 g / L, 60 g / L, 61 g / L, 62 g / L, 63 g / L, 64 g / L, 65 g / L, 66 g / L, 67 g / L, 68 g / L, 69 g / L, 70 g / L, 71 g / L, 72 g / L, 73 g / L, 74 g / L g / L, 72g / L, 73g / L, 74g / L, 75g / L, 76g / L, 77g / L, 78g / L, 79g / L, 80g / L, 81g / L, 82g / L, 83g / L, 84g / L, 85g / L or more than 85g / L of said sugar.

[0163] In another and / or additional preferred embodiment of the methods and / or cells of the present invention, the functionally impaired or knocked-out synthesis of LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA confers unaffected and / or enhanced i) sugar formation, ii) productivity, iii) biomass production, iv) cell growth, and / or v) yield of produced sugars relative to the corresponding unmodified cells.

[0164] In another aspect of the invention, the cell produces a sugar as described herein.

[0165] In a preferred embodiment of the methods and / or cells of the invention, the cells produce a mixture comprising, consisting of, or consisting essentially of a sugar as described herein and any one or more of LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA, and / or a sialylated LBA. In another and / or additional preferred embodiment, the cells produce a mixture comprising, consisting of, or consisting essentially of a sugar as described herein and any one or more of LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA, and / or a sialylated LBA, wherein the mixture comprises ≤ 10% by weight of the LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA, and / or a sialylated LBA; preferably ≤ 9% by weight of the LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA, and / or a sialylated LBA. % by weight of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA; more preferably ≤8 wt% of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA; even more preferably ≤7 wt% of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA; even more preferably ≤6 wt% of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA A; more preferably ≤5 wt% of said LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA; more preferably ≤4 wt% of said LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA; more preferably ≤3 wt% of said LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA; more preferably ≤2 wt% of said LBA, modified forms of LBA, LBA % by weight of said LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA; more preferably ≤1 wt% of said LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA; more preferably ≤0.5 wt% of said LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA; most preferably ≤0.1 wt% of said LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA.

[0166] In another and / or additional preferred embodiment, the cell does not produce LBA.

[0167] In another aspect of the invention, the sugars produced by the cells of the invention are recovered from the culture or incubation medium and / or the cells. In a preferred embodiment, the sugars are purified. In another and / or additional preferred embodiment, the sugars are purified from the LBA. In another and / or additional embodiment, the sugars are purified from modified forms of the LBA, glycosylated forms of the LBA, fucosylated LBA, and / or sialylated LBA.

[0168] The term "separated from said culture or incubation" means harvesting, collecting or recovering said sugar from the cells and / or the medium in which they are grown.

[0169] The sugar can be separated from the aqueous culture medium or culture medium in which the cells are grown in a conventional manner. Where the sugar is still present in the cells that produced it, conventional means for releasing or extracting the sugar 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. The culture medium or culture medium and / or cell extract can then be used together or separately to further separate the sugar.

[0170] This preferably involves clarifying the sugar to remove suspended particles and contaminants, particularly cells, cellular components, insoluble metabolites produced by culturing genetically modified cells, and debris. In this step, the sugar can be clarified in a conventional manner. Preferably, the sugar is clarified by centrifugation, flocculation, decantation, and / or filtration. The second step of separating the sugar preferably involves removing substantially all remaining proteins, peptides, amino acids, RNA, DNA, endotoxins, and glycolipids from the sugar, which could interfere with the subsequent separation step, preferably after it has been clarified. In this step, remaining proteins and related impurities can be removed from the sugar in a conventional manner. Preferably, residual proteins, salts, byproducts, color, endotoxins and other related impurities are removed from the sugars 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. In addition to size exclusion chromatography, residual proteins and related impurities are retained by the chromatography medium or the selected membrane.

[0171] In a further preferred embodiment, the methods described herein also provide for further purification of the sugars of the present invention. Further purification of the sugars can be accomplished, for example, by using (activated) charcoal or carbon, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment, or ion exchange 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 sugars. Another purification step is drying, for example, spray drying or freeze drying the resulting sugars.

[0172] In an exemplary embodiment, the separation and purification of the sugars is carried out in a process comprising the following steps in any order:

[0173] a) contacting the culture 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 sugars and allowing passage of at least a portion of the proteins, salts, by-products, colors and other related impurities;

[0174] b) subjecting the retentate from step a) to a diafiltration process using said membrane with an aqueous solution of an inorganic electrolyte, followed by optional diafiltration with pure water to remove excess electrolyte,

[0175] c) and collecting the retentate enriched in said sugars in the form of salts of cations from said electrolyte.

[0176] In an alternative exemplary embodiment, the separation and purification of the sugars is performed in a process comprising the following steps in any order: subjecting the culture or a clarified form thereof to two membrane filtration steps using different membranes, wherein

[0177] - a membrane having a molecular weight cut-off of about 300 to about 500 Daltons, and

[0178] - Another membrane has a molecular weight cut-off of about 600 to about 800 Daltons.

[0179] In an alternative exemplary embodiment, the separation and purification of the sugars is carried out in a process comprising the following steps, in any order: treating the culture or a clarified form thereof with a strong cation exchange resin in H+-form and a weak anion exchange resin in free base form.

[0180] In an alternative exemplary embodiment, the separation and purification of the sugars is performed in the following manner. The culture comprising the produced sugars, biomass, media components and contaminants (wherein the purity of the sugars produced in the culture is <80%) is subjected to the following purification steps:

[0181] i) separating the biomass from the culture,

[0182] ii) cation exchanger treatment to remove positively charged materials,

[0183] iii) anion exchanger treatment to remove negatively charged materials,

[0184] iv) a nanofiltration step and / or an electrodialysis step,

[0185] A purified solution is provided, comprising the produced sugars at a purity greater than or equal to 80%. Optionally, the purified solution is spray-dried.

[0186] In an alternative exemplary embodiment, the separation and purification of the sugars is performed in a process comprising the following steps, in any order: enzymatic treatment of the culture; removal of biomass from the culture; ultrafiltration; nanofiltration; and a column chromatography step. Preferably, such column chromatography is a single column or multiple columns. Further preferably, the column chromatography step is a simulated moving bed chromatography. Such simulated moving bed chromatography preferably comprises: i) at least four columns, at least one of which comprises 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 comprising water; and / or iv) an operating temperature of 15 to 60 degrees Celsius.

[0187] In a particular embodiment, the invention provides the produced sugar spray-dried to a powder, wherein the spray-dried powder comprises <15 wt% water, preferably <10 wt% water, more preferably <7 wt% water, most preferably <5 wt% water.

[0188] To identify the sugars described herein, monomer building blocks (e.g., monosaccharide or polysaccharide unit composition), side chain anomeric configurations, the presence and position of substituent groups, degree of polymerization / molecular weight, and linkage patterns 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 solved using, for example, solid-state NMR, FT-IR (Fourier transform infrared spectroscopy), and WAXS (wide angle X-ray scattering). The degree of polymerization (DP), DP distribution, and polydispersity can be determined, for example, by viscometry and SEC (SEC-HPLC, high performance size exclusion chromatography). To identify the monomeric components of the sugar, methods such as acid-catalyzed hydrolysis, HPLC (high performance liquid chromatography), or GLC (gas-liquid chromatography) (after conversion to alditol acetate) can be used. To determine glycosidic bonds, the sugar is methylated with methyl iodide and a strong base in DMSO, hydrolyzed, reduced to partially methylated alditol, acetylated to methylated alditol acetate, and analyzed by GLC / MS (gas-liquid chromatography-mass spectrometry). To determine the sequence of the glycan, partial depolymerization is performed using an acid or enzyme to determine the structure. To identify the anomeric configuration, the sugar is subjected to enzymatic analysis, for example, by contacting it with an enzyme specific for a particular type of linkage (e.g., β-galactosidase or α-glucosidase, etc.), and the product can be analyzed using NMR.

[0189] In another aspect, the present invention provides the use of a cell as described herein for producing a sugar, wherein the sugar is selected from the list comprising, consisting of or consisting essentially of: a monosaccharide; a phosphorylated monosaccharide; an activated monosaccharide; a disaccharide; an oligosaccharide; a neutral (uncharged) oligosaccharide; a negatively charged, preferably sialylated, oligosaccharide; a milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO); a sialylated milk oligosaccharide; a neutral (uncharged) milk oligosaccharide; a negatively charged, preferably sialylated, oligosaccharide; a milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO); a milk oligosaccharide; ... Sugars; fucosylated milk oligosaccharides; nonfucosylated neutral (uncharged) milk oligosaccharides; sialylated mammalian milk oligosaccharides; neutral (uncharged) mammalian milk oligosaccharides; fucosylated mammalian milk oligosaccharides; nonfucosylated neutral (uncharged) mammalian milk oligosaccharides; sialylated human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; nonfucosylated neutral (uncharged) human milk oligosaccharides; O-antigen; enterobacterial common antigen (ECA); in capsular polysaccharides oligosaccharide repeats present in the sugar; peptidoglycan; amino sugar; Lewis type antigenic oligosaccharide; antigens of the human ABO blood group system; animal oligosaccharides, which are preferably selected from the list consisting of N-glycans and O-glycans; plant oligosaccharides, which are preferably selected from the list consisting of N-glycans and O-glycans; fucosylated oligosaccharides, which are preferably selected from the list comprising, consisting of or essentially consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (5FL), 5-fucosyllactose (6FL), 6-fucosyllactose (7FL), 7-fucosyllactose (8FL), 8-fucosyllactose (9FL), 9-fucosyllactose (10FL), 10-fucosyllactose (11FL), 12-fucosyllactose (13FL), 14-fucosyllactose (15FL), 16-fucosyllactose (17FL), 18-fucosyllactose (19FL), 19-fucosyllactose (20FL), 20-fucosyllactose (21FL), 22-fucosyllactose (23FL), 24-fucosyllactose (24FL), 25-fucosyllactose (25FL), 26-fucosyllactose (27FL), 28-fucosyllactose (29FL), 29-fucosyllactose (30FL), 30-fucosyllactose (31FL), 31-fucosyllactose (32FL), 32-fucosyllactose (33FL), 33-fucosyllactose (34FL), 35-fucosyllactose (35FL), 36-fu Fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lact-N-hexose, and difucosyl-lact-N-neohexose;Sialyl oligosaccharides, preferably selected from the list comprising, consisting of or consisting essentially of: 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), disialyllacto-N-tetraose, disialyllacto- N-neotetraose, monosialyllactose-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllactose-N-neohexose I, monosialyllactose-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllactose-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose Saccharide II and monofucosyldisialyllacto-N-tetraose; neutral (uncharged) saccharides containing N-acetylglucosamine, preferably selected from the list comprising, consisting of or essentially consisting of: lacto-N-biose (LNB), N-acetyllactosamine (LacNAc), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactose lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose; sugars containing N-acetylglucosamine; sugars containing N-acetyllactosamine; sugars containing lacto-N-biose; non-fucosylated neutral (uncharged) sugars; chitosan; oligosaccharides containing chitosan; heparosan; chondroitin sulfate; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; dermatan sulfate; hyaluronan; hyaluronic acid; and keratan sulfate.

[0190] In another aspect, the invention provides the use of the methods described herein for producing a saccharide, wherein the saccharide is selected from the list comprising, consisting of, or consisting essentially of: a monosaccharide; a phosphorylated monosaccharide; an activated monosaccharide; a disaccharide; an oligosaccharide; a neutral (uncharged) oligosaccharide; a negatively charged, preferably sialylated, oligosaccharide; a milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO); a sialylated milk oligosaccharide; a neutral (uncharged) milk oligosaccharide; a negatively charged, preferably sialylated, oligosaccharide; a milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO); a sialylated milk oligosaccharide; a neutral (uncharged) milk oligosaccharide; a negatively charged, preferably sialylated, oligosaccharide; a milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO); a milk oligosaccharide; ... Sugars; fucosylated milk oligosaccharides; nonfucosylated neutral (uncharged) milk oligosaccharides; sialylated mammalian milk oligosaccharides; neutral (uncharged) mammalian milk oligosaccharides; fucosylated mammalian milk oligosaccharides; nonfucosylated neutral (uncharged) mammalian milk oligosaccharides; sialylated human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; nonfucosylated neutral (uncharged) human milk oligosaccharides; O-antigen; enterobacterial common antigen (ECA); in capsular polysaccharides oligosaccharide repeats present in the sugar; peptidoglycan; amino sugar; Lewis type antigenic oligosaccharide; antigens of the human ABO blood group system; animal oligosaccharides, which are preferably selected from the list consisting of N-glycans and O-glycans; plant oligosaccharides, which are preferably selected from the list consisting of N-glycans and O-glycans; fucosylated oligosaccharides, which are preferably selected from the list comprising, consisting of or essentially consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (5FL), 5-fucosyllactose (6FL), 6-fucosyllactose (7FL), 7-fucosyllactose (8FL), 8-fucosyllactose (9FL), 9-fucosyllactose (10FL), 10-fucosyllactose (11FL), 12-fucosyllactose (13FL), 14-fucosyllactose (15FL), 16-fucosyllactose (17FL), 18-fucosyllactose (19FL), 19-fucosyllactose (20FL), 20-fucosyllactose (21FL), 22-fucosyllactose (23FL), 24-fucosyllactose (24FL), 25-fucosyllactose (25FL), 26-fucosyllactose (27FL), 28-fucosyllactose (29FL), 29-fucosyllactose (30FL), 30-fucosyllactose (31FL), 31-fucosyllactose (32FL), 32-fucosyllactose (33FL), 33-fucosyllactose (34FL), 35-fucosyllactose (35FL), 36-fu Fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lact-N-hexose, and difucosyl-lact-N-neohexose;Sialyl oligosaccharides, preferably selected from the list comprising, consisting of or consisting essentially of: 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), disialyllacto-N-tetraose, disialyllacto- N-neotetraose, monosialyllactose-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllactose-N-neohexose I, monosialyllactose-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllactose-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose Saccharide II and monofucosyldisialyllacto-N-tetraose; neutral (uncharged) saccharides containing N-acetylglucosamine, preferably selected from the list comprising, consisting of or essentially consisting of: lacto-N-biose (LNB), N-acetyllactosamine (LacNAc), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactose lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose; sugars containing N-acetylglucosamine; sugars containing N-acetyllactosamine; sugars containing lacto-N-biose; non-fucosylated neutral (uncharged) sugars; chitosan; oligosaccharides containing chitosan; heparosan; chondroitin sulfate; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; dermatan sulfate; hyaluronan; hyaluronic acid; and keratan sulfate.

[0191] In another aspect, the present invention provides a purified sugar or purified sugar mixture as described herein for use in medicine, preferably for use in the prevention or treatment of gastrointestinal disorders.

[0192] In another aspect, the invention provides the purposes of the purified sugar obtained by the method described herein in food or feed preparations, in dietary supplements, in cosmetic ingredients or in pharmaceutical ingredients. In some embodiments, the sugar is mixed with one or more ingredients suitable for food, feed, dietary supplements, pharmaceutical ingredients, cosmetic ingredients or medicines. The purified sugar can be used to manufacture preparations, as food additives, prebiotics, symbiotics, for the supplementation of infant food, adult food, young animal feed, adult animal feed, or as therapeutically or pharmaceutically active compounds, or used in cosmetic applications. In another aspect, the invention provides sugar or sugar mixtures as described herein as additives in food, preferably as additives in human food and / or pet food, more preferably as the purposes of additives in human infant food. In the context of the present invention, the food is human food, preferably baby food, human infant food and / or infant formula or infant supplement, and the feed is pet food, animal milk substitutes, veterinary products, veterinary feed supplements, nutritional supplements, weaning feed or creep feed.

[0193] 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. "Prebiotics" are substances that promote the growth of microorganisms (particularly microorganisms in the gastrointestinal tract) that are beneficial to the host. In some embodiments, dietary supplements provide a variety of prebiotics, including the sugar, which is a prebiotic purified by the method disclosed in this specification, to promote the growth of one or more beneficial microorganisms. Examples of prebiotic ingredients for dietary supplements include other prebiotic molecules (such as HMOs) and plant polysaccharides (such as inulin, pectin, b-glucans and xylo-oligosaccharides). "Prebiotic" products typically include live microorganisms that replace or are added to the gastrointestinal microbial flora for the benefit of the recipient. Examples of such microorganisms include Lactobacillus species (e.g., Lactobacillus acidophilus and Lactobacillus bulgaricus), Bifidobacterium species (e.g., B. animalis, B. longum, and B. infantis (e.g., Bi-26), and Saccharomyces boulardii. In some embodiments, the saccharides produced and / or purified by the methods of the present disclosure are orally administered in combination with such microorganisms. Examples of further ingredients for dietary supplements include oligosaccharides (e.g., 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyl lactose, 6'-sialyl lactose), disaccharides (e.g., lactose), monosaccharides (e.g., glucose, galactose, L-fucose, sialic acid, glucosamine, and N-acetylglucosamine), thickeners (e.g., gum arabic), acidity regulators (e.g., trisodium citrate), water, skim milk, and flavorings.

[0194] In some embodiments, the sugar purified by the method described herein is incorporated into people's infant food (for example, infant formula).Infant formula is generally for feeding the artificial food of baby as the complete or partial substitute of human breast milk.In some embodiments, infant formula is sold with powder, and is ready to carry out bottle or cup feeding to baby by mixing with water.The composition of infant formula is typically designed to simulate human breast milk in general.In some embodiments, the sugar purified by the method described herein is included in infant formula to provide those similar nutritional benefits provided by the oligosaccharides in human breast milk.In some embodiments, the purified sugar is mixed with one or more components of 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, rapeseed 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 (HMOs). 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. In some embodiments, the one or more infant formula ingredients include lactose, whey protein concentrate, and / or high oleic safflower oil. In some embodiments, the concentration of the oligosaccharides in the infant formula is about the same concentration as that typically found in human breast milk. In some embodiments, sugars purified by the methods described herein are added to the infant formula at a concentration that is about the same as the concentration at which the compound is normally found in human breast milk.

[0195] In another aspect, the present application provides a mixture comprising, consisting of, or consisting essentially of: (i) a sugar, and (ii) LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA, and / or a sialylated LBA, wherein the sugar, LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA, and / or a sialylated LBA is obtainable or obtained by the methods described herein.

[0196] In another and / or additional particular embodiment, the present application provides a mixture comprising, consisting of, or consisting essentially of: (i) a sugar, and (ii) ≤10 wt% LBA, ≤9 wt% LBA, ≤8 wt% LBA, ≤7 wt% LBA, ≤6 wt% LBA, ≤5 wt% LBA, ≤4 wt% LBA, ≤3 wt% LBA, ≤2 wt% LBA, ≤1 wt% LBA, ≤0.5 wt% LBA, and / or ≤0.1 wt% LBA, wherein the sugar and LBA are obtainable or obtained by the methods described herein.

[0197] In another and / or additional particular embodiment, the present application provides a mixture comprising, consisting of, or consisting essentially of: a sugar, and (i) ≤10 wt% LBA, ≤9 wt% LBA, ≤8 wt% LBA, ≤7 wt% LBA, ≤6 wt% LBA, ≤5 wt% LBA, ≤4 wt% LBA, ≤3 wt% LBA, ≤2 wt% LBA, ≤1 wt% LBA, ≤0.5 wt% LBA, and / or ≤0.1 wt% LBA, and / or (ii) ) ≤10 wt % of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤9 wt % of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤8 wt % of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤7 wt % of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤6 wt % of LBA %, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤5 wt% of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤4 wt% of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤2 wt% of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤1 wt% of modified forms of LBA, LBA % of a glycosylated form, fucosylated LBA and / or sialylated LBA, ≤0.5 wt. % of a modified form of LBA, a glycosylated form of LBA, fucosylated LBA and / or sialylated LBA, and / or ≤0.1 wt. % of a modified form of LBA, a glycosylated form of LBA, fucosylated LBA and / or sialylated LBA, wherein the sugar, LBA, modified form of LBA, glycosylated form of LBA, fucosylated LBA and / or sialylated LBA is obtainable or obtained by the methods described herein.

[0198] Unless otherwise defined, all technical and scientific terms used in this article generally have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used in this article 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 used in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, purification steps are carried out according to the manufacturer's instructions.

[0199] Further advantages can be derived from the specific embodiments and examples. It goes without saying that the features mentioned above and those still to be explained below can be used not only in the respectively specified combination, but also in other combinations or on their own without departing from the scope of the present invention.

[0200] Furthermore, the present invention relates to the following particular embodiments:

[0201] 1. A cell capable of synthesizing, preferably synthesizing, lactobionic acid (4-O-β-galactopyranosyl-D-gluconate, LBA), said cell being genetically modified for the production of a sugar, said cell comprising a pathway for the production of said sugar, characterized in that LBA synthesis is functionally impaired or knocked out in said cell.

[0202] 2. The cell according to embodiment 1, wherein the pathway for producing sugars is selected from the list consisting of a fucosylation pathway, a sialylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N-acetylgalactosaminylation pathway, a mannosylation pathway and an 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.

[0203] 3. The cell according to any one of embodiments 1 or 2, wherein the cell:

[0204] - has, preferably expresses, more preferably overexpresses, one or more glycosyltransferases selected from the list consisting of fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosamine transferase, N-acetylgalactosamine transferase, N-acetylmannosamine transferase, xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosamine transferase, N-glycolylneuraminic acid transferase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine transaminase, UDP-N-acetylglucosamine enolpyruvyltransferase and fucosamine transferase, and / or

[0205] - capable of producing, preferably producing, one or more nucleotide activating sugars, preferably the cell is genetically engineered for the production of one or more of said nucleotide activating sugars.

[0206] 4. The cell according to any one of the preceding embodiments, wherein the cell comprises a pathway for synthesizing a nucleotide activated sugar 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-acetylamino-2,6-dideoxy-L-arabino-4-hexulose, UDP-2-acetylamino-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetylamino-2,6-dideoxy-L-mannose), d TDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetylamino-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-phospho-L-galactose (UDP-L-PneNAc or UDP-2-acetylamino-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-isorhamnosamine (UDP-L-QuiNAc or UDP- 2-acetylamino-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-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), GDP-rhamnose, and UDP-xylose.

[0207] 5. The cell according to any one of the preceding embodiments, wherein the cell has, preferably expresses, more preferably overexpresses one or more genes selected from the list comprising: mannose-6-phosphate isomerase, phosphomannose mutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, fucose-1-phosphate guanylyltransferase, 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 uridyltransferase, glucosamine-1-phosphate acetyltransferase, Neu5Ac synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase, and UDP-N-acetylgalactosamine pyrophosphorylase.

[0208] 6. A cell according to any of the preceding embodiments, wherein the LBA synthesis is obtained by expressing at least one gene selected from the list comprising genes encoding carbohydrate oxidases, dehydrogenases, lactonases, oxygen-dependent FAD-linked oxidoreductases and pyrroloquinoline quinone (PQQ) oxidoreductases, and wherein the at least one gene is functionally impaired or knocked out.

[0209] 7. The cell of embodiment 6, wherein:

[0210] - the dehydrogenase is selected from the list comprising lactose dehydrogenase, quinoline glucose dehydrogenase, aldose dehydrogenase, glucose / fructose dehydrogenase, glucose / sorbosone dehydrogenase, cellobiose dehydrogenase, pyrroloquinoline quinone (PQQ) dehydrogenase and malate dehydrogenase, and / or

[0211] - The carbohydrate oxidase is lactose oxidase.

[0212] 8. The cell of embodiment 6 or 7, wherein the at least one gene encodes an enzyme, wherein the enzyme:

[0213] - an enzyme class selected from the list consisting of EC: 1.1.3.-, EC: 1.1.3.5, EC: 1.1.3.4, EC: 1.1.5.-, EC: 1.1.5.2 and EC: 1.1.99.18,

[0214] - comprising a polypeptide sequence comprising an IPR domain selected from the list consisting of: IPR000172, IPR001479, IPR002372, IPR006094, IPR007867, IPR011041, IPR011042, IPR011047, IPR012132, IPR012938, IPR012951, IPR013154, IPR013428, IPR015402, IPR015920, IPR016166, IPR016169, IPR017511, IPR017512, IPR018391, IPR027424, IPR029056, IPR031640, IPR036188, and IPR036318, as defined in 90.0.

[0215] - comprising a polypeptide sequence comprising a Panther domain selected from the list consisting of PTHR11552, PTHR13460, PTHR32303:SF4, PTHR42973 and PTHR47190 as defined by InterPro 90.0 released on August 4, 2022,

[0216] comprising a polypeptide sequence comprising a PFAM domain selected from the list consisting of PF00732, PF01011, PF05199, PF07995, PF08031, PF08240, PF13360, PF13570, PF01565, PF16010 and PF16912 as defined by InterPro 90.0 released on August 4, 2022,

[0217] - comprising a polypeptide sequence comprising the conserved protein domain cd10280 as defined by InterPro 90.0 released on August 4, 2022,

[0218] - is part of a NOG family selected from the list consisting of: COG0277, COG2133, COG2303 and COG4993 as defined by eggNOG5.0 released in 2019, and / or

[0219] - Using cofactors selected from the list including: pyrroloquinoline quinone (PQQ), FAD, divalent metal cations, Ca 2+ 、Cu 2+ Mg 2+ 、Zn 2+ , heme, heme C and pyridoxal 5'-phosphate.

[0220] 9. A cell according to any one of the preceding embodiments, wherein at least one gene involved in the synthesis and / or import of a cofactor involved in LBA synthesis is functionally impaired or knocked out, preferably the cofactor is selected from the group consisting of pyrroloquinoline quinone (PQQ), FAD, divalent metal cations, Ca 2+ 、Cu 2+ Mg 2+ 、Zn 2+ , heme, heme C, and pyridoxal 5'-phosphate.

[0221] 10. The cell according to any one of embodiments 6 to 9, wherein the at least one gene is functionally impaired by insertion, deletion and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from the list comprising: a promoter sequence, a ribosome binding site, an untranslated region, a coding sequence and a transcription terminator sequence of the at least one gene.

[0222] 11. The cell according to any one of the preceding embodiments, wherein the sugar is selected from the list comprising: monosaccharides; phosphorylated monosaccharides; activated monosaccharides; disaccharides; oligosaccharides; neutral (uncharged) oligosaccharides; negatively charged, preferably sialylated oligosaccharides; milk oligosaccharides, preferably mammalian milk oligosaccharides (MMOs), more preferably human milk oligosaccharides (HMOs); sialylated milk oligosaccharides; neutral (uncharged) milk oligosaccharides; fucosylated milk oligosaccharides; non-fucosylated neutral (uncharged) milk oligosaccharides; sialylated mammalian milk oligosaccharides; neutral (uncharged) mammalian milk oligosaccharides; fucosylated mammalian milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides; sialylated Human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; O-antigens; enterobacterial common antigens (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigenic 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; fucosylated oligosaccharides, which are preferably selected from the group consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose ( 6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated oligosaccharides, which are preferably selected from the group comprising: 3' sialyl lactose (3'SL), 6' sialyl lactose (6'SL), sialyl lacto-N-tetraose a (LSTa), sialyl lacto-N-tetraose b (LSTb), Sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, and monofucosyldisialyllacto-N-tetraose;Neutral (uncharged) sugars containing N-acetylglucosamine, preferably selected from the group comprising: lacto-N-biose (LNB), N-acetyllactosamine (LacNAc), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose; sugars containing N-acetylglucosamine; sugars containing N-acetyllactosamine; sugars containing lacto-N-biose; non-fucosylated neutral (uncharged) sugars; chitosan; oligosaccharides containing chitosan; heparosan; chondroitin sulfate; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; dermatan sulfate; hyaluronan; hyaluronic acid; and keratan sulfate. ;

[0223] 12. The cell according to any one of the preceding embodiments, wherein the cell is capable of producing the sugar from one or more precursors, preferably producing the sugar from one or more precursors, preferably the precursor is lactose.

[0224] 13. The cell according to embodiment 12, 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.

[0225] 14. The cell according to any one of embodiments 12 or 13, 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.

[0226] 15. The cell of any one of embodiments 12 to 14, wherein at least one of the one or more precursors is internalized into the cell via one or more membrane proteins.

[0227] 16. The cell according to any one of the preceding embodiments, wherein the cell comprises an at least partially inactivated catabolic pathway for a selected mono-, di- or oligosaccharide involved in and / or required for the synthesis of said sugar.

[0228] 17. The cell according to any one of the preceding 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, algae cells, plant cells and fungal cells.

[0229] 18. The cell according to any one of the preceding embodiments, wherein the cell is a bacterial, fungal, yeast or plant cell,

[0230] - 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, Lactobacillus, Corynebacterium and Vibrio; even more preferably, the bacterium is selected from the list consisting of Escherichia coli strains, Bacillus subtilis strains, Vibrio natriuresis strains; even more preferably, the Escherichia coli strain is K12 strain; most preferably, the Escherichia coli K12 strain is Escherichia coli MG1655,

[0231] - preferably, the fungus belongs to a genus selected from the group consisting of Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus,

[0232] - preferably, the yeast belongs to a genus selected from the group consisting of Saccharomyces, Zygosaccharomyces, Pichia, Komagata, Hansenula, Yarrowia, Stamosaccharomyces, Kluyveromyces, Debaryomyces, Candida, Schizosaccharomyces, Schwannella or Torulospora; 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, Schwannella occidentalis, Torulospora delavirdii, Yarrowia lipolytica, Zygosaccharomyces rouxii and Zygosaccharomyces bailii,

[0233] Preferably, the plant cell is an algae cell or is derived from a rose, tobacco, alfalfa, rice, tomato, cotton, oilseed rape, soybean, maize or corn plant.

[0234] 19. The cell according to any one of the preceding 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.

[0235] 20. A method for producing sugars, the method comprising:

[0236] i. cultivating and / or incubating the cell according to any one of the preceding embodiments in a culture and / or incubation medium under conditions allowing the production of said sugar and said LBA,

[0237] ii. preferably, separating said sugar from said culturing and / or incubation,

[0238] iii. Preferably, the sugars are separated from the LBA.

[0239] 21. The method of embodiment 20, wherein the culture or incubation medium comprises one or more precursors for producing the sugar and / or the LBA.

[0240] 22. The method of any one of embodiments 20 or 21, wherein the cells produce 30 g / L or more of the sugar in the whole broth and / or supernatant, and / or wherein the sugar in the whole broth and / or supernatant has a purity of at least 80%, measured based on the total amount of sugar and its precursors produced by the cells in the whole broth and / or supernatant, respectively.

[0241] 23. The method of any one of embodiments 20 to 22, wherein the functionally degraded or knocked-out synthesis of LBA confers unaffected and / or enhanced i) sugar formation, ii) productivity, iii) biomass production, iv) cell growth, and / or v) yield of produced sugars relative to a corresponding unmodified cell.

[0242] 24. The method of any one of embodiments 20 to 23, wherein the cell produces a mixture comprising the sugar and LBA, wherein the mixture comprises ≤10 wt% LBA, preferably ≤9 wt% LBA, more preferably ≤8 wt% LBA, even more preferably ≤7 wt% LBA, even more preferably ≤6 wt% LBA, even more preferably ≤5 wt% LBA, even more preferably ≤4 wt% LBA, even more preferably ≤3 wt% LBA, even more preferably ≤2 wt% LBA, even more preferably ≤1 wt% LBA, even more preferably ≤0.5 wt% LBA, and most preferably ≤0.1 wt% LBA.

[0243] 25. The method of any one of embodiments 20 to 24, wherein the cell produces a modified form of LBA, preferably wherein the modified form of LBA is a glycosylated LBA, more preferably wherein the modified form of LBA comprises a fucosylated LBA and a sialylated LBA.

[0244] 26. The method of any one of embodiments 20 to 23 or 25, wherein the cells do not produce free LBA.

[0245] 27. The method according to any one of embodiments 20 to 26, wherein the sugar is recovered from the culture or incubation medium and / or the cells, more preferably purified.

[0246] 28. The method of any one of embodiments 20 to 27, wherein the sugar is purified from the LBA and / or from a modified form of the LBA.

[0247] 29. Use of a cell according to any one of embodiments 1 to 19 for producing a sugar, wherein the sugar is selected from the list comprising: a monosaccharide; a phosphorylated monosaccharide; an activated monosaccharide; a disaccharide; an oligosaccharide; a neutral (uncharged) oligosaccharide; a negatively charged, preferably sialylated oligosaccharide; a milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO); a sialylated milk oligosaccharide; a neutral (uncharged) milk oligosaccharide; a fucosylated milk oligosaccharide; a non-fucosylated neutral (uncharged) milk oligosaccharide; a sialylated mammalian milk oligosaccharide; a neutral (uncharged) mammalian milk oligosaccharide; a fucosylated mammalian milk oligosaccharide; a non-fucosylated neutral (uncharged) mammalian milk oligosaccharide; Milk oligosaccharides; sialylated human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; O-antigens; enterobacterial common antigens (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis type antigenic 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; fucosylated oligosaccharides, which are preferably selected from the group consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6-fucosyllactose), ... Sialyl lactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated oligosaccharides, preferably selected from the group comprising 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllactose-N-tetraose a (LSTa), sialyllactose-N-tetraose b (LSTb) ), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, and monofucosyldisialyllacto-N-tetraose;Neutral (uncharged) sugars containing N-acetylglucosamine, preferably selected from the group comprising: lacto-N-biose (LNB), N-acetyllactosamine (LacNAc), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose; sugars containing N-acetylglucosamine; sugars containing N-acetyllactosamine; sugars containing lacto-N-biose; non-fucosylated neutral (uncharged) sugars; chitosan; oligosaccharides containing chitosan; heparosan; chondroitin sulfate; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; dermatan sulfate; hyaluronan; hyaluronic acid; and keratan sulfate. ;

[0248] 30. Use of the method according to any one of embodiments 20 to 28 for producing a sugar, wherein the sugar is selected from the list comprising: monosaccharides; phosphorylated monosaccharides; activated monosaccharides; disaccharides; oligosaccharides; neutral (uncharged) oligosaccharides; negatively charged, preferably sialylated oligosaccharides; milk oligosaccharides, preferably mammalian milk oligosaccharides (MMO), more preferably human milk oligosaccharides (HMO); sialylated milk oligosaccharides; neutral (uncharged) milk oligosaccharides; fucosylated milk oligosaccharides; non-fucosylated neutral (uncharged) milk oligosaccharides; sialylated mammalian milk oligosaccharides; neutral (uncharged) mammalian milk oligosaccharides; fucosylated mammalian milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides Milk oligosaccharides; sialylated human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; O-antigens; enterobacterial common antigens (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis type antigenic 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; fucosylated oligosaccharides, which are preferably selected from the group consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6-fucosyllactose), ... Sialyl lactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated oligosaccharides, preferably selected from the group comprising 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllactose-N-tetraose a (LSTa), sialyllactose-N-tetraose b (LSTb) ), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, and monofucosyldisialyllacto-N-tetraose;Neutral (uncharged) sugars containing N-acetylglucosamine, preferably selected from the group comprising: lacto-N-biose (LNB), N-acetyllactosamine (LacNAc), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose; sugars containing N-acetylglucosamine; sugars containing N-acetyllactosamine; sugars containing lacto-N-biose; non-fucosylated neutral (uncharged) sugars; chitosan; oligosaccharides containing chitosan; heparosan; chondroitin sulfate; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; dermatan sulfate; hyaluronan; hyaluronic acid; and keratan sulfate. ;

[0249] More particularly, the present invention relates to the following preferred particular embodiments:

[0250] 1. A cell capable of synthesizing and / or synthesizing lactobionic acid (4-O-β-galactopyranosyl-D-gluconic acid, LBA), modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA in the presence of lactose in the culture or incubation medium of the cell, wherein the synthesis of LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA is obtained by expressing at least one gene selected from the list consisting of genes encoding carbohydrate oxidase, desmin, pyridoxine kinase, oligosaccharide ... Hydrogenase, lactonase, oxygen-dependent FAD-linked oxidoreductase and pyrroloquinoline quinone (PQQ) oxidoreductase, the cell is genetically modified for the production of sugars, the cell comprises a pathway for the production of said sugars, characterized in that the synthesis of said LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA is functionally degraded or knocked out in the cell by degrading or knocking out said at least one gene, two or more of said genes or all of said genes.

[0251] 2. The cell according to preferred embodiment 1, wherein:

[0252] - the pathway for producing sugars is selected from the list consisting of or consisting essentially of a fucosylation pathway, a sialylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N-acetylgalactosaminylation pathway, a mannosylation pathway, and an N-acetylmannosaminylation pathway, and / or

[0253] - the pathway for producing sugars is selected from the list consisting of or consisting essentially of a fucosylation pathway, a sialylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N-acetylgalactosaminylation pathway, a mannosylation pathway, and an N-acetylmannosaminylation pathway, and wherein the cell is genetically engineered to comprise at least one of said pathways, and / or the cell comprises at least one of said pathways, wherein at least one of said pathways has been genetically engineered.

[0254] 3. The cell according to any one of preferred embodiments 1 or 2, wherein the cell:

[0255] - having, expressing and / or overexpressing one or more glycosyltransferases selected from the list consisting of or consisting essentially of: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosamine transferase, N-acetylgalactosamine transferase, N-acetylmannosamine transferase, xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosamine transferase, N-glycolylneuraminic acid transferase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine transaminase, UDP-N-acetylglucosamine enolpyruvyltransferase and fucosamine transferase,

[0256] - is capable of producing and / or produces one or more nucleotide activating sugars,

[0257] - genetically engineered to produce one or more of the nucleotide activated sugars,

[0258] - comprising a pathway for synthesizing a nucleotide-activated sugar selected from the list consisting of or consisting essentially 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-acetylamino-2,6-dideoxy-L-arabino-4-hexulose, UDP-2-acetylamino-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetylamino-2,6-dideoxy-L-mannose), dTDP-N- Acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetylamino-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-l-phospho-glucosamine (UDP-L-PneNAc or UDP-2-acetylamino-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-isorhamnosamine (UDP-L-QuiNAc or UDP-2-acetylamino-2,6-dideoxy-L-talose), CMP-Neu5Ac, CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-Neu5Gc, GDP-rhamnose, and UDP-xylose,

[0259] - having, expressing and / or overexpressing one or more genes selected from the list consisting of or consisting essentially of: mannose-6-phosphate isomerase, phosphomannose mutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, fucose-1-phosphate guanylyltransferase, 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 uridyltransferase, glucosamine-1-phosphate acetyltransferase, Neu5Ac synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase, and UDP-N-acetylgalactosamine pyrophosphorylase, and / or

[0260] - comprising at least partially inactivated catabolic pathways for selected mono-, di- or oligosaccharides involved in and / or required for the synthesis of said saccharides.

[0261] 4. A cell according to any one of the preceding preferred embodiments, wherein:

[0262] - the dehydrogenase is selected from the list consisting of or consisting essentially of lactose dehydrogenase, quinoline glucose dehydrogenase, aldose dehydrogenase, glucose / fructose dehydrogenase, glucose / sorbosone dehydrogenase, cellobiose dehydrogenase, pyrroloquinoline quinone (PQQ) dehydrogenase and malate dehydrogenase, and / or

[0263] - The carbohydrate oxidase is lactose oxidase.

[0264] 5. The cell according to any one of the preceding preferred embodiments, wherein the at least one gene encodes an enzyme, wherein the enzyme:

[0265] - an enzyme class selected from the list consisting of or consisting essentially of EC: 1.1.3.-, EC: 1.1.3.5, EC: 1.1.3.4, EC: 1.1.5.-, EC: 1.1.5.2 and EC: 1.1.99.18,

[0266] - comprising a polypeptide sequence comprising an IPR domain selected from the list consisting of or consisting essentially of: IPR000172, IPR001479, IPR002372, IPR006094, IPR007867, IPR011041, IPR011042, IPR011047, IPR012132, IPR012938, IPR012951, IPR013154, IPR013428, IPR015402, IPR015920, IPR016166, IPR016169, IPR017511, IPR017512, IPR018391, IPR027424, IPR029056, IPR031640, IPR036188, and IPR036318, as defined in 90.0.

[0267] - comprising a polypeptide sequence comprising a Panther domain selected from the list consisting of or consisting essentially of PTHR11552, PTHR13460, PTHR32303:SF4, PTHR42973 and PTHR47190 as defined by InterPro 90.0 released on August 4, 2022,

[0268] - comprising a polypeptide sequence comprising a PFAM domain selected from the list consisting of or consisting essentially of PF00732, PF01011, PF05199, PF07995, PF08031, PF08240, PF13360, PF13570, PF01565, PF16010 and PF16912 as defined by InterPro 90.0 released on August 4, 2022,

[0269] - comprising a polypeptide sequence comprising the conserved protein domain cd10280 as defined by InterPro 90.0 released on August 4, 2022,

[0270] - is part of a NOG family selected from the list consisting of or consisting essentially of: COG0277, COG2133, COG2303 and COG4993 as defined by eggNOG5.0 released in 2019, and / or

[0271] - using a cofactor selected from the list consisting of or consisting essentially of: pyrroloquinoline quinone (PQQ), FAD, a divalent metal cation, Ca 2+ 、Cu 2+ Mg 2+ 、Zn 2+ , heme, heme C and pyridoxal 5'-phosphate.

[0272] 6. The cell according to any of the preceding preferred embodiments, wherein at least one gene involved in the synthesis and / or import of the following cofactors is functionally impaired or knocked out:

[0273] - cofactors involved in the synthesis of LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, and / or

[0274] - a cofactor selected from the list consisting of or consisting essentially of and involved in the synthesis of LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA: pyrroloquinoline quinone (PQQ), FAD, a divalent metal cation, Ca 2+ 、Cu 2+ Mg 2+ 、Zn 2+ , heme, heme C and pyridoxal 5'-phosphate.

[0275] 7. The cell according to any one of the preceding preferred embodiments, wherein the at least one gene is functionally impaired by insertion, deletion and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from the list consisting of or essentially consisting of: a promoter sequence, a ribosome binding site, an untranslated region, a coding sequence and a transcription terminator sequence of the at least one gene.

[0276] 8. A cell according to any one of the aforementioned preferred embodiments, wherein the cell comprises a quinone protein glucose dehydrogenase gene, which is functionally deteriorated by insertion, deletion and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from a list consisting of or essentially consisting of the following: the promoter sequence, ribosome binding site, untranslated region, coding sequence and transcription terminator sequence of the quinone protein glucose dehydrogenase gene.

[0277] 9. The cell according to preferred embodiment 8, wherein the cell further comprises an aldose sugar dehydrogenase gene, which is rendered functionally impaired by insertion, deletion and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from the list consisting of or essentially consisting of the following: a promoter sequence, a ribosome binding site, an untranslated region, a coding sequence and a transcription terminator sequence of the aldose sugar dehydrogenase gene.

[0278] 10. The cell according to any one of preferred embodiments 1 to 7, wherein the cell comprises an aldose sugar dehydrogenase gene that is rendered functionally impaired by insertion, deletion and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from the list consisting of or essentially consisting of the following: a promoter sequence, a ribosome binding site, an untranslated region, a coding sequence and a transcription terminator sequence of the aldose sugar dehydrogenase gene.

[0279] 11. A cell according to preferred embodiment 10, wherein the cell further comprises a quinone protein glucose dehydrogenase gene, which is rendered functionally impaired by the insertion, deletion and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from a list consisting of or essentially consisting of the following: a promoter sequence, a ribosome binding site, an untranslated region, a coding sequence and a transcription terminator sequence of the quinone protein glucose dehydrogenase gene.

[0280] 12. A cell according to any one of the preceding preferred embodiments, wherein the sugar is selected from the list consisting of or consisting essentially of: monosaccharides; phosphorylated monosaccharides; activated monosaccharides; disaccharides; oligosaccharides; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; sialylated oligosaccharides; milk oligosaccharides; mammalian milk oligosaccharides (MMOs); human milk oligosaccharides (HMOs); sialylated milk oligosaccharides; neutral (uncharged) milk oligosaccharides; fucosylated milk oligosaccharides; non-fucosylated neutral (uncharged) milk oligosaccharides; sialylated mammalian milk oligosaccharides; neutral (uncharged) mammalian milk oligosaccharides; fucosylated mammalian milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides; sialylated human milk oligosaccharides ; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; O-antigens; enterobacterial common antigens (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigenic oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides; animal oligosaccharides selected from the list consisting of N-glycans and O-glycans; plant oligosaccharides; plant oligosaccharides selected from the list consisting of N-glycans and O-glycans; fucosylated oligosaccharides; fucosylated oligosaccharides selected from the list consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose ( 6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated oligosaccharides selected from the list consisting of 3' sialyl lactose (3'SL), 6' sialyl lactose (6'SL), sialyl lacto-N-tetraose a (LSTa), sialyl lacto-N-tetraose b (LSTb), sialyl lacto-N-tetraose c ( LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, and monofucosyldisialyllacto-N-tetraose; neutral (uncharged) sugars containing N-acetylglucosamine;A neutral (uncharged) sugar containing N-acetylglucosamine selected from the list consisting of: lacto-N-biose (LNB), N-acetyllactosamine (LacNAc), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose; sugars containing N-acetylglucosamine; sugars containing N-acetyllactosamine; sugars containing lacto-N-biose; non-fucosylated neutral (uncharged) sugars; chitosan; oligosaccharides containing chitosan; heparosan; chondroitin sulfate; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; dermatan sulfate; hyaluronan; hyaluronic acid; and keratan sulfate.

[0281] 13. The cell according to any one of the preceding preferred embodiments, wherein the cell:

[0282] - is capable of being produced from one or more precursors and / or produces said sugar, LBA, modified form of LBA, glycosylated form of LBA, fucosylated LBA and / or sialylated LBA from one or more precursors,

[0283] - is capable of producing and / or produces said sugar, LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA from lactose,

[0284] - is capable of producing and / or produces at least one precursor for the production of said sugar, LBA, modified form of LBA, glycosylated form of LBA, fucosylated LBA and / or sialylated LBA,

[0285] - is capable of producing and / or produces all precursors for the production of said sugars, LBAs, modified forms of LBAs, glycosylated forms of LBAs, fucosylated LBAs and / or sialylated LBAs,

[0286] - genetically engineered to produce at least one precursor for producing said sugar, LBA, modified form of LBA, glycosylated form of LBA, fucosylated LBA and / or sialylated LBA, and / or

[0287] - Genetically engineered for the production of all precursors for the production of said sugars, LBAs, modified forms of LBAs, glycosylated forms of LBAs, fucosylated LBAs and / or sialylated LBAs.

[0288] 14. The cell of preferred embodiment 13, wherein at least one of the one or more precursors is internalized into the cell via one or more membrane proteins.

[0289] 15. The cell according to any one of the preceding preferred embodiments, wherein the cell:

[0290] - selected from the group consisting of prokaryotic cells and eukaryotic cells,

[0291] - is selected from the group consisting of yeast cells, bacterial cells, archaeal cells, algae cells, plant cells and fungal cells,

[0292] - is Escherichia coli (E. coli) or yeast with a lactose permease-positive phenotype, and / or

[0293] - an Escherichia coli or yeast having a lactose permease-positive phenotype, wherein the lactose permease is encoded by the gene LacY or LAC12, respectively.

[0294] 16. A method for producing sugars, the method comprising:

[0295] i. culturing and / or incubating the cell according to any one of the preceding preferred embodiments in a culture and / or incubation medium under conditions that allow the production of said sugar and any one or more of lactobionic acid (LBA), modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, and

[0296] ii. (1) separating the sugar from the culture and / or incubation, and / or

[0297] (2) separating the sugar from the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA.

[0298] 17. The method according to preferred embodiment 16, wherein the culture or incubation medium comprises one or more precursors for producing the sugar, LBA, modified form of LBA, glycosylated form of LBA, fucosylated LBA and / or sialylated LBA.

[0299] 18. The method according to any one of preferred embodiments 16 or 17, wherein the cells produce 30 g / L or more of the sugar in the whole broth and / or supernatant, and / or wherein the sugar in the whole broth and / or supernatant has a purity of at least 80%, measured based on the total amount of sugar and its precursors produced by the cells in the whole broth and / or supernatant, respectively.

[0300] 19. The method according to any one of preferred embodiments 16 to 18, wherein the functionally impaired or knocked-out synthesis of LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA confers unaffected and / or enhanced i) sugar formation, ii) productivity, iii) biomass production, iv) cell growth, and / or v) yield of produced sugars relative to corresponding unmodified cells.

[0301] 20. The method according to any one of preferred embodiments 16 to 19, wherein the cell produces a mixture comprising the sugar and any one or more of the LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA, wherein the mixture comprises ≤ 10 wt % of the LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA, ≤ 9 wt % of the LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA % of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤ 8 wt% of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤ 7 wt% of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤ 6 wt% of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤ 5% by weight of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤4% by weight of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤3% by weight of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤2% by weight of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA % of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤1 wt% of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤0.5 wt% of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, and / or ≤0.1 wt% of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA.

[0302] 21. The method according to any one of preferred embodiments 16 to 19, wherein the cell does not produce LBA.

[0303] 22. The method according to any one of preferred embodiments 16 to 21, wherein the sugar is recovered from the culture or incubation medium and / or the cells, and / or wherein the sugar is purified.

[0304] 23. A method according to any one of preferred embodiments 16 to 22, wherein the sugar is (i) purified from the LBA and / or (ii) purified from a modified form of the LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA.

[0305] 24. Use of a cell according to any one of preferred embodiments 1 to 15 for producing a sugar, wherein the sugar is selected from the list consisting of or consisting essentially of: monosaccharides; phosphorylated monosaccharides; activated monosaccharides; disaccharides; oligosaccharides; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; sialylated oligosaccharides; milk oligosaccharides; mammalian milk oligosaccharides (MMO); human milk oligosaccharides (HMO); sialylated milk oligosaccharides; neutral (uncharged) milk oligosaccharides; fucosylated milk oligosaccharides; non-fucosylated neutral (uncharged) milk oligosaccharides; sialylated mammalian milk oligosaccharides; neutral (uncharged) mammalian milk oligosaccharides; fucosylated mammalian milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides; Sialyl human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; O-antigens; enterobacterial common antigens (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigenic oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides; animal oligosaccharides selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides; plant oligosaccharides selected from the group consisting of N-glycans and O-glycans; fucosylated oligosaccharides; fucosylated oligosaccharides selected from the group consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucose , lactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated oligosaccharides selected from the group consisting of 3' sialyl lactose (3'SL), 6' sialyl lactose (6'SL), sialyl lacto-N-tetraose a (LSTa), sialyl lacto-N-tetraose b (LSTb), sialyl lacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, and monofucosyldisialyllacto-N-tetraose; neutral (uncharged) sugars containing N-acetylglucosamine;Neutral (uncharged) sugars containing N-acetylglucosamine selected from the group consisting of lacto-N-biose (LNB), N-acetyllactosamine (LacNAc), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose; sugars containing N-acetylglucosamine; sugars containing N-acetyllactosamine; sugars containing lacto-N-biose; non-fucosylated neutral (uncharged) sugars; chitosan; oligosaccharides containing chitosan; heparosan; chondroitin sulfate; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; dermatan sulfate; hyaluronan; hyaluronic acid; and keratan sulfate.

[0306] 25. Use of the method according to any one of preferred embodiments 16 to 23 for producing a sugar, wherein the sugar is selected from the list consisting of or consisting essentially of: monosaccharides; phosphorylated monosaccharides; activated monosaccharides; disaccharides; oligosaccharides; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; sialylated oligosaccharides; milk oligosaccharides; mammalian milk oligosaccharides (MMO); human milk oligosaccharides (HMO); sialylated milk oligosaccharides; neutral (uncharged) milk oligosaccharides; fucosylated milk oligosaccharides; non-fucosylated neutral (uncharged) milk oligosaccharides; sialylated mammalian milk oligosaccharides; neutral (uncharged) mammalian milk oligosaccharides; fucosylated mammalian milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides Animal milk oligosaccharides; sialylated human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; O-antigens; enterobacterial common antigens (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigenic oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides; animal oligosaccharides selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides; plant oligosaccharides selected from the group consisting of N-glycans and O-glycans; fucosylated oligosaccharides; fucosylated oligosaccharides selected from the group consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated oligosaccharides selected from the group consisting of 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllactose-N-tetraose a (LSTa), sialyllactose-N-tetraose b ( LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, and monofucosyldisialyllacto-N-tetraose;A neutral (uncharged) sugar containing N-acetylglucosamine; a neutral (uncharged) sugar containing N-acetylglucosamine selected from the group consisting of lacto-N-biose (LNB), N-acetyllactosamine (LacNAc), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose , lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose; sugars containing N-acetylglucosamine; sugars containing N-acetyllactosamine; sugars containing lacto-N-biose; non-fucosylated neutral (uncharged) sugars; chitosan; oligosaccharides containing chitosan; heparosan; chondroitin sulfate; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; dermatan sulfate; hyaluronan; hyaluronic acid; and keratan sulfate.

[0307] 26. A mixture comprising, consisting of or essentially consisting of: (i) a sugar, and (ii) LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA, wherein the sugar, LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA is obtainable or obtained by a method according to any one of preferred embodiments 16 to 23.

[0308] 27. A mixture comprising, consisting of or consisting essentially of (i) sugars, and (ii) ≤10 wt% LBA, ≤9 wt% LBA, ≤8 wt% LBA, ≤7 wt% LBA, ≤6 wt% LBA, ≤5 wt% LBA, ≤4 wt% LBA, ≤3 wt% LBA, ≤2 wt% LBA, ≤1 wt% LBA, ≤0.5 wt% LBA, and / or ≤0.1 wt% LBA, wherein the sugars and LBA are obtainable or obtained by a process according to any one of preferred embodiments 16 to 20, 22 or 23.

[0309] 28. A mixture comprising, consisting of, or consisting essentially of a sugar and (i) ≤10 wt% LBA, ≤9 wt% LBA, ≤8 wt% LBA, ≤7 wt% LBA, ≤6 wt% LBA, ≤5 wt% LBA, ≤4 wt% LBA, ≤3 wt% LBA, ≤2 wt% LBA, ≤1 wt% LBA, ≤0.5 wt% LBA, and / or ≤0.1 wt% LBA, and / or (ii) ≤10 wt% of a modified form of LBA, Glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤9 wt% of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤8 wt% of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤7 wt% of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤6 wt% of modified forms of LBA, glycosylated forms of LBA , fucosylated LBA and / or sialylated LBA, ≤5 wt% of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤4 wt% of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤2 wt% of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤1 wt% of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA A and / or sialylated LBA, ≤0.5 wt. % of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, and / or ≤0.1 wt. % of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, wherein the sugar, LBA, modified form of LBA, glycosylated form of LBA, fucosylated LBA and / or sialylated LBA is obtainable or obtained by the method according to any one of preferred embodiments 16 to 23.

[0310] The present invention will be described in more detail in the examples.The following figures and examples will serve as further illustration and clarification of the present invention and are not intended to be limiting.

[0311] Description of the drawings

[0312] Figure 1: Growth rates expressed in relative percentages (%) obtained in growth experiments with modified E. coli strains modified as described in Example 1 for the production of 2'FL, LNT, LNnT, 3'SL, or 6'SL, and with native gcd and ylil expression (reference strain, gcd+ylil+) or with gcd genomic knockout (gcd KO, ylil+) or with gcd genomic knockout and ylil genomic knockout (gcd KO, ylilKO). The growth experiments were performed according to the culture conditions provided in Example 1, wherein the substratum or culture medium was supplemented with 30 g / L sucrose, 20 g / L lactose, and 3.30E-4 g / L pyrroloquinoline quinone (PQQ). The horizontal dotted line indicates the set point to which all adaptations were normalized. Example

[0313] Example 1. Materials and methods

[0314] A. Escherichia coli

[0315] Culture medium and cultivation

[0316] Luria Broth (LB) medium consisted of 1% tryptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco), and 0.5% sodium chloride (VWR, Leuven, Belgium). The minimal medium used for culture experiments in 96-well plates or shake flasks contained 2.00 g / L NH4Cl, 5.00 g / L (NH4)2SO4, 2.993 g / L KH2PO4, 7.315 g / L K2HPO4, 8.372 g / L MOPS, 0.5 g / L NaCl, 0.5 g / L MgSO4.7H2O, 30 g / L sucrose or 30 g / L glycerol, 1 ml / L vitamin solution, 100 μl / L molybdate solution, and 1 mL / L selenium solution. As precursors and / or acceptors for sugar synthesis, compounds such as galactose, glucose, fructose, fucose, lactose, sialic acid, N-acetyllactosamine (LacNAc), lacto-N-biose (LNB) can be added to the culture medium. For LBA production, cofactors such as pyrroloquinoline quinone (PQQ), FAD, divalent metal cations, Ca 2+ 、Cu 2+ Mg 2+ 、Zn 2+, heme, heme C, and pyridoxal 5'-phosphate. Minimal medium was set to a pH of 7 with 1M KOH. The vitamin solution consisted of 3.6 g / L FeCl2.4H2O, 5.0 g / L CaCl2.2H2O, 1.3 g / L MnCl2.2H2O, 0.38 g / L CuCl2.2H2O, 0.5 g / L CoCl2.6H2O, 0.94 g / L ZnCl2, 0.0311 g / L H3BO4, 0.4 g / L Na2EDTA.2H2O, and 1.01 g / L thiamine hydrochloride. The molybdate solution contained 0.967 g / L NaMoO4.2H2O. The selenium solution contained 42 g / L SeO2. The minimal medium that is used for fermentation comprises 6.75g / L NH Cl, 1.25g / L (NH ) SO , 2.93g / L KH PO And 7.31g / L KH PO , 0.5g / L NaCl, 0.5g / L MgSO .7H O, 30g / L sucrose or 30g / L glycerol, 1mL / L vitamin solution, 100 μ L / L molybdate solution and 1mL / L selenium solution, have the identical composition described above.As specifically indicated in each example, 20g / L lactose is added to the substratum in addition.Compound medium is sterilized by autoclaving (121 ℃, 21 minutes), and minimal medium is sterilized by filtration (0.22 μ m Sartorius). The culture medium is prepared by adding antibiotics. When needed, culture medium is made to have selectivity: for example, chloramphenicol (20mg / L), carbenicillin (100mg / L), spectinomycin (40mg / L) and / or kanamycin (50mg / L). From frozen vials, in 150 μ L LB, start the pre-culture of 96-well microtiter plate experiment, and it is incubated overnight with 800rpm on orbital shaking table at 37 ℃. This culture is used as the inoculum for 96-well square microtiter plates (having 400 μ L minimal medium) by diluting 400x. Then, these final 96-well culture plates are incubated 72 hours, or shorter, or longer, at 37 ℃ on orbital shaking table with 800rpm. In order to measure sugar concentration when the culture experiment is finished, by boiling culture fluid at 60 ℃ for 15 minutes from each hole, taking full culture fluid sample (=mean value of intracellular and extracellular sugar concentration). Precultures for bioreactors were started from an entire 1 mL frozen vial of a strain, inoculated into 250 mL or 500 mL of minimal medium in 1 L or 2.5 L shake flasks, and incubated at 37°C on an orbital shaker at 200 rpm for 24 hours.A 5-L bioreactor was then inoculated (250 mL inoculum in 2 L batch medium); the process was controlled by MFCS control software (Sartorius Stedim Biotech, Melsungen, Germany). Culture conditions were set at 37°C and maximum agitation; the pressure gas flow rate depended on the strain and bioreactor. The pH was controlled at 6.8 using 0.5 M H2SO4 and 20% NH4OH. The exhaust gas was cooled. If foaming occurred during fermentation, a 10% silicone antifoam solution was added.

[0317] Strains and mutations

[0318] Escherichia coli K12 MG1655[λ - ,F - ,rph-1] was obtained from Coli Genetic Stock Center (US), CGSC strain #: 7740 in March 2007. Gene disruption, gene introduction and gene replacement were performed by using the technique published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645). All constitutive promoter, UTR and terminator sequences were derived from libraries described by Cambray et al. (Nucleic Acids Res. 2013, 41(9), 5139-5148), Dunn et al. (Nucleic Acids Res. 1980, 8, 2119-2132), Edens et al. (Nucleic Acids Res. 1975, 2, 1811-1820), Kim and Lee (FEBS Letters 1997, 407, 353-356) and Mutalik et al. (Nat. Methods 2013, No. 10, 354-360). Genes were synthesized and ordered at Twist Bioscience (twistbioscience.com) or IDT (eu.idtdna.com) and codon usage was adjusted using the supplier's tools. All strains were stored at -80°C in cryovials (overnight LB cultures mixed 1:1 with 70% glycerol).

[0319] In one example for GDP-fucose production, the mutant strain is derived from E. coli K12 MG1655, which comprises a knockout of the E. coli wcaJ gene and a genomic knockin of a constitutive transcription unit comprising a sucrose transporter, such as CscB from E. coli W (UniProt ID E0IXR1); a fructokinase, such as Frk from Zymomonas mobilis (UniProt ID Q03417); and a sucrose phosphorylase, such as BaSP from Bifidobacterium adolescentis (UniProt ID DAOZZH6). In the mutant E. coli strain, GDP-fucose production can be further optimized by genomic knockout of any one or more of the E. coli genes including glgC, agp, pfkA, pfkB, pgi, arcA, iclR, pgi and lon, as described in WO2016075243 and WO2012007481. GDP-fucose production can be additionally optimized, including genomic knock-in of constitutive transcription units for: mannose-6-phosphate isomerase, e.g., manA from E. coli (UniProt ID P00946); phosphomannose mutase, e.g., manB from E. coli (UniProt ID P24175); mannose-1-phosphate guanylyltransferase, e.g., manC from E. coli (UniProt ID P24174); GDP-mannose 4,6-dehydratase, e.g., gmd from E. coli (UniProt ID P0AC88); and GDP-L-fucose synthase, e.g., fcl from E. coli (UniProt ID P32055). GDP-fucose production can also be obtained by genomic knockout of the E. coli fucK and fucI genes and genomic knockin of a constitutive transcription unit comprising a fucose permease, such as fucP from E. coli (UniProt ID P11551); and a bifunctional enzyme with fucokinase / fucose-1-phosphate guanylyltransferase activity, such as fkp from Bacteroides fragilis (UniProt ID SUV40286.1). All mutant strains can be additionally modified with genomic knockout of the E. coli LacZ, LacY and LacA genes and with genomic knockin of a constitutive transcription unit for a lactose permease, such as E. coli LacY (UniProt ID P02920).To produce fucosylated sugars, the mutant GDP-fucose producing strain is additionally modified with an expression plasmid containing constitutive transcription units for: a fucosyltransferase, such as α-1,2-fucosyltransferase HpFutC from H. pylori (UniProt ID Q9X435), to produce 2'-fucosyllactose (2'FL); or α-1,3-fucosyltransferase HpFucT from H. pylori (UniProt ID DO30511), to produce 3-fucosyllactose (3-FL); or α-1,2-fucosyltransferase HpFutC from H. pylori (UniProt ID Q9X435) and α-1,3-fucosyltransferase HpFucT from H. pylori (UniProt ID O30511) to produce difucosyllactose (DiFL).

[0320] In one example for the production of lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc), the mutant strain is derived from Escherichia coli K12 MG1655 and is modified with knockouts of the E. coli lacZ, lacY, lacA, and nagB genes and with genomic knock-ins of constitutive transcription units for: a lactose permease, such as E. coli LacY (UniProt ID P02920); and a galactoside β-1,3-N-acetylglucosaminyltransferase, such as lgtA from Neisseria meningitidis (UniProt ID Q9JXQ6). In one example for the production of LN3-derived oligosaccharides such as lacto-N-tetraose (LNT, Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc), the mutant LN3-producing strain is further modified with a constitutive transcription unit for N-acetylglucosamine β-1,3-galactosyltransferase (e.g., wbgO from Escherichia coli O55:H7 (Uniprot ID D3QY14)) delivered to the strain via genomic knock-in or from an expression plasmid. In one example for the production of LN3-derived oligosaccharides such as lacto-N-neotetraose (LNnT, Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc), the mutant LN3-producing strain is further modified with a constitutive transcription unit for N-acetylglucosamine β-1,4-galactosyltransferase (e.g., LgtB from Neisseria meningitidis (UniprotID Q51116, sequence version 02, December 1, 2000)) delivered to the strain via genomic knock-in or from an expression plasmid. In the mutant E. coli strain, LN3, LNT, and / or LNnT production can be further optimized using genomic knockouts of any one or more E. coli genes including galT, ushA, ldhA, and agp. Optionally, the mutant LN3, LNT and / or LNnT producing strains can also be modified with a genomic knock-in of a constitutive transcription unit for L-glutamine-D-fructose-6-phosphate aminotransferase, such as E. coli glmS (UniProt ID P17169, sequence version 04, January 23, 2007), for enhanced UDP-GlcNAc production.Optionally, the mutant E. coli strain can also be adapted with genomic knock-in of the constitutive transcription units for UDP-glucose-4-epimerase, such as galE from E. coli (UniProt ID P09147), phosphoglucosamine mutase, such as glmM from E. coli (UniProt ID P31120, sequence version 03, January 23, 2007), and N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase, such as glmU from E. coli (UniProt ID P0ACC7). Optionally, the mutant LN3, LNT and / or LNnT producing E. coli strains can also be adapted to grow on sucrose via genomic knock-in of constitutive transcription units comprising a sucrose transporter, such as CscB from E. coli W (UniProtID E0IXR1); a fructokinase, such as Frk from Zymomonas mobilis (UniProtID Q03417); and a sucrose phosphorylase, such as BaSP from Bifidobacterium adolescentis (UniProtID A0ZZH6).

[0321] In one example for sialic acid production, the mutant strain is derived from Escherichia coli K12 MG1655 containing a genomic knock-in of constitutive transcription units comprising one or more copies of glucosamine 6-phosphate N-acetyltransferase, such as GNA1 from Saccharomyces cerevisiae (UniProt ID P43577); N-acetylglucosamine 2-epimerase, such as AGE from Bacteroides ovatus (UniProt ID A7LVG6); and N-acetylneuraminic acid synthase, such as NeuB from Neisseria meningitidis (UniProt ID EONCD4).

[0322] Alternatively and / or additionally, sialic acid production can be obtained by genomic knock-in of constitutive transcription units comprising a UDP-N-acetylglucosamine 2-epimerase, such as NeuC from Campylobacter jejuni (C. jejuni) (UniProt ID Q93MP8); and an N-acetylneuraminic acid synthase, such as NeuB from Neisseria meningitidis (UniProt ID EONCD4). Alternatively and / or additionally, sialic acid production can be obtained by genomic knock-in of constitutive transcription units comprising a phosphoglucosamine mutase, e.g., glmM from Escherichia coli (UniProt ID P31120, sequence version 03, January 23, 2007); an N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase, e.g., glmU from Escherichia coli (UniProt ID POACC7); a UDP-N-acetylglucosamine 2-epimerase, e.g., NeuC from Campylobacter jejuni (UniProt ID Q93MP8); and an N-acetylneuraminic acid synthase, e.g., NeuB from Neisseria meningitidis (UniProt ID EONCD4). Alternatively and / or additionally, sialic acid production can be obtained by genomic knock-in of a constitutive transcription unit comprising a bifunctional UDP-GlcNAc 2-epimerase / N-acetylmannosamine kinase, e.g., from mouse (strain C57BL / 6J) (UniProt ID Q91WG8); an N-acylneuraminic acid-9-phosphate synthase, e.g., from Pseudomonas sp. UW4 (UniProt ID K9NPH9); and an N-acylneuraminic acid-9-phosphatase, e.g., from Bacteroides thetaiotaomicron (UniProt ID Q8A712).

[0323] In the mutant E. coli strain, sialic acid production can be further optimized with genomic knockouts of E. coli genes including any one or more of nagA, nagB, nagC, nagD, nagE, nanA, nanE, nanK, manX, manY and manZ (as described in WO18122225) and / or genomic knockouts of E. coli genes including any one or more of nanT, poxB, ldhA, adhE, aldB, pflA, pflC, ybiY, ackA and / or pta, and with genomic knockin of a constitutive transcription unit comprising one or more copies of L-glutamine-D-fructose-6-phosphate aminotransferase, such as E. coli glmS (UniProt ID P17169, sequence version 04, January 23, 2007); preferably, a phosphatase, such as any one of the E. coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU, or PsMupP from Pseudomonas putida, ScDOG1 from Saccharomyces cerevisiae, or BsAraL from Bacillus subtilis, as described in WO18122225; and an acetyl-CoA synthetase, such as acs from E. coli (UniProtID P27550).

[0324] For sialylated oligosaccharide production, the sialic acid producing strain is further modified to express an N-acylneuraminic acid cytidylyltransferase, such as the NeuA enzyme from Pasteurella multocida (UniProt ID A0A849CI62), and a sialyltransferase, such as the α-2,3-sialyltransferase PmultST3 from Pasteurella multocida (UniProt ID Q9CLP3) to produce 3'SL, or the α-2,6-sialyltransferase PdST6 from Photobacterium damselae (UniProt ID O66375) to produce 6'SL. The constitutive transcription units of the N-acylneuraminic acid cytidylyltransferase and the sialyltransferase can be delivered to the mutant strain via genomic knock-in or via expression plasmids. If the mutant strain producing sialic acid and CMP-sialic acid is intended to produce sialylated lactose structures, the strain is additionally modified with genomic knockouts of the E. coli LacZ, LacY, and LacA genes and with genomic knock-in of a constitutive transcription unit for a lactose permease, such as E. coli LacY (UniProt ID P02920). All mutant strains producing sialic acid, CMP-sialic acid, and / or sialylated sugars can optionally be adapted to grow on sucrose via genomic knock-in of a constitutive transcription unit comprising a sucrose transporter, such as CscB from E. coli W (UniProt ID E0IXR1); a fructokinase, such as Frk from Zymomonas mobilis (UniProt ID Q03417); and a sucrose phosphorylase, such as BaSP from Bifidobacterium adolescentis (UniProt ID A0ZZH6).

[0325] In another example, the mutant E. coli strain adapted for LNT production described herein can be further modified with one or more copies of a glucosamine 6-phosphate N-acetyltransferase, such as GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), an N-acetylglucosamine 2-epimerase, such as AGE from Bacteroides ovatus (UniProt ID A7LVG6), and an N-acetylneuraminic acid synthase, such as NeuB from Neisseria meningitidis (UniProt ID E0NCD4); and an expression plasmid comprising constitutive expression cassettes for: N-acylneuraminic acid cytidylyltransferase (NeuA) from Pasteurella multocida (UniProt ID A0A849CI62), and 1) an α-2,3-sialyltransferase PmultST3 from Pasteurella multocida (UniProt ID Q9CLP3), or 2) α-2,6-sialyltransferase from Photorhabditis mermanni (PdST6) (UniProtID O66375), to produce 1) LSTa(Neu5Ac-α2,3-Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc) or 2) LSTb(Gal-β1,3-(Neu5Ac-α2,6)-GlcNAc-β1,3-Gal-β1,4-Glc), respectively. In another example, the mutant E. coli strain adapted for LNnT production described herein can be further modified with one or more copies of glucosamine 6-phosphate N-acetyltransferase, such as GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), N-acetylglucosamine 2-epimerase, such as AGE from Bacteroides ovatus (UniProt ID A7LVG6), and N-acetylneuraminic acid synthase, such as NeuB from Neisseria meningitidis (UniProt ID EONCD4); and an expression plasmid comprising a constitutive expression cassette for N-acylneuraminic acid cytidylyltransferase (NeuA) from Pasteurella multocida (UniProt ID The enzymes were selected from the group consisting of 1) an α-2,3-sialyltransferase PmultST3 from Pasteurella multocida (UniProtID Q9CLP3) or 2) an α-2,6-sialyltransferase PdST6 from Photorhabdus mermanii (UniProtID O66375), to produce 1) LSTd (Neu5Ac-α2,3-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc) or 2) LSTc (Neu5Ac-α2,6-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc), respectively.

[0326] B. Saccharomyces cerevisiae

[0327] Culture medium and cultivation

[0328] Strains were grown on synthetic defined yeast medium (SD CSM) or CSM drop-out (SD CSM-Ura, SD CSM-Trp, SD CSM-His) with a complete supplement mixture containing 6.7 g / L Yeast Nitrogen Base (YNB w / o AA, Difco), 20 g / L agar (Difco) (solid culture), 22 g / L glucose monohydrate or 20 g / L lactose, and 0.79 g / L CSM or 0.77 g / L CSM-Ura, 0.77 g / L CSM-Trp, or 0.77 g / L CSM-His (MP Biomedicals). Compounds such as galactose, glucose, fructose, fucose, lactose, sialic acid, N-acetyllactosamine (LacNAc), and lacto-N-biose (LNB) can be added to the culture medium as precursors and / or acceptors for sugar synthesis. For LBA production, cofactors such as pyrroloquinoline quinone (PQQ), FAD, divalent metal cations, Ca 2+ 、Cu 2+ Mg 2+ 、Zn 2+ , heme, heme C and pyridoxal 5'-phosphate.

[0329] Typically, yeast strains are initially grown on SD CSM plates to obtain single colonies. These plates are grown at 30°C for 2-3 days. Starting from a single colony, a preculture is grown overnight in 5 mL at 30°C with shaking at 200 rpm. Subsequently, 2% of this preculture is used to inoculate 125 mL shake flasks in 25 mL of culture medium. These shake flasks are incubated at 30°C with orbital shaking at 200 rpm.

[0330] Strains, plasmids, and mutations

[0331] Saccharomyces cerevisiae BY4742, created by Brachmann et al. (Yeast (1998) 14: 115-32), available at the Euroscarf culture collection, was used. All mutant strains were created by homologous recombination or plasmid transformation using the method of Gietz (Yeast 11: 355-360, 1995). Genes were expressed using synthetic constitutive promoters, such as those described by, for example, Blazeck (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012), Redden and Alper (Nat. Commun. 2015, 6, 7810), Liu et al. (Microb. Cell Fact. 2020, 19, 38), Xu et al. (Microb. Cell Fact. 2021, 20, 148), and Lee et al. (ACS Synth. Biol. 2015, 4 (9), 975-986).

[0332] In an example for producing GDP-fucose, yeast expression plasmids such as p2a_2μ_Fuc (Chan 2013, Plasmid 70, 2-17) can be used to express foreign genes in Saccharomyces cerevisiae. The plasmid contains an ampicillin resistance gene and a bacterial replication origin (to allow selection and maintenance in E. coli) as well as 2μ yeast ori and Ura3 selection markers (for selection and maintenance in yeast). The plasmid is further modified with constitutive transcription units for the following: lactose permease, such as LAC12 from Kluyveromyces lactis (UniProt ID P07921); GDP-mannose 4,6-dehydratase, such as gmd from E. coli (UniProt ID P0AC88); and GDP-L-fucose synthase, such as fcl from E. coli (UniProt ID P32055). Yeast expression plasmid p2a_2μ_Fuc2 can be used as an alternative expression plasmid to plasmid p2a_2μ_Fuc, which contains constitutive transcription units for the following: lactose permease, such as LAC12 from Kluyveromyces lactis (UniProt ID P07921); fucose permease, such as fucP from Escherichia coli (UniProt ID P11551); and a bifunctional enzyme with fucokinase / fucose-1-phosphate guanylyltransferase activity, such as fkp from Bacteroides fragilis (UniProt ID SUV40286.1). To further produce fucosylated sugars, p2a_2μ_Fuc and its variant p2a_2μ_Fuc2 additionally contain constitutive transcription units for fucosyltransferase.

[0333] In one example for producing UDP-galactose, a yeast expression plasmid can be derived from the pRS420-plasmid series (Christianson et al., 1992, Gene 110: 119-122), which contains a HIS3 selection marker and a constitutive transcription unit for a UDP-glucose-4-epimerase, such as galE from Escherichia coli (UniProt ID P09147). This plasmid can be further modified with constitutive transcription units for a lactose permease, such as LAC12 from Kluyveromyces lactis (UniProt ID P07921), and a galactoside β-1,3-N-acetylglucosaminyltransferase activity, such as lgtA from Neisseria meningitidis (UniProt ID Q9JXQ6). In one example for further producing LN3-derived oligosaccharides such as LNT, the mutant LN3-producing strain is further modified with a constitutive transcription unit for N-acetylglucosamine β-1,3-galactosyltransferase (e.g., WbgO from Escherichia coli O55:H7 (Uniprot ID D3QY14)). In one example for producing LN3-derived oligosaccharides such as lacto-N-neotetraose (LNnT, Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc), the mutant LN3-producing strain is further modified with a constitutive transcription unit for N-acetylglucosamine β-1,4-galactosyltransferase (e.g., LgtB from Neisseria meningitidis (Uniprot ID Q51116, sequence version 02, December 1, 2000)).

[0334] In one example for the production of sialic acid and CMP-sialic acid, the yeast expression plasmid is derived from the pRS420-plasmid series (Christianson et al., 1992, Gene 110: 119-122), which contains the TRP1 selection marker and constitutive transcription units for: L-glutamine-D-fructose-6-phosphate aminotransferase, such as E. coli glmS (UniProt ID P17169, sequence version 04 (January 23, 2007)); phosphatase, such as SurE from E. coli (UniProt ID P0A840); N-acylglucosamine 2-epimerase, such as AGE from Bacteroides ovatus (UniProt ID A7LVG6); N-acetylneuraminic acid synthase, such as NeuB from Neisseria meningitidis (UniProt ID IDEONCD4); and N-acylneuraminic acid cytidylyltransferase, such as NeuA from Pasteurella multocida (UniProt A0A849C162). Optionally, a constitutive transcription unit for glucosamine 6-phosphate N-acetyltransferase, such as GNA1 from Saccharomyces cerevisiae (UniProt ID P43577) is also added. In one example for producing sialylated sugars, the plasmid further comprises constitutive transcription units for the following: lactose permease, such as LAC12 from Kluyveromyces lactis (UniProt ID P07921); and sialyltransferase.

[0335] Preferably, but not necessarily, any one or more of the glycosyltransferases and / or proteins involved in nucleotide activated sugar synthesis are fused at the N-terminus and / or C-terminus to a SUMOstar tag (e.g., obtained from pYSUMOstar, Life Sensors, Malvern, PA) to enhance their solubility.

[0336] Optionally, the mutant yeast strain is modified with a genomic knock-in of a constitutive transcription unit encoding a chaperone protein, such as Hsp31, Hsp32, Hsp33, Sno4, Kar2, Ssb1, Sse1, Sse2, Ssa1, Ssa2, Ssa3, Ssa4, Ssb2, Ecm10, Ssc1, Ssq1, Ssz1, Lhs1, Hsp82, Hsc82, Hsp78, Hsp104, Tcp1, Cct4, Cct8, Cct2, Cct3, Cct5, Cct6, or Cct7 (Gong et al., 2009, Mol. Syst. Biol. 5:275). Plasmids were maintained in the host E. coli DH5α (F -,phi80dlacZdeltaM15,delta(lacZYA-argF)U169,deoR,recA1,endA1,hsdR17(rk - ,mk + ),phoA,supE44,lambda - ,thi-1,gyrA96,relA1).

[0337] C. Bacillus subtilis

[0338] Culture medium and cultivation

[0339] Two media were used to culture Bacillus subtilis: complex media such as rich Luria Broth (LB) and minimal media for shake flask cultures. LB media consisted of 1% tryptone (Difco), 0.5% yeast extract (Difco), and 0.5% sodium chloride (VWR). Luria Broth agar (LBA) plates consisted of LB media supplemented with 12 g / L agar (Difco). Minimal media contained 2.00 g / L (NH4)2SO4, 7.5 g / L KH2PO4, 17.5 g / L K2HPO4, 1.25 g / L sodium citrate, 0.25 g / L MgSO4.7H2O, 0.05 g / L tryptophan, 10 to 30 g / L glucose (or another carbon source, including but not limited to fructose, maltose, sucrose, glycerol, and maltotriose), 10 mL / L trace element mix, and 10 mL / L ferric citrate solution. The pH of the culture medium was set to 7 with 1 M KOH. As precursors and / or acceptors for sugar synthesis, compounds such as galactose, glucose, fructose, fucose, lactose, sialic acid, LacNAc, LNB can be added to the culture medium. For LBA production, cofactors such as PQQ, FAD, divalent metal cations, Ca 2+ 、Cu 2+ Mg 2+ 、Zn 2+The present invention provides the method for the preparation of the present invention.The present invention provides the method for the preparation of the present invention.The present invention provides the method for the preparation of the present invention.The present invention provides the method for the preparation of the present invention.The present invention provides the method for the preparation of the present invention.The present invention provides the method for the preparation of the present invention. When necessary, the medium is made selective by adding antibiotics.

[0340] Subtilis bacterial strain is initially grown on LB agar to obtain single bacterium colony.These flat plates are grown overnight at 37 ℃.Starting from single bacterium colony, pre-culture is grown overnight in 5mL at 37 ℃, wherein shakes with 200rpm.Subsequently, in 25mL substratum, 125mL shake flask experiment is inoculated with 2% this pre-culture.These shake flasks are incubated 72 hours at 37 ℃, or shorter, or longer, wherein have the fixed track of 200rpm and shake.When the culture experiment finishes, take a sample to measure supernatant concentration (extracellular sugar concentration, after making cell spin centrifugation 5 minutes), perhaps by before making cell spin centrifugation, culture fluid is boiled 15 minutes at 90 ℃ or boiled 60 minutes at 60 ℃ (=full culture fluid concentration, i.e. intracellular and extracellular sugar concentration)).

[0341] Strains, plasmids, and mutations

[0342] Using Bacillus subtilis 168, it is available at the Bacillus Genetic Stock Center (Bacillus Genetic Stock Center) (Ohio, USA). As described by Yan et al. (Appl & Environm.Microbial., September 2008, pp. 5556-5562), a plasmid for gene deletion via Cre / lox is constructed. Gene disruption is carried out by homologous recombination with linear DNA and transformation via electroporation, as described by Xue et al. (J.microb.Meth.34 (1999) 183-191). The method for gene knockout is described by Liu et al. (Metab.Engine.24 (2014) 61-69). The integration vector described by Popp et al. (Sci.Rep., 2017, 7, 15158) is used as an expression vector and, if necessary, can be further used for genomic integration. Suitable promoters for expression can be derived from the partial repository (iGem): sequence id: BBa_K143012, BBa_K823000, BBa_K823002 or BBa_K823003. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.

[0343] In one example for the production of lactose-based oligosaccharides, a mutant strain of B. subtilis was created to contain a gene encoding a lactose importer (eg, E. coli lacY having UniProtID P02920).

[0344] In one example for producing LN3, the Bacillus subtilis strain is modified with a genomic knock-in comprising a constitutive transcription unit for a lactose importer (e.g., E. coli lacY with UniProt ID P02920) and a galactoside β-1,3-N-acetylglucosamine transferase (e.g., LgtA from Neisseria meningitidis (UniProt ID Q9JXQ6)). For LNT production, the LN3-producing strain is further modified with a constitutive transcription unit for an N-acetylglucosamine β-1,3-galactosyltransferase (e.g., WbgO from E. coli O55:H7 (UniProt ID D3QY14)). For LNnT production, the LN3-producing strain is further modified with a constitutive transcription unit for N-acetylglucosamine β-1,4-galactosyltransferase (e.g., LgtB from Neisseria meningitidis (UniProtID Q51116, sequence version 02, December 1, 2000). N-acetylglucosamine β-1,3-galactosyltransferase and N-acetylglucosamine β-1,4-galactosyltransferase can be delivered to the strain via genomic knock-in or from an expression plasmid.

[0345] To produce fucosylated sugars, the B. subtilis strain was modified with a constitutive transcription unit for a fucosyltransferase.

[0346] In one example for sialic acid production, a mutant Bacillus subtilis strain was created by overexpressing a fructose-6-P-aminotransferase, such as the native fructose-6-P-aminotransferase glmS (UniProtID POCI73), to enhance the intracellular glucosamine-6-phosphate pool. Furthermore, the enzymatic activities of the genes nagA, nagB, and gamA were disrupted by genetic knockout, and glucosamine-6-P-aminotransferase (e.g., GNA1 from Saccharomyces cerevisiae (UniProtID P43577)), N-acetylglucosamine 2-epimerase (e.g., from Bacteroides ovatus (UniProtID A7LVG6)), and N-acetylneuraminic acid synthase (e.g., from Neisseria meningitidis (UniProtID ONENCD4)) were overexpressed genomically. To allow for the production of sialylated sugars, the sialic acid-producing strain is further modified with a constitutive transcription unit comprising an N-acylneuraminic acid cytidylyltransferase, such as NeuA from Pasteurella multocida (UniProt ID A0A849CI62); and a sialyltransferase.

[0347] For growth on sucrose, the mutant strain can be additionally modified with genomic knock-ins comprising the constitutive transcription units of the following: sucrose transporter (CscB) from Escherichia coli W (UniProt ID E0IXR1), fructokinase (Frk) from Zymomonas mobilis (UniProt ID Q03417), and sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (UniProt ID A0ZZH6).

[0348] D. Corynebacterium glutamicum

[0349] Culture medium and cultivation

[0350] Use two different substratums, i.e., complex medium such as enriching tryptone yeast extract (TY) substratum and minimal medium (MMsf) for shaking flask. Minimal medium uses 1000x reserve trace element mixture. Trace element mixture is by 10g / L CaCl , 10g / L FeSO .7H 2 O, 10g / L MnSO .H 2 O, 1g / L ZnSO .7H 2 O, 0.2g / L CuSO , 0.02g / L NiCl .6H 2 O, 0.2g / L biotin (pH 7) and 0.03g / L protocatechuic acid composition. Minimal medium (MMsf) experiments for shake flasks contain 20 g / L (NH4)2SO4, 5 g / L urea, 1 g / L KH2PO4, 1 g / L K2HPO4, 0.25 g / L MgSO4.7H2O, 42 g / L MOPS, from 10 up to 30 g / L glucose or another carbon source (including but not limited to fructose, maltose, sucrose, glycerol and maltotriose) (when specifically specified in the examples) and 1 mL / L trace element mixture. As precursors and / or acceptors for sugar synthesis, compounds such as galactose, glucose, fructose, fucose, lactose, sialic acid, LacNAc, LNB can be added to the culture medium. For LBA production, cofactors such as pyrroloquinoline quinone (PQQ), FAD, divalent metal cations, Ca 2+ 、Cu 2+ Mg 2+ 、Zn 2+, heme, heme C and pyridoxal 5'-phosphate. TY medium is composed of 1.6% tryptone (Difco, Erembodegem, Belgium), 1% yeast extract (Difco) and 0.5% sodium chloride (VWR.Leuven, Belgium). TY agar (TYA) plates are composed of TY medium supplemented with 12 g / L agar (Difco, Erembodegem, Belgium). Complex medium (such as TY) is sterilized by autoclaving (121° C., 21 minutes) and minimal medium is sterilized by filtration (0.22 μm Sartorius). When necessary, the medium is made selective by adding antibiotics.

[0351] In 6mL TY, start preculture from freezing bottle or from the single bacterium colony of TY flat board, and with it under 37 ℃ on fixed orbit shaking table with 200rpm incubation overnight.Subsequently, in 25mL MMsf substratum, inoculate 125mL shake flask experiment with 2% this preculture.With these shake flasks 37 ℃ of incubations 72 hours, perhaps shorter, perhaps longer, wherein have the fixed orbit of 200rpm and shake.When the culture experiment finishes, take sample to measure supernatant concentration (extracellular sugar concentration, after making cell rotation centrifugal 5 minutes), perhaps by before making cell rotation centrifugal, culture fluid was boiled 15 minutes at 90 ℃ or boiled 60 minutes at 60 ℃ (=full culture fluid concentration, i.e. intracellular and extracellular sugar concentration)).

[0352] Strains and mutations

[0353] Use Corynebacterium glutamicum, it is available (ATCC 13032) at American Type Culture Collection.By using by the Cre / loxP technology described by people such as Suzuki (Appl.Microbiol.Biotechnol., 2005Apr, 67 (2): 225-33) prepare integration plasmid vector, and construct by the temperature-sensitive shuttle vector described by people such as Okibe (Journal of Microbiological Methods 85,2011,155-163) for gene deletion, sudden change and insertion.The promotor suitable for (heterologous) gene expression can be derived from people such as Yim (Biotechnol.Bioeng., 2013Nov, 110 (11): 2959-69).Clone can be carried out by using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction connection.

[0354] In one example for the production of lactose-based oligosaccharides, a mutant strain of C. glutamicum was created to contain a gene encoding a lactose importer (eg, E. coli lacY having UniProt ID P02920).

[0355] In one example for producing LN3, the C. glutamicum strain was modified with a genomic knock-in comprising a constitutive expression unit for a lactose importer (e.g., E. coli lacY with UniProt ID P02920) and a galactoside β-1,3-N-acetylglucosamine transferase (e.g., LgtA from Neisseria meningitidis (UniProt ID Q9JXQ6)). For LNT production, the LN3-producing strain was further modified with a constitutive transcription unit for N-acetylglucosamine β-1,3-galactosyltransferase (e.g., WbgO from E. coli O55:H7 (UniProt ID D3QY14)). For LNnT production, the LN3-producing strain is further modified with a constitutive transcription unit for N-acetylglucosamine β-1,4-galactosyltransferase (e.g., LgtB from Neisseria meningitidis (UniProtID Q51116, sequence version 02, December 1, 2000). N-acetylglucosamine β-1,3-galactosyltransferase and N-acetylglucosamine β-1,4-galactosyltransferase can be delivered to the strain via genomic knock-in or from an expression plasmid.

[0356] To further produce fucosylated sugars, the mutant C. glutamicum strain was further modified with a constitutive transcription unit for a fucosyltransferase.

[0357] In one example for sialic acid production, a mutant Corynebacterium glutamicum strain was created by overexpressing a fructose-6-P-aminotransferase, for example the native fructose-6-P-aminotransferase glmS (UniProt ID Q8NND3, sequence version 03, January 23, 2007), to enhance the intracellular glucosamine-6-phosphate pool. Furthermore, the enzymatic activities of the genes nagA, nagB, and gamA were disrupted by genetic knockout, and glucosamine-6-P-aminotransferase (e.g., GNA1 from Saccharomyces cerevisiae (UniProt ID P43577)), N-acetylglucosamine 2-epimerase (e.g., from Bacteroides ovatus (UniProt ID A7LVG6)), and N-acetylneuraminic acid synthase (e.g., from Neisseria meningitidis (UniProt ID EONCD4)) were overexpressed on the genome. To allow for the production of sialylated sugars, the sialic acid-producing strain is further modified with a constitutive transcription unit comprising an N-acylneuraminic acid cytidylyltransferase, such as NeuA from Pasteurella multocida (UniProtID A0A849CI62); and a sialyltransferase.

[0358] For growth on sucrose, the mutant strain can be additionally modified with genomic knock-ins comprising the constitutive transcription units of the following: sucrose transporter (CscB) from Escherichia coli W (UniProt ID E0IXR1), fructokinase (Frk) from Zymomonas mobilis (UniProt ID Q03417), and sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (UniProt ID A0ZZH6).

[0359] E. Optical density

[0360] The cell density of the cultures was monitored regularly by measuring the optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium, or with Spark 10M microplate reader, Tecan, Switzerland). The maximum growth rate (mumax) was calculated based on the observed optical density at 600 nm using the R package grofit.

[0361] F. Growth rate / velocity measurements

[0362] The maximum growth rate (μMax) was calculated based on the observed optical density at 600 nm by using the R package grofit.

[0363] G. Heterologous and homologous expression

[0364] The gene to be expressed, whether it comes from a plasmid or from the genome, is synthesized synthetically by one of the following companies: IDT or Twist Bioscience. The proteins described in this disclosure are summarized in Table 1. Unless otherwise stated, the UniProt ID of the described protein corresponds to its sequence version 01 as present in the UniProt database version release 2021_03 on June 9, 2021. Expression can be further promoted by optimizing the codon usage to that of the expression host. The supplier's tools are used to optimize the genes. Table 1. Overview of proteins with corresponding UniProt IDs described in this disclosure (sequence version 01, UniProt database 2021_03 on June 9, 2021)

[0365]

[0366]

[0367] *Sequence version 02 (November 1, 1997) present in the UniProt database 2021_03 on June 9, 2021;

[0368] **Sequence version 03 (January 23, 2007) present in the UniProt database 2021_03 on June 9, 2021;

[0369] ***Sequence version 03 (December 2, 2020) present in the UniProt database 2021_03 on June 9, 2021;

[0370] ****Sequence version 03 (January 23, 2007) present in the UniProt database 2021_03 on June 9, 2021;

[0371] *****Sequence version 02 (January 23, 2007) present in the UniProt database 2021_03 on June 9, 2021;

[0372] °Sequence version 04 (January 23, 2007) present in the UniProt database 2021_03 on June 9, 2021;

[0373] °°Sequence version 02 (December 1, 2000) present in the UniProt database 2021_03 on June 9, 2021;

[0374] °°°Sequence version 03 (July 19, 2003) present in the UniProt database 2021_03 on June 9, 2021;

[0375] °°°°Sequence version 02 (February 1, 2005) present in the UniProt database 2021_03 on June 9, 2021.

[0376] H. Analytical Analysis

[0377] Standards such as, but not limited to, sucrose, lactose, 3'SL, 6'SL, lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LSTa, LSTc, and LSTd were purchased from Carbosynth (UK), Elicityl (France), and IsoSep (Sweden). Other compounds were analyzed using standards prepared in-house.

[0378] Neutral oligosaccharides were analyzed on a Waters Acquity H-class UPLC with evaporative light scattering detector (ELSD) or refractive index (RI) detection. A volume of 0.7 μL of sample was injected onto an Acquity UPLC BEH Amide VanGuard column ( Waters Acquity UPLC BEH Amide column (2.1x 5mm) (2.1x 100mm; The column temperature was 50°C. The mobile phase consisted of a 1 / 4 water and 3 / 4 acetonitrile solution to which 0.2% triethylamine was added. The method was isocratic with a flow rate of 0.130 mL / min. The ELSD detector had a drift tube temperature of 50°C, an N2 gas pressure of 50 psi, a gain of 200, and a data transfer rate of 10 pps. The RI detector temperature was set at 35°C.

[0379] Sialyl oligosaccharides were analyzed on a Waters Acquity H-class UPLC with refractive index (RI) detection. A volume of 0.5 μL of sample was injected onto a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm; The column temperature was 50°C. The mobile phase consisted of a mixture of 70% acetonitrile, 26% ammonium acetate buffer (150 mM), and 4% methanol to which 0.05% pyrrolidine was added. The method was isocratic with a flow rate of 0.150 mL / min. The temperature of the RI detector was set at 35°C.

[0380] Both neutral and sialylated sugars were analyzed on a Waters Acquity H-class UPLC with refractive index (RI) detection. A volume of 0.5 μL of sample was injected onto a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm; The column temperature was 50° C. The mobile phase consisted of a mixture of 72% acetonitrile and 28% ammonium acetate buffer (100 mM) to which 0.1% triethylamine was added. The method was isocratic with a flow rate of 0.260 mL / min. The temperature of the RI detector was set at 35° C.

[0381] For analysis on a mass spectrometer, a Waters Xevo TQ-MS with electrospray ionization (ESI) was used, with a desolvation temperature of 450° C., a nitrogen desolvation gas flow rate of 650 L / h, and a cone voltage of 20 V. The MS was operated in negative mode with selected ion monitoring (SIM) for all oligosaccharides. Separation was performed on a Waters Acquity UPLC with a Thermo Hypercarb column (2.1×100 mm; 3 μm) at 35° C. A gradient was used in which eluent A was ultrapure water with 0.1% formic acid and eluent B was acetonitrile with 0.1% formic acid. The oligosaccharides were separated over 55 minutes using the following gradient: an initial increase in eluent B from 2 to 12% in 21 minutes, a second increase in eluent B from 12 to 40% in 11 minutes, and a third increase in eluent B from 40 to 100% in 5 minutes. As a washing step, 100% eluent B was used for 5 minutes. For column equilibration, the initial conditions of 2% eluent B were restored within 1 minute and maintained for 12 minutes.

[0382] Neutral sugars and sialylated sugars at low concentrations (less than 50 mg / L) were analyzed on a Dionex HPAEC system with pulsed amperometric detection (PAD). A volume of 5 μL of sample was injected onto a Dionex CarboPac PA200 column 4 x 250 mm with a Dionex CarboPac PA200guard column 4 x 50 mm. The column temperature was set to 30° C. A gradient was used where eluent A was deionized water, eluent B was 200 mM sodium hydroxide, and eluent C was 500 mM sodium acetate. Oligosaccharides were separated over 60 minutes while maintaining a constant proportion of 25% eluent B using the following gradient: an initial isocratic step (75% eluent A) with a hold of 10 minutes, an initial increase from 0 to 4% eluent C over 8 minutes, a second isocratic step (71% eluent A and 4% eluent C) with a hold of 6 minutes, a second increase from 4 to 12% eluent C over 2.6 minutes, a third isocratic step (63% eluent A and 12% eluent C) with a hold of 3.4 minutes, and a third increase from 12 to 48% eluent C over 5 minutes. As a wash step, 48% eluent C was used for 3 minutes. For column equilibration, the initial conditions of 75% eluent A and 0% eluent C were restored over 1 minute and held for 11 minutes. The applied flow rate was 0.5 mL / min.

[0383] Lactobionic acid is analyzed on the Dionex HPAEC system with pulsed current detection (PAD). The volume of 5 μL sample is injected on the Dionex CarboPac PA01 post 2x250mm with Dionex CarboPac PA01 guard post 4x 50mm. Column temperature is set to 30 ℃. Use gradient, wherein eluent A is ultrapure water, eluent B is 200mM sodium hydroxide, and eluent C is 500mM sodium acetate. The total gradient time is 41 minutes, and in the first 7 minutes, starts with 50% B and 5% C. During the next 18 minutes, the concentration slowly changes to 50% B and 40% C at 25 minutes, followed by a rinsing step with 100% C for 7 minutes. For column balance, the initial condition with 50% B and 5% C is recovered within 9 minutes. The flow rate applied is 0.25mL / minute.

[0384] Example 2. Reduced lactobionic acid (LBA) production in a modified E. coli host in which LBA synthesis is knocked out

[0385] Mutant E. coli strains for producing 2'FL, LNT, LNnT, 3'SL, or 6'SL were engineered as described in Example 1. These strains were further modified with a genomic knockout of the native quinone protein glucose dehydrogenase gene (GeneID: 944830), encoding the gcd enzyme (UniProt ID P15877, sequence version 03 (December 2, 2020)). In the next step, these mutant strains were further modified with a genomic knockout of the native aldose sugar dehydrogenase gene (GeneID: 945467), encoding the ylil enzyme (UniProt ID P75804).

[0386] According to the culture conditions provided in Example 1, all new bacterial strains and corresponding reference strains with the same genetic makeup but still having native gcd and ylil genes were evaluated in a growth experiment, wherein the bacterial strains were cultured in a minimal medium supplemented with 30 g / L sucrose, 20 g / L lactose, and 3.30E-4 g / L pyrroloquinoline quinone (PQQ). The bacterial strains were grown in three biological replicates in 96-well plates. After 72 hours of incubation, the cultures were harvested and lactobionic acid (LBA) was analyzed on UPLC to produce LBA. In addition, the maximum growth rate (Mumax, h) was determined as provided in Example 1. -1 ).

[0387] As shown in Table 2, each bacterial strain with gcd knocks out and natural ylil gene shows the LBA generation that reduces compared to its reference strain with identical genetic makeup and naturally expresses gcd and ylil separately.For LNT, LNnT, 3 ' SL and 6 ' SL production bacterial strain with gcd knocks out, LBA generation is even reduced to below the detection limit.For 2 ' FL bacterial strain, produce, on gcd knocks out, add ylil and knock out and cause the LBA generation that further reduces compared to when only carrying out gcd knocks out.The knockout of gcd gene or the knockout of gcd and ylil gene do not influence 2 ' FL, LNT, LNnT, 3 ' SL or 6 ' SL in bacterial strain separately and produce (result not shown). Figure 1 It was demonstrated that genomic knockout of gcd and genomic knockout of both gcd and ylil had little effect on the growth rate of the mutant strains compared to a reference strain expressing native gcd and ylil genes.

[0388] Table 1. Average (n=3) production of lactobionic acid (LBA) in modified E. coli strains engineered as described in Example 1 for production of 2'FL, LNT, LNnT, 3'SL, or 6'SL and having native gcd and ylil expression (reference strain, gcd+ylil+) or having a gcd genomic knockout (gcd KO, ylil+) or having both a gcd genomic knockout and a ylil genomic knockout (gcd KO, ylilKO) in growth experiments according to the culture conditions provided in Example 1, wherein the culture medium was supplemented with 30 g / L sucrose, 20 g / L lactose, and 3.30E-4 g / L pyrroloquinoline quinone (PQQ).

[0389]

[0390] *ND=Not Detectable

[0391] Example 3. 3'SL or 6'SL production in modified E. coli hosts when evaluated in fed-batch fermentations with sucrose and lactose

[0392] The mutant E. coli strain modified to produce 3'SL or 6'SL and having the genome knockout (gcd KO, ylilKO) of both gcd and ylil genes described in Example 2 is selected for further evaluation in fed-batch fermentation process. In addition, a fed-batch fermentation process is set up with a reference strain having the same genetic makeup but expressing gcd and ylil genes (gcd+, ylil+). Fed-batch fermentation with bioreactor scale is carried out as described in Example 1. Sucrose is used as a carbon source, and lactose is added in the batch culture medium. During the fed-batch fermentation, sucrose is added via other feeding. Contrary to the culture experiment described in this article and wherein only at the end of cultivation (i.e., 72 hours described in this article) taking final sample, broth samples are regularly taken out at several time points during the fermentation process, and UPLC is used to measure the 3'SL or 6'SL produced, as described in Example 2. For each strain tested, two independent fermentations were performed, and the measured 3'SL or 6'SL concentrations were averaged across all biological replicates and then normalized to the mean 3'SL or 6'SL concentration of the respective reference strain.

[0393] This experiment demonstrated that the average 3'SL titer measured at the end of fed-batch fermentation with a 3'SL strain with gcd and ylil knockouts (gcd KO, ylilKO) was 97.1% of the average 3'SL titer measured at the end of fed-batch fermentation with a 3'SL reference strain (gcd+, ylil+). Furthermore, the average 6'SL titer measured at the end of fed-batch fermentation with a 6'SL strain with gcd and ylil knockouts (gcd KO, ylilKO) was 99.7% of the average 6'SL titer measured at the end of fed-batch fermentation with a 6'SL reference strain (gcd+, ylil+). In summary, the 3'SL or 6'SL titer was not affected by knockout of the gcd and ylil genes in the respective 3'SL or 6'SL production strains.

[0394] Example 4. Oligosaccharide production in a modified E. coli host when evaluated in a fed-batch fermentation with sucrose and lactose

[0395] The mutant E. coli strain modified to produce 2'FL, LNT or LNnT and having both the gcd and ylil genes knocked out (gcd KO, ylil KO) described in Example 2 was selected for further evaluation in a fed-batch fermentation process. In addition, a fed-batch fermentation process was established using a reference strain with the same genetic makeup but expression of the gcd and ylil genes (gcd+, ylil+). Fed-batch fermentation was performed on a bioreactor scale as described in Example 1. Sucrose was used as a carbon source, and lactose was added to the batch culture medium. During the fed-batch fermentation, sucrose was added via another feed. In contrast to the culture experiments described herein and in which a final sample was taken only at the end of the culture (i.e., 72 hours described herein), broth samples were regularly taken out at several time points during the fermentation process, and UPLC was used to measure the 2'FL, LNT or LNnT titer produced in each strain, as described in Example 1.

[0396] Example 5. Oligosaccharide production in a modified E. coli host when evaluated in a fed-batch fermentation process with sucrose and lactose

[0397] Further modify the mutant E. coli strain modified for the production of 3-FL, DiFL, LSTa, LSTb, LSTc or LSTd described in Example 1 to have both genetic knockouts (gcd KO, ylilKO) of gcd and ylil genes. Each new bacterial strain can produce 3-FL, diFL, LSTa, LSTb, LSTc or LSTd with an amount similar to the reference strain still having the expression of gcd and ylil genes separately. Select the new bacterial strain for the further evaluation in fed-batch fermentation process. In addition, set up fed-batch fermentation process with the reference strain having the expression of gcd and ylil genes (gcd+, ylil+) separately. Carry out fed-batch fermentation with bioreactor scale as described in Example 1. Use sucrose as carbon source, and add lactose in batch culture medium. During fed-batch, add sucrose via other feeding. In contrast to the cultivation experiments described herein, in which only a final sample was taken at the end of the cultivation (i.e., 72 hours as described herein), broth samples were taken regularly at several time points during the fermentation process and the titers of 3-FL, diFL, LSTa, LSTb, LSTc, or LSTd produced in the respective strains were measured using UPLC, as described in Example 1. Example 6. Evaluation of the production of glycosylated forms of LBA in modified E. coli hosts

[0398] The mutant E. coli strain modified for production of 2'FL, 3'SL or 6'SL and modified with genomic knockout of both gcd and ylil genes (gcd KO, ylil KO) as described in Example 2 and the mutant E. coli strain modified for production of 3-FL, DiFL, LSTa, LSTb, LSTc or LSTd and modified with genomic knockout of both gcd and ylil genes (gcd KO, ylil KO) as described in Example 5 were selected for further evaluation in fed-batch fermentation. Fed-batch fermentation at bioreactor scale was performed as described in Example 1. Sucrose was used as the carbon source, and lactose and PQQ were added to the batch culture medium. During the fed-batch period, sucrose was added via an additional feed. In contrast to the cultivation experiments described herein and in which a final sample was taken only at the end of the cultivation (i.e., 72 hours as described herein), culture broth samples were taken regularly at several time points during the fermentation process and the 2'FL, 3'SL, 6'SL, 3-FL, diFL, LSTa, LSTb, LSTc, LSTd, LBA, fucosylated LBA or sialylated LBA titers produced in the respective strains were measured using UPLC as described in Example 1.

[0399] Example 7. Functional degradation of genes in modified hosts

[0400] Making gene function worse is a common practice in biotechnology. As described above, there are several techniques for reducing expression or making gene function worse (e.g., using siRNA, CrispR interference, RNAi, miRNA, asRNA, mutant genes, knockout genes, transposon mutagenesis, etc.).

[0401] CrispR interference is one of the latest technologies in making genes worse in terms of function. It requires designing sgRNA that recognizes the target gene (gene to be made to worse in terms of function) and expressing a DNA endonuclease (e.g., dCas9 protein) guided by a mutated RNA that loses its endonuclease activity. sgRNA is composed of a base pairing region that is mainly present in the downstream or upstream 20 nucleotides of the PAM area (e.g., NGG, in the case of dCas9). The base pairing region is complementary to the region in the target gene. The tighter the base pairing region is combined with the 5' end of the gene target, the better the suppression. In order to avoid off-target suppression, BLAST is performed on the base pairing region for the genome to ensure that there are no other regions complementary to the base pairing region except the target gene. Examples of design tools are described by Doench et al. (2016) [Nature Biotechnology, Vol. 34, pp. 184-191 (2016)] or Labun et al. (2019) [Nucleic Acids Research, Vol. 47, pp. W171-W174 (2019)], but are also provided by most synthetic DNA providers.

[0402] Both dCas9 and sgRNA are expressed in cells according to the methods described in Example 1. Preferably, both are expressed from the genome, thereby ensuring stable expression over several generations.

[0403] Example 8. Evaluation of Lactobionic Acid (LBA) Production in a Modified E. coli Host in Which LBA Synthesis is Functionally Impaired

[0404] Mutant E. coli strains for producing 2'FL, LNT, LNnT, 3'SL, or 6'SL were engineered as described in Example 1. These strains were further modified to functionally degrade the quinone protein glucose dehydrogenase gene (GeneID: 944830) encoding the gcd enzyme (UniProt ID P15877) and the aldose sugar dehydrogenase gene (GeneID: 945467) encoding the ylil enzyme (UniProt ID P75804) in their cells to reduce or eliminate the production of lactobionic acid (LBA) when grown on a medium containing lactose and pyrroloquinoline quinone (PQQ). As an example, the gcd gene and the ylil gene were functionally degraded using the CrispRi technology described in Example 7. Both the sgRNA for the gcd gene (SEQ ID NO: 01) and the sgRNA for the ylil gene (SEQ ID NO: 02) had CGG as the PAM sequence. The sequence was expressed using the constitutive promoter sequence described in Example 1. The novel strain was evaluated in a growth experiment according to the culture conditions provided in Example 1, wherein the strain was cultured in minimal medium supplemented with 30 g / L sucrose, 20 g / L lactose, and 3.30E-4 g / L pyrroloquinoline quinone (PQQ). The strain was grown in three biological replicates in 96-well plates. After 72 hours of incubation, the maximum growth rate was determined, the culture fluid was harvested, and LBA production was analyzed on a UPLC.

[0405] Example 9. Reduced Lactobionic Acid (LBA) Production in a Modified E. coli Host in Which LBA Synthesis is Reduced

[0406] As described in Example 1, the mutant Escherichia coli strains for producing 2'FL, LNT, LNnT, 3'SL or 6'SL were transformed. These strains were further modified by knocking out the genome of the natural aldose dehydrogenase gene (GeneID:945467) encoding the ylil enzyme (UniProt ID P75804). In the next step, these mutant strains were further modified by knocking out the genome of the natural quinone protein glucose dehydrogenase gene (GeneID:944830) encoding the gcd enzyme (UniProt ID P15877, sequence version 03 (December 2, 2020)). According to the culture conditions provided in Example 1, all new strains and the corresponding reference strains with the same genetic composition but still having natural gcd gene and ylil gene were evaluated in a growth experiment, wherein the strain was cultured in a minimal medium supplemented with 30g / L sucrose, 20g / L lactose and 3.30E-4g / L pyrroloquinoline quinone (PQQ). The strains were grown in three biological replicates in 96-well plates. After 72 hours of incubation, the cultures were harvested and analyzed on UPLC for lactobionic acid (LBA) production in strains with (1) ylil knockout and the natural gcd gene or (2) knockout of both ylil and gcd genes compared to a reference strain still having the natural ylil gene and gcd gene.

Claims

1. A cell capable of synthesizing and / or synthesizing lactobionic acid (4-O-β-galactopyranosyl-D-gluconic acid, LBA), modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA in the presence of lactose in the culture or incubation medium of the cell, wherein the synthesis of LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA is obtained by expressing at least one gene selected from the list consisting of genes encoding carbohydrate oxidases, dehydrogenases, lactonases, oxygen-dependent FAD-linked oxidoreductases and pyrroloquinoline quinone (PQQ) oxidoreductases, the cell being genetically engineered for the production of a sugar, the cell comprising a pathway for the production of the sugar, characterized in that The synthesis of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA is functionally impaired or knocked out in the cell by impairing the function of at least one gene, two or more of the genes or all of the genes.

2. The cell according to claim 1, wherein: - the pathway for producing sugars is selected from the list consisting of or consisting essentially of a fucosylation pathway, a sialylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N-acetylgalactosaminylation pathway, a mannosylation pathway, and an N-acetylmannosaminylation pathway, and / or - the pathway for producing sugars is selected from the list consisting of or consisting essentially of a fucosylation pathway, a sialylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N-acetylgalactosaminylation pathway, a mannosylation pathway, and an N-acetylmannosaminylation pathway, and wherein the cell is genetically engineered to comprise at least one of said pathways, and / or the cell comprises at least one of said pathways, wherein at least one of said pathways has been genetically engineered.

3. The cell according to any one of claims 1 or 2, wherein the cell: - having, expressing and / or overexpressing one or more glycosyltransferases selected from the list consisting of or consisting essentially of: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosamine transferase, N-acetylgalactosamine transferase, N-acetylmannosamine transferase, xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosamine transferase, N-glycolylneuraminic acid transferase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine transaminase, UDP-N-acetylglucosamine enolpyruvyltransferase and fucosamine transferase, - is capable of producing and / or produces one or more nucleotide activating sugars, - genetically engineered to produce one or more of the nucleotide activated sugars, - comprising a pathway for synthesizing a nucleotide-activated sugar selected from the list consisting of or consisting essentially 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-acetylamino-2,6-dideoxy-L-arabino-4-hexulose, UDP-2-acetylamino-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetylamino-2,6-dideoxy-L-mannose), dTDP-N- Acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetylamino-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-l-phospho-glucosamine (UDP-L-PneNAc or UDP-2-acetylamino-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-isorhamnosamine (UDP-L-QuiNAc or UDP-2-acetylamino-2,6-dideoxy-L-talose), CMP-Neu5Ac, CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-Neu5Gc, GDP-rhamnose, and UDP-xylose, - having, expressing and / or overexpressing one or more genes selected from the list consisting of or consisting essentially of: mannose-6-phosphate isomerase, phosphomannose mutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, fucose-1-phosphate guanylyltransferase, 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 uridyltransferase, glucosamine-1-phosphate acetyltransferase, Neu5Ac synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase, and UDP-N-acetylgalactosamine pyrophosphorylase, and / or - comprising at least partially inactivated catabolic pathways for selected mono-, di- or oligosaccharides involved in and / or required for the synthesis of said saccharides.

4. The cell according to any one of the preceding claims, wherein: - the dehydrogenase is selected from the list consisting of or consisting essentially of lactose dehydrogenase, quinoline glucose dehydrogenase, aldose dehydrogenase, glucose / fructose dehydrogenase, glucose / sorbosone dehydrogenase, cellobiose dehydrogenase, pyrroloquinoline quinone (PQQ) dehydrogenase and malate dehydrogenase, and / or - The carbohydrate oxidase is lactose oxidase.

5. The cell of any one of the preceding claims, wherein the at least one gene encodes an enzyme, wherein the enzyme: - an enzyme class selected from the list consisting of or consisting essentially of EC: 1.1.3.-, EC: 1.1.3.5, EC: 1.1.3.4, EC: 1.1.5.-, EC: 1.1.5.2 and EC: 1.1.99.18, - comprising a polypeptide sequence comprising an IPR domain selected from the list consisting of or consisting essentially of: IPR000172, IPR001479, IPR002372, IPR006094, IPR007867, IPR011041, IPR011042, IPR011047, IPR012132, IPR012938, IPR012951, IPR013154, IPR013428, IPR015402, IPR015920, IPR016166, IPR016169, IPR017511, IPR017512, IPR018391, IPR027424, IPR029056, IPR031640, IPR036188, and IPR036318, as defined in 90.

0. - comprising a polypeptide sequence comprising a Panther domain selected from the list consisting of or consisting essentially of PTHR11552, PTHR13460, PTHR32303:SF4, PTHR42973 and PTHR47190 as defined by InterPro 90.0 released on August 4, 2022, - comprising a polypeptide sequence comprising a PFAM domain selected from the list consisting of or consisting essentially of PF00732, PF01011, PF05199, PF07995, PF08031, PF08240, PF13360, PF13570, PF01565, PF16010 and PF16912 as defined by InterPro 90.0 released on August 4, 2022, - comprising a polypeptide sequence comprising the conserved protein domain cd10280 as defined by InterPro 90.0 released on August 4, 2022, - is part of a NOG family selected from the list consisting of or consisting essentially of: COG0277, COG2133, COG2303 and COG4993 as defined by eggNOG5.0 released in 2019, and / or - using a cofactor selected from the list consisting of or consisting essentially of: pyrroloquinoline quinone (PQQ), FAD, a divalent metal cation, Ca 2+ 、Cu 2+ Mg 2+ 、Zn 2+ , heme, heme C and pyridoxal 5'-phosphate.

6. The cell according to any one of the preceding claims, wherein at least one gene involved in the synthesis and / or import of the following cofactors is functionally impaired or knocked out: - cofactors involved in the synthesis of LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, and / or - a cofactor selected from the list consisting of or consisting essentially of and involved in the synthesis of LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA: pyrroloquinoline quinone (PQQ), FAD, a divalent metal cation, Ca 2+ 、Cu 2+ Mg 2+ 、Zn 2+ , heme, heme C and pyridoxal 5'-phosphate.

7. The cell according to any one of the preceding claims, wherein the at least one gene is functionally impaired by insertion, deletion and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from the list consisting of or essentially consisting of: a promoter sequence, a ribosome binding site, an untranslated region, a coding sequence and a transcription terminator sequence of the at least one gene.

8. The cell according to any one of the preceding claims, wherein the cell comprises a quinone protein glucose dehydrogenase gene that is rendered functionally impaired by insertion, deletion and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from a list consisting of or essentially consisting of: a promoter sequence, a ribosome binding site, an untranslated region, a coding sequence and a transcription terminator sequence of the quinone protein glucose dehydrogenase gene.

9. The cell according to claim 8, wherein the cell further comprises an aldose sugar dehydrogenase gene that is rendered functionally impaired by insertion, deletion and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from the list consisting of or essentially consisting of: a promoter sequence, a ribosome binding site, an untranslated region, a coding sequence and a transcription terminator sequence of the aldose sugar dehydrogenase gene.

10. The cell according to any one of claims 1 to 7, wherein the cell comprises an aldose sugar dehydrogenase gene that is rendered functionally impaired by insertion, deletion and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from the list consisting of or essentially consisting of: a promoter sequence, a ribosome binding site, an untranslated region, a coding sequence and a transcription terminator sequence of the aldose sugar dehydrogenase gene.

11. The cell according to claim 10, wherein the cell further comprises a quinone protein glucose dehydrogenase gene, which is rendered functionally impaired by insertion, deletion and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from the list consisting of or essentially consisting of the following: a promoter sequence, a ribosome binding site, an untranslated region, a coding sequence and a transcription terminator sequence of the quinone protein glucose dehydrogenase gene.

12. The cell of any one of the preceding claims, wherein the sugar is selected from the list consisting of or consisting essentially of: a monosaccharide; a phosphorylated monosaccharide; an activated monosaccharide; a disaccharide; an oligosaccharide; a neutral (uncharged) oligosaccharide; a negatively charged oligosaccharide; a sialylated oligosaccharide; a milk oligosaccharide; a mammalian milk oligosaccharide (MMO); a human milk oligosaccharide (HMO); a sialylated milk oligosaccharide; a neutral (uncharged) milk oligosaccharide; a fucosylated milk oligosaccharide; a non-fucosylated neutral (uncharged) milk oligosaccharide; a sialylated mammalian milk oligosaccharide; a neutral (uncharged) mammalian milk oligosaccharide; a fucosylated mammalian milk oligosaccharide; a non-fucosylated neutral (uncharged) mammalian milk oligosaccharide; a sialylated human milk oligosaccharide; a neutral (uncharged) mammalian milk oligosaccharide; a fucosylated mammalian milk oligosaccharide; a non-fucosylated neutral (uncharged) mammalian milk oligosaccharide; a neutral (uncharged) mammalian milk oligosaccharide; a sialylated human milk oligosaccharide; a neutral (uncharged) mammalian milk oligosaccharide; a fucosylated mammalian milk oligosaccharide; a neutral (uncharged) mammalian milk oligosaccharide; a sialylated human ... neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; O-antigens; enterobacterial common antigens (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigenic oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides; animal oligosaccharides selected from the list consisting of N-glycans and O-glycans; plant oligosaccharides; plant oligosaccharides selected from the list consisting of N-glycans and O-glycans; fucosylated oligosaccharides; fucosylated oligosaccharides selected from the list consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6 FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated oligosaccharides selected from the list consisting of 3' sialyl lactose (3'SL), 6' sialyl lactose (6'SL), sialyl lacto-N-tetraose a (LSTa), sialyl lacto-N-tetraose b (LSTb), sialyl lacto-N-tetraose c ( LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, and monofucosyldisialyllacto-N-tetraose; neutral (uncharged) sugars containing N-acetylglucosamine;A neutral (uncharged) sugar containing N-acetylglucosamine selected from the list consisting of: lacto-N-biose (LNB), N-acetyllactosamine (LacNAc), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose; sugars containing N-acetylglucosamine; sugars containing N-acetyllactosamine; sugars containing lacto-N-biose; non-fucosylated neutral (uncharged) sugars; chitosan; oligosaccharides containing chitosan; heparosan; chondroitin sulfate; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; dermatan sulfate; hyaluronan; hyaluronic acid; and keratan sulfate.

13. The cell of any one of the preceding claims, wherein the cell: - is capable of producing and / or produces said sugar, LBA, modified form of LBA, glycosylated form of LBA, fucosylated LBA and / or sialylated LBA from one or more precursors, - is capable of producing and / or produces said sugar, LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA from lactose, - is capable of producing and / or produces at least one precursor for the production of said sugar, LBA, modified form of LBA, glycosylated form of LBA, fucosylated LBA and / or sialylated LBA, - is capable of producing and / or produces all precursors for the production of said sugars, LBAs, modified forms of LBAs, glycosylated forms of LBAs, fucosylated LBAs and / or sialylated LBAs, - genetically engineered to produce at least one precursor for producing said sugar, LBA, modified form of LBA, glycosylated form of LBA, fucosylated LBA and / or sialylated LBA, and / or - Genetically engineered for the production of all precursors for the production of said sugars, LBAs, modified forms of LBAs, glycosylated forms of LBAs, fucosylated LBAs and / or sialylated LBAs.

14. The cell of claim 13, wherein at least one of the one or more precursors is internalized into the cell via one or more membrane proteins.

15. The cell of any one of the preceding claims, wherein the cell: - selected from the group consisting of prokaryotic cells and eukaryotic cells, - is selected from the group consisting of yeast cells, bacterial cells, archaeal cells, algae cells, plant cells and fungal cells, - is Escherichia coli (E. coli) or yeast with a lactose permease-positive phenotype, and / or - an Escherichia coli or yeast having a lactose permease-positive phenotype, wherein the lactose permease is encoded by the gene LacY or LAC12, respectively.

16. A method for producing sugars, the method comprising: i. culturing and / or incubating a cell according to any one of the preceding claims in a culture and / or incubation medium under conditions that allow the production of said sugar and any one or more of lactobionic acid (LBA), modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, and ii. (1) separating the sugar from the culture and / or incubation, and / or (2) separating the sugar from the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA.

17. The method of claim 16, wherein the culture or incubation medium comprises one or more precursors for producing the sugar, LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA.

18. The method of any one of claims 16 or 17, wherein the cells produce 30 g / L or more of the sugar in the whole broth and / or supernatant, and / or wherein the sugar in the whole broth and / or supernatant has a purity of at least 80%, measured based on the total amount of sugar and its precursors produced by the cells in the whole broth and / or supernatant, respectively.

19. The method of any one of claims 16 to 18, wherein functionally impaired or knocked-out synthesis of LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA confers unaffected and / or enhanced i) sugar formation, ii) productivity, iii) biomass production, iv) cell growth, and / or v) yield of produced sugars relative to corresponding unmodified cells.

20. The method of any one of claims 16 to 19, wherein the cell produces a mixture comprising the sugar and any one or more of the LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA, and / or a sialylated LBA, wherein the mixture comprises ≤ 10 wt% of the LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA, and / or a sialylated LBA, ≤ 9 wt% of the LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA, and / or a sialylated LBA, % of the glycosylated form of LBA, fucosylated LBA and / or sialylated LBA, ≤8 wt% of the LBA, modified form of LBA, glycosylated form of LBA, fucosylated LBA and / or sialylated LBA, ≤7 wt% of the LBA, modified form of LBA, glycosylated form of LBA, fucosylated LBA and / or sialylated LBA, ≤6 wt% of the LBA, modified form of LBA, glycosylated form of LBA, fucosylated LBA and / or sialylated LBA, ≤5 % by weight of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤4 % by weight of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤3 % by weight of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤2 % by weight of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA % of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤1 wt% of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤0.5 wt% of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, and / or ≤0.1 wt% of the LBA, modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA.

21. The method of any one of claims 16 to 19, wherein the cells do not produce LBA.

22. The method according to any one of claims 16 to 21, wherein the sugar is recovered from the culture or incubation medium and / or the cells, and / or wherein the sugar is purified.

23. The method of any one of claims 16 to 22, wherein the sugar is purified (i) from the LBA and / or (ii) from a modified form of the LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA.

24. Use of a cell according to any one of claims 1 to 15 for the production of a sugar, wherein the sugar is selected from the list consisting of or consisting essentially of: a monosaccharide; a phosphorylated monosaccharide; an activated monosaccharide; a disaccharide; an oligosaccharide; a neutral (uncharged) oligosaccharide; a negatively charged oligosaccharide; a sialylated oligosaccharide; a milk oligosaccharide; a mammalian milk oligosaccharide (MMO); a human milk oligosaccharide (HMO); a sialylated milk oligosaccharide; a neutral (uncharged) milk oligosaccharide; a fucosylated milk oligosaccharide; a non-fucosylated neutral (uncharged) milk oligosaccharide; a sialylated mammalian milk oligosaccharide; a neutral (uncharged) mammalian milk oligosaccharide; a fucosylated mammalian milk oligosaccharide; a non-fucosylated neutral (uncharged) mammalian ...sialylated mammalian milk oligosaccharide; a neutral (uncharged) mammalian milk oligosaccharide; a sialylated mammalian milk oligosaccharide; a neutral (uncharged) mammalian milk oligosaccharide; a sialylated mammalian milk oligosaccharide; acidified human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; O-antigens; enterobacterial common antigens (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigenic oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides; animal oligosaccharides selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides; plant oligosaccharides selected from the group consisting of N-glycans and O-glycans; fucosylated oligosaccharides; fucosylated oligosaccharides selected from the group consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose Lactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated oligosaccharides selected from the group consisting of 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), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, and monofucosyldisialyllacto-N-tetraose; neutral (uncharged) sugars containing N-acetylglucosamine;Neutral (uncharged) sugars containing N-acetylglucosamine selected from the group consisting of lacto-N-biose (LNB), N-acetyllactosamine (LacNAc), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose; sugars containing N-acetylglucosamine; sugars containing N-acetyllactosamine; sugars containing lacto-N-biose; non-fucosylated neutral (uncharged) sugars; chitosan; oligosaccharides containing chitosan; heparosan; chondroitin sulfate; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; dermatan sulfate; hyaluronan; hyaluronic acid; and keratan sulfate.

25. Use of the method according to any one of claims 16 to 23 for producing a sugar, wherein the sugar is selected from the list consisting of or consisting essentially of: monosaccharides; phosphorylated monosaccharides; activated monosaccharides; disaccharides; oligosaccharides; neutral (uncharged) oligosaccharides; negatively charged oligosaccharides; sialylated oligosaccharides; milk oligosaccharides; mammalian milk oligosaccharides (MMO); human milk oligosaccharides (HMO); sialylated milk oligosaccharides; neutral (uncharged) milk oligosaccharides; fucosylated milk oligosaccharides; non-fucosylated neutral (uncharged) milk oligosaccharides; sialylated mammalian milk oligosaccharides; neutral (uncharged) mammalian milk oligosaccharides; fucosylated mammalian milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides; sialylated mammalian milk oligosaccharides; neutral (uncharged) mammalian milk oligosaccharides; fucosylated mammalian milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides; sialylated mammalian milk oligosaccharides; acidified human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; O-antigens; enterobacterial common antigens (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigenic oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides; animal oligosaccharides selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides; plant oligosaccharides selected from the group consisting of N-glycans and O-glycans; fucosylated oligosaccharides; fucosylated oligosaccharides selected from the group consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose Lactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated oligosaccharides selected from the group consisting of 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), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, and monofucosyldisialyllacto-N-tetraose; neutral (uncharged) sugars containing N-acetylglucosamine;Neutral (uncharged) sugars containing N-acetylglucosamine selected from the group consisting of lacto-N-biose (LNB), N-acetyllactosamine (LacNAc), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose; sugars containing N-acetylglucosamine; sugars containing N-acetyllactosamine; sugars containing lacto-N-biose; non-fucosylated neutral (uncharged) sugars; chitosan; oligosaccharides containing chitosan; heparosan; chondroitin sulfate; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; dermatan sulfate; hyaluronan; hyaluronic acid; and keratan sulfate.

26. A mixture comprising, consisting of or essentially consisting of: (i) a sugar, and (ii) LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA, wherein the sugar, LBA, a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or a sialylated LBA is obtainable or obtained by the method according to any one of claims 16 to 23.

27. A mixture comprising, consisting of or consisting essentially of (i) sugars, and (ii) ≤10 wt% LBA, ≤9 wt% LBA, ≤8 wt% LBA, ≤7 wt% LBA, ≤6 wt% LBA, ≤5 wt% LBA, ≤4 wt% LBA, ≤3 wt% LBA, ≤2 wt% LBA, ≤1 wt% LBA, ≤0.5 wt% LBA, and / or ≤0.1 wt% LBA, wherein the sugars and LBA are obtainable or obtained by the process according to any one of claims 16 to 20, 22 or 23.

28. A mixture comprising, consisting of, or consisting essentially of a sugar and (i) ≤10 wt% LBA, ≤9 wt% LBA, ≤8 wt% LBA, ≤7 wt% LBA, ≤6 wt% LBA, ≤5 wt% LBA, ≤4 wt% LBA, ≤3 wt% LBA, ≤2 wt% LBA, ≤1 wt% LBA, ≤0.5 wt% LBA, and / or ≤0.1 wt% LBA, and / or (ii) ≤10 wt% of a modified form of LBA, Glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤9 wt% of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤8 wt% of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤7 wt% of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤6 wt% of modified forms of LBA, glycosylated forms of LBA Formula, fucosylated LBA and / or sialylated LBA, ≤5 wt% of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤4 wt% of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤2 wt% of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤1 wt% of modified forms of LBA, glycosylated forms of LBA, fucosylated LBA and / or sialylated LBA, ≤1 wt% of modified forms of LBA, BA and / or sialylated LBA, ≤0.5 wt. % of a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or sialylated LBA, and / or ≤0.1 wt. % of a modified form of LBA, a glycosylated form of LBA, a fucosylated LBA and / or sialylated LBA, wherein the sugar, LBA, modified form of LBA, glycosylated form of LBA, fucosylated LBA and / or sialylated LBA is obtainable or obtained by the method according to any one of claims 16 to 23.

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