Fermentative production of oligosaccharides by total fermentation using a mixed raw material

Abstract

Abstract Genetically modified microbial cells for the production of oligosaccharides comprising a galactose-β1,4-glucose moiety at their reducing end are disclosed, wherein said microbial cells are capable of producing said oligosaccharides in the absence of exogenously added lactose, and a method for producing said oligosaccharides using said microbial cells is described.

Description

Fermentative production of oligosaccharides by total fermentation using a mixed raw material The present invention relates to genetically modified microbial cells for the production of lactose or an oligosaccharide of interest comprising a galactose-p1,4-glucose structural unit at its reducing end, as well as to a method for producing lactose or an oligosaccharide of interest comprising a galactose-p1,4-glucose terminal structural unit at its reducing end. Background Breast milk comprises a complex mixture of carbohydrates, fats, proteins, vitamins, minerals, and trace elements. The most predominant fraction of human milk consists of carbohydrates. The carbohydrate fraction of human milk can be further divided into (i) lactose and (ii) oligosaccharides (human milk oligosaccharides, HMOs). While lactose (galactose-p1, 4-glucose) is used as an energy source, the infant does not metabolize oligosaccharides. The oligosaccharide fraction represents up to 1 / 10 of the total carbohydrate fraction and likely consists of more than 150 different oligosaccharides. The occurrence and concentration of these complex oligosaccharides are specific to humans and, therefore, are not found in large quantities in the milk of other mammals, including dairy farm animals. The most prominent oligosaccharides in human milk are 2'-fucosillactose and 3'-fucosillactose, which together can contribute up to one-third of the total HMO fraction. Additional prominent HMOs present in human milk include lacto-N-tetraose, lacto-N-neotetraose, and lacto-N-fucopentaose I. In addition to these neutral oligosaccharides, acidic HMOs such as 3'-sialillactose, 6'-sialillactose, 3-fucosyl-3'-sialillactose, sialyl-lacto-W-tetraose, and disialyl-lacto-W-tetraose can also be found in human milk. Notably, the vast majority of HMOs comprise a galactose-p1,4-glucose structural unit at their reducing end. HMO structures are closely related to the epitopes of glycoconjugates on the surface of epithelial cells, the Lewis blood group antigens, such as Lewis x (LeX).The structural similarity of HMOs to epithelial epitopes explains the protective properties of HMOs against bacterial pathogens. The presence of oligosaccharides in human milk has been known for a long time, and the physiological functions of these oligosaccharides have been the subject of medical research for many decades. Specific functions have already been identified for some of the most abundant oligosaccharides in human milk. In addition to the local effects on the intestinal tract mentioned earlier in this document, HMOs have also been shown to cause systemic effects in infants by entering their systemic circulation. Furthermore, the impact of HMOs on protein-carbohydrate interactions, such as leukocyte-selectin binding, can modulate immune responses and reduce inflammation. Moreover, HMOs are increasingly recognized as a key substrate for the development of infant microbiomes. Due to the well-studied beneficial properties of prebiotic oligosaccharides, particularly HMOs, but their limited availability from natural sources, efficient commercial production of HMOs, i.e., on a large scale, is highly desirable. In attempts to produce individual oligosaccharides from human milk on a large scale, chemical routes were developed for some of these oligosaccharides. However, such methods involve the use of various harmful chemicals, which pose a risk of contaminating the final product. To date, it is not possible to obtain them through chemical synthesis in sufficient quantities or with qualities suitable for food applications. To avoid the drawbacks associated with the chemical synthesis of human milk oligosaccharides (HMOs), several enzymatic methods and fermentative approaches have been developed for their production. Fermentative production procedures have been developed for various HMOs, such as 2'-fucosillactose, 3-fucosillactose, lacto-W-tetraose, lacto-W-neotetraose, 3'-sialillactose, and 6'-sialillactose. These production procedures typically use genetically modified bacterial strains, such as recombinant Escherichia coli. Today, all fermentative production procedures, as well as biocatalytic reactions for producing HMOs, are based exclusively on exogenously added lactose as the starting substrate. One or more monosaccharides are added to the lactose in the procedures (US 7, 521, 212 B1; Albermann et al., (2001) Carbohydr. Res. 334 (2) p 97-103). The addition of monosaccharides to lactose can be catalyzed by either glycosyltransferases or glycosidases using appropriate activated monosaccharide substrates. In addition, further monosaccharides can be added to lactose by transglucosidase reactions. In particular, the fermentative production of HMOs proved effective because the necessary but difficult-to-synthesize nucleotide-activated monosaccharides are provided by the metabolism of the microbial cells employed. However, the use of whole cells for HMO synthesis also presents—compared to the biocatalytic approach—several significant disadvantages. These relate to transport procedures across the cell membrane, secondary metabolic reactions, and the need to purify the oligosaccharides synthesized by the microbial cells from a complex mixture containing, among other things, various polyols (e.g., carbohydrates), nucleic acids, polypeptides, inorganic material, etc. A technical problem related to the use of lactose in fermentation processes that must be overcome, particularly when the resulting oligosaccharide is intended for human consumption, is the rearrangement of lactose (beta-D-galactopyranosyl-(1⁴)-D-glucose) to lactulose (beta-D-galactopyranosyl-(1⁴)-D-fructofuranose) following heat treatment. This rearrangement can occur extensively through thermal sterilization of lactose, leading to the conversion of various percentages of the lactose present in the fermentation medium or feed to lactulose. However, lactulose is a non-digestible sugar for humans and is widely used as a laxative in the treatment of chronic constipation. The conversion of lactose to lactulose not only leads to the generation of unwanted lactulose but also provides an undesirable substrate for glycosylation reactions in microbial cells. This results in the generation of more complex oligosaccharides (e.g., 2'-fucosylctulose) as byproducts. Thus, the generation of lactulose from lactose leads to the contamination of the desired product with closely related oligosaccharides, which are difficult or even impossible to separate from the desired product. Additionally, lactose can be converted into allolactose (beta-D-galactopyranosyl-(1^6)-D-glucopyranose), another unwanted contaminant (Huber et al., "Efflux of beta-galactosidase products from Escherichia coli' (1980) J. Bacteriol. 141, 528-533), if supplied to a beta-galactosidase-positive E. coli strain. Furthermore, the addition of lactose can cause a well-documented effect known as "lactose-induced cell death." This effect is likely due to the excessive uptake of lactose by the microbial cell and the associated collapse of the proton gradient across the bacterial membrane. In particular, overexpression of the lactose permease gene (e.g., lacY from E. coli) in combination with exposure of the recombinant microbial cell to excess lactose can lead to a considerable delay in the growth of the recombinant strain and an increase in the synthesis of cellular polysaccharides (Grube et al., "Hydrogen-producing Escherichia coli strains overexpressing lactose permease: FT-IR analysis of the lactose-induced stress" (2013) Biotechnol. Appl. Biochem. 5, 31). Additionally, any commercially available lactose today is derived from whey, a waste product of the dairy industry. Whey is produced in enormous quantities in the manufacture of cheese and casein. Thus, because it comes from the dairy industry, concerns remain regarding potential contamination of lactose with prion proteins, the causative agent of bovine spongiform encephalopathy (BSE), also known as mad cow disease. BSE is a fatal neurodegenerative disease in cattle, causing spongy degeneration of the brain and spinal cord. BSE can be transmitted to humans, where it is known as variant Creutzfeldt-Jakob disease. Above all, lactose remains one of the most expensive components of the fermentation medium, and replacing it with glucose, glycerol, sucrose, etc. would lead to a more cost-effective production of HMOs. To overcome the aforementioned drawbacks, improved means and methods for HMO production were developed. For example, WO 2015 / 150328 A1 discloses bacterial host cells capable of producing oligosaccharides comprising a terminal galactose-(1^4)-glucose disaccharide, wherein said bacterial host cell expresses at least one recombinant nucleic acid sequence encoding a p-1,4-galactosyltransferase capable of galactosylating a free glucose monosaccharide to generate lactose intracellularly, and containing and expressing at least one recombinant nucleic acid sequence encoding a fucosyltransferase, a sialyltransferase, a glucosamyltransferase, or a galactosyltransferase.This bacterial host cell is capable of generating the oligosaccharide without the exogenous addition of lactose, meaning that the bacterial host cell can be cultured in a culture medium without exogenous lactose to produce said oligosaccharide. More specifically, WO 2015 / 150328 A1 discloses a genetically modified strain of E. coli for the production of 2'-fucosillactose using sucrose or a combination of glucose and sucrose as a carbon source. For the use of sucrose, this E. coli strain was genetically modified to express the four genes of the E. coli W csc gene group, namely the genes encoding sucrose permease (cscB), fructokinase (cscK), sucrose hydrolase (cscA), and a transcriptional repressor (cscR). However, producing 2'-FL using this genetically modified E. coli strain that uses sucrose as the sole carbon and energy source has its drawbacks because it is also difficult to heat-sterilize sucrose without a considerable degree of hydrolysis and the formation of unwanted byproducts. Sterile filtration of the sucrose solution can be used as an alternative, but sterile filtration carries a high risk of contamination of the fermentation by extraneous growths, particularly in industrial-scale fermentation. Furthermore, culturing a microbial cell for the production of an HMO in the presence of sucrose as a carbon source, wherein said microbial cell has been genetically modified to possess a split metabolism so that the monomers that constitute sucrose are used in different metabolic pathways, leads to undesirable growth characteristics of the bacterial cell culture, presumably due to the stoichiometry of the monomers generated from intracellular sucrose hydrolysis not matching the quantitative requirements of the different monomers in the different pathways. To overcome the aforementioned drawbacks, a genetically modified microbial cell is provided that is capable of producing an oligosaccharide of interest comprising a galactose-p1,4-glucose structural unit at its reducing end when grown on a mixed monosaccharide feedstock as the main source of carbon and energy, but in the absence of exogenously added lactose. Summary The invention is set forth in the attached set of claims. Brief description of the drawings Figure 1 shows a schematic drawing of an example embodiment of a genetically modified microbial cell according to the invention for the production of 2'-fucosillactose. Figure 2 shows a schematic drawing of another example of embodiment of a genetically modified microbial cell according to the invention for the production of 2'-fucosillactose. Figure 3 shows a schematic drawing of another example of embodiment of a genetically modified microbial cell according to the invention for the production of 2'-fucosillactose. Figure 4 shows the growth characteristics of E. coli strains during cultivation on glucose (A) or a mixed monosaccharide feedstock consisting of glucose and fructose (B) as the sole source of carbon and energy. Detailed description A method for producing lactose or an oligosaccharide of interest is provided, comprising a galactose-p1, 4-glucose structural unit at its reducing end, the method comprising the steps of: a) provide a genetically modified microbial cell as described in this document; b) cultivating the microbial cell in a culture medium and under conditions that are permissive for the production of said lactose or oligosaccharide of interest, wherein the culture medium contains a mixture of glucose and at least one additional compound selected from the group consisting of fructose, galactose, mannose, xylose, rhamnose, glycerol, succinate, pyruvate and malate as the main carbon source; and c) recover the lactose or oligosaccharide of interest from the culture medium and / or the microbial cell. The genetically modified microbial cell for the production of lactose or an oligosaccharide of interest comprising a galactose-p1,4-glucose structural unit at its reducing end possesses at least one glucose transporter for translocating glucose from the culture medium to the cytoplasm of the microbial cell, a UDP-galactose biosynthesis pathway for the intracellular biosynthesis of UDP-galactose, and at least one galactosyltransferase that is capable of galactosylating free intracellular glucose to produce lactose intracellularly. The genetically modified microbial cell is capable of producing lactose. In certain embodiments, the microbial cell can use the lactose it produces to manufacture an oligosaccharide of interest that carries a galactose-p1,4-glucose structural unit at its reducing end. The production of this oligosaccharide of interest does not require an exogenous supply of lactose to the microbial cell. The genetically modified microbial cell possesses at least one glucose transporter to translocate glucose from the culture medium in which the microbial cell is grown to the cytoplasm of the microbial cell so that free glucose is available for intracellular lactose biosynthesis. Typically, the genetically modified microbial cell comprises at least one functional gene that encodes such a glucose transporter that is capable of translocating glucose (Glu) from the culture medium to the cell's cytoplasm. The term "functional gene," as used herein, refers to a nucleic acid molecule comprising a nucleotide sequence encoding a protein or polypeptide, and also containing regulatory sequences operatively linked to that protein-coding nucleotide sequence such that the protein- or polypeptide-coding nucleotide sequence can be expressed in / by the microbial cell bearing that functional gene. Thus, when cultured under conditions permissive to the expression of the functional gene, that functional gene is expressed, and the microbial cell expressing that functional gene typically comprises the protein or polypeptide encoded by the protein-coding region of the functional gene.As used in this document, the terms "nucleic acid" and "polynucleotide" refer to a deoxyribonucleotide or ribonucleotide polymer in either single-stranded or double-stranded form and, unless otherwise limited, encompass known analogues of naturally occurring nucleotides that hybridize with nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise stated, a particular nucleic acid sequence includes its complementary sequence. The term "operatively linked" as used herein means a functional link between a nucleic acid expression control sequence (such as a promoter, signal sequence, or series of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence affects the transcription and / or translation of the nucleic acid corresponding to the second sequence. Accordingly, the term "Promoter" designates DNA sequences that typically "precede" a gene in a DNA polymer and provide a site for the initiation of mRNA transcription. "Regulatory" DNA sequences, which are also typically "upstream" of (i.e., before) a gene in a given DNA polymer, bind to proteins that determine the frequency (or rate) of transcriptional initiation.Collectively called "promoter / regulatory" or "control" DNA sequences, these sequences that precede a selected gene (or set of genes) in a functional DNA polymer cooperate to determine whether transcription (and eventual expression) of a gene will occur. DNA sequences that follow a gene in a DNA polymer and provide a signal for the termination of mRNA transcription are called transcription "terminator" sequences. The term "recombinant," as used herein with reference to a bacterial host cell, indicates that the bacterial cell replicates a heterologous nucleic acid or expresses a peptide or protein encoded by a heterologous nucleic acid (i.e., a sequence "foreign to that cell"). Recombinant cells may contain genes not found in the cell's native (non-recombinant) form. Recombinant cells may also contain genes found in the cell's native form, in which the genes are modified and reintroduced into the cell by artificial means. The term also encompasses cells containing an endogenous nucleic acid that has been modified without removing the nucleic acid from the cell; such modifications include those achieved through gene replacement, site-specific mutation, and related techniques.Accordingly, a "recombinant polypeptide" is one that has been produced by a recombinant cell. A "heterologous sequence" or "heterologous nucleic acid," as used herein, is one that originates from a source foreign to the particular host cell (e.g., from a different species) or, if it comes from the same source, is modified from its original form. Thus, a heterologous nucleic acid operatively linked to a promoter comes from a different source than the one from which the promoter is derived, or, if it comes from the same source, is modified from its original form. The heterologous sequence can be stably introduced, for example, by transfection, transformation, conjugation, or transduction, into the genome of the host cell, with the techniques used depending on the host cell into which the sequence is to be introduced.A person skilled in the technique knows several techniques and they are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory and Manual, 2nd Ed., Cold Spring Harbor Laboratory and Press, Cold Spring Harbor, NY (1989). According to the above, a "genetically modified microbial cell" is understood to be a bacterial cell that has been transformed or transfected, or is capable of being transformed or transfected by an exogenous polynucleotide sequence. Thus, the nucleic acid sequences as used in the present invention may be comprised, for example, in a vector that is to be stably transformed / transfected or otherwise introduced into host microorganism cells. A wide variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, but are not limited to, chromosomal, episomal, and virus-derived vectors, for example, vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression system constructs may contain control regions that regulate, in addition to generating, expression. Generally, any system or vector suitable for maintaining, propagating, or expressing polynucleotides and synthesizing a polypeptide 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 routine and well-known techniques, such as, for example, those set out in Sambrook et al., supra. The technique is well-documented in patents and related literature concerning recombinant DNA methodologies for the isolation, synthesis, purification, and amplification of genetic materials for use in the transformation of selected host organisms. Thus, it is common knowledge to transform host organisms with viral plasmid DNA or circular "hybrid" DNA that includes selected exogenous (i.e., foreign or "heterologous") DNA sequences. Known procedures in the technique first involve generating a transformation vector by enzymatically cleaving viral or circular plasmid DNA to form linear DNA strands. The selected foreign DNA strands, which typically include sequences encoding the desired protein product, are then prepared in linear form using identical or similar enzymes.The linear viral or plasmid DNA is incubated with the foreign DNA in the presence of ligating enzymes capable of performing a restoration procedure and "hybrid" vectors are formed that include the selected exogenous DNA segment "spliced" into the viral or circular DNA plasmid. The term "nucleotide sequence that codes for..." generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA, and generally represents the portion of a gene that codes for a particular polypeptide or protein. The term includes, without limitation, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions or single-stranded, double-stranded, and triple-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded regions or, more generally, double-stranded or triple-stranded regions, or a mixture of single-stranded and double-stranded regions.The term also encompasses polynucleotides that include a single continuous region or discontinuous regions that encode the polypeptide (e.g., interrupted by an integrated phage or an insertion or editing sequence) along with additional regions that may also contain coding and / or non-coding sequences. At least one glucose transporter is selected from the group consisting of glucose-facilitated diffusion proteins and glucose translocating permeases. A suitable glucose-facilitated diffusion protein is encoded by the glf gene of Zymomonas mobilis subsp. mobilis (ATCC 31821 / ZM4 / CP4 strain). A suitable glucose translocating permease is encoded by the galp gene of E. coli K-12. The glucose translocating permease is also known as a galactose-proton symporter or galactose permease, but it also imports glucose across the cell membrane. Thus, in a further and / or alternative embodiment, the genetically modified microbial cell comprises and expresses at least one gene comprising the protein-coding region of the glf gene of Zymomonas mobilis subsp. mobilis (ATCC 31821 / ZM4 / CP4 strain), the galp gene of E. coli K-12, or functional variants thereof. The term "variant(s)," as used herein, refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains the essential (enzymatic) properties of the reference polynucleotide or polypeptide. A typical polynucleotide variant differs in its nucleotide sequence from another reference polynucleotide. Changes in the variant's nucleotide sequence may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in substitutions, additions, deletions, fusions, and truncations of amino acids in the polypeptide encoded by the reference sequence, as discussed later. A typical polypeptide variant differs in its amino acid sequence from another reference polypeptide.Generally, the differences are limited, so the sequences of the reference polypeptide and the variant are very similar overall and, in many regions, identical. A variant and a reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination. A substituted or inserted amino acid residue may or may not be encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring allelic variant, or it may be a variant not known to occur naturally. Non-natural 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. Within the scope of the present invention are also included polymorphic nucleic acid / polynucleotide and polypeptide variants, alleles, mutants, and cross-species homologs, having an amino acid sequence having an amino acid sequence identity greater than approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater amino acid sequence identity, preferably over a region of at least approximately 25, 50, 100, 200, 500, 1000 or more amino acids, to a polypeptide encoded by a wild-type protein. According to the above, a "functional variant" of any of the genes / proteins disclosed herein is intended to designate sequence variants of the genes / proteins that still retain the same or somewhat less activity of the gene or protein from which the respective fragment is derived. The genetically modified microbial cell possesses a UDP-galactose biosynthesis pathway for the intracellular formation of GDP-galactose (GDP-Gal), because an efficient supply of UDP-galactose is needed for the intracellular biosynthesis of lactose. In a further and / or alternative embodiment, UDP-galactose can be obtained from the metabolism of microbial cells themselves, i.e., from the activity of a phosphoglucomutase, a UTP-glucose-1-phosphate-uridyltransferase, and a UDP-glucose-4-epimerase. The intracellular supply of GDP-galactose can be enhanced by genetic modifications such as the expression or overexpression of one or more of the genes encoding polypeptides that exhibit phosphoglucomutase activity, UDP-glucose-1-phosphate-uridyltransferase activity, and UDP-glucose-4-epimerase activity, respectively. The term "overexpression" or "overexpressed" as used herein refers to an enzyme or polypeptide expression level that is higher than that measured in a wild-type cell of the same species as the host cell that has not been genetically altered. Phosphoglucomutase is an enzyme that facilitates the interconversion of glucose-1-phosphate to glucose-6-phosphate in an α-D-glucose monomer from the 1' to the 6' position or from the 6' to the 1' position. An example gene encoding a suitable phosphoglucomutase is the pgm gene of E. coli K-12 (GenBank: U08369.1). Thus, in a further and / or alternative embodiment, the genetically modified microbial cell comprises and expresses / overexpresses a gene encoding a phosphoglucomutase, the gene preferably comprising the protein-coding region of the pgm gene of E. coli or a variant thereof. UTP-glucose-1-phosphate uridyltransferase, such as GalU or a functional variant thereof, catalyzes the conversion of α-D-glucose-1-phosphate to UDP-glucose using UTP. An example gene encoding a suitable UTP-glucose-1-phosphate uridyltransferase is the galU gene of E. coli K-12 (GenBank: M98830.1). Thus, in a further and / or alternative embodiment, the genetically modified microbial cell comprises and expresses / overexpresses a gene encoding a UTP-glucose-1-phosphate uridyltransferase, the gene preferably comprising the protein-coding region of the galU gene of E. coli or a variant thereof. UDP-glucose-4-epimerase, such as GalE or a functional variant thereof, catalyzes the epimerization of UDP-glucose to UDP-galactose. An example gene encoding a UDP-glucose-4-epimerase is the galE gene of E. coli K-12. Thus, in a further and / or alternative embodiment, the genetically modified microbial cell comprises and expresses / overexpresses a gene encoding a UDP-glucose-4-epimerase, the gene preferably comprising the protein-coding region of the galE gene of E. coli or a variant thereof. In a further and / or alternative embodiment, the UDP-galactose biosynthesis pathway further comprises the enzymatic activity of a glucose-6-phosphate isomerase that converts fructose-6-phosphate to glucose-6-phosphate and vice versa. An example gene encoding a glucose-6-phosphate isomerase is the pgi gene of E. coli K-12. Thus, in a further and / or alternative embodiment, the genetically modified microbial cell comprises and expresses / overexpresses a gene encoding a glucose-6-phosphate isomerase, the gene preferably comprising the protein-coding region of the pgi gene of E. coli or a variant thereof. Alternatively, UDP-galactose can be obtained by feeding microbial cells galactose through the culture medium. The galactose is absorbed by the cell and phosphorylated to galactose-1-phosphate, which is then converted to UDP-galactose. The genes encoding the enzymes with the required enzymatic activities are known in the literature (Groissoird et al., "Characterization, Expression, and Mutation of the Lactococcus lactis galPMKTE Genes, Involved in Galactose Utilization via the Leloir Pathway (2003) J. Bacteriol. 185 (3) 870-878). The genetically modified microbial cell comprises a p-1,4-galactosyltransferase capable of galactosylating free glucose monosaccharide. In a further and / or alternative embodiment, a suitable p-1,4-galactosyltransferase is derived from Neisseria menningitidis, Aggregatibacter aphrophilus, or Pasteurella multocida, preferably a p-1,4-galactosyltransferase encoded by the IgtB gene of Neisseria menningitidis, the lex-1 gene of Aggregatibacter aphrophilus, or the galTpm1141 gene of Pasteurella multocida (GenBank: AEC04686).Thus, in a further and / or alternative embodiment, the genetically modified microbial cell comprises and expresses / overexpresses a gene encoding a p-1,4-galactosyltransferase, the gene preferably comprising the protein-coding region of the IgtB gene of Neisseria menningitidis, the lex-1 gene of Aggregatibacter aphrophilus, the galTpm1141 gene of Pasteurella multocida, or a variant thereof. p-1,4-galactosyltransferase uses UDP-galactose as a substrate for the transfer of the galactose structural unit to the free glucose monosaccharide, thus synthesizing a galactose-p1,4-glucose disaccharide, i.e., lactose. In a further and / or alternative embodiment, the genetically modified microbial cell comprises at least one additional glycosyltransferase, i.e., in addition to said p-1,4-galactosyltransferase. Generally, and throughout this disclosure, the term "glycosyltransferase activity" or "glycosyltransferase" designates and encompasses enzymes that are responsible for the biosynthesis of disaccharides, oligosaccharides, and polysaccharides, and catalyze the transfer of monosaccharide structural units from an activated monosaccharide / nucleotide sugar (the "glycosyl donor") to a glycosyl acceptor molecule. In a preferred embodiment, the at least one additional glycosyltransferase is a fucosyltransferase, a sialyltransferase, a glucosamineltransferase or a galactosyltransferase; more preferably, the at least one additional glycosyltransferase is selected from at least one of the following: alpha-1,2-fucosyltransferase, alpha-1,3-fucosyltransferase, beta-1,3-N-acetylglucosamyltransferase, beta-1,3-galactosyltransferase, alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase, beta-1,4-galactosyltransferase or beta-1,6-galactosyltransferase. The enzymatic activity of at least one additional glycosyltransferase allows the production of oligosaccharides of interest comprising a galactose-p-1,4-glucose structural unit at their reducing end, using lactose as an acceptor for the activity of the additional glycosyltransferase. Table 1 identifies the most abundant HMOs that can be produced by microbial cells and the methods disclosed herein as oligosaccharides of interest. Table 1: List of oligosaccharides of interest that can be produced using a genetically modified microbial cell and / or a method as described in this document. In a further and / or alternative embodiment, the microbial cell comprises a glucose translocating phosphotransferase (PtsG) system. The glucose translocating phosphotransferase system catalyzes the phosphorylation of incoming glucose simultaneously with its translocation across the cell membrane. The general mechanism of the Pts system is as follows: a phosphoryl group from phosphoenolpyruvate (PEP) is transferred, via a signal transduction pathway, to enzyme I (EI), which in turn transfers it to a phosphoryl carrier, the histidine protein (HPr). Phospho-HPr then transfers the phosphoryl group to a sugar-specific permease, a membrane-bound complex known as enzyme 2 (Ell), which transports the sugar into the cell. Ell consists of at least three structurally distinct domains: IIA, IIB, and IIC. These can either fuse into a single polypeptide chain or exist as two or three interacting chains, formerly called enzymes II (EII) and III (EIII). The first domain (IIA or EIIA) carries the permease's first specific phosphorylation site, a histidine phosphorylated by phospho-HPr. The second domain (IIB or EllB) is phosphorylated by phospho-IIA at a cysteinyl or histidyl residue, depending on the transported sugar. Finally, the phosphoryl group is transferred from domain IIB to the sugar substrate concurrently with the sugar uptake processed by domain IIC. This third domain (IIC or ElIC) forms the translocation channel and the specific substrate binding site. In this way, the PtsG system acquires exogenous glucose and provides glucose-6-phosphate to the microbial cell. Glucose-6-phosphate can be used in the UDP-galactose biosynthesis pathway and / or converted to fructose-6-phosphate, which in turn can be used to generate energy-rich triphosphates in central metabolism and / or, for example, in the biosynthesis of nucleotide-activated saccharides such as GDP-fucose. In a further and / or alternative embodiment, the glucokinase genes of the microbial cell have been deleted or functionally inactivated so that the microbial cell does not possess any polypeptide with glucokinase activity. Glucokinase (Glk) b phosphorylates free glucose at its carbon 6 atom to generate glucose phosphate. In the absence of glucokinase activity, the free glucose that translocates to the cytoplasm of the microbial cell becomes available as a substrate for p1,4-galactosyltransferase to produce lactose, while the glucose-6-phosphate obtained from PtsG activity can be used for the formation of UDP-galactose or other metabolic pathways. In a further and / or alternative embodiment, the genetically modified microbial cell comprises a fructose transporter for translocating fructose (Fru) from the culture medium to the cytoplasm of the microbial cell. A fructose transporter suitable for the uptake of free fructose is an isoform (PtsG-F) as described by Kornberg et al. PNAS 97: 1808-1812 (2000). Internalized fructose can then be phosphorylated by fructokinase (FrK) to provide fructose-6-phosphate (Fru-6-P). Fructose-6-phosphate can be used in the UDP-galactose biosynthesis pathway and / or in other metabolic pathways such as the generation of energy-rich triphosphates in central metabolism and / or, for example, in the biosynthesis of nucleotide-activated saccharides such as GDP-fucose. In a further and / or alternative embodiment, the genetically modified microbial cell comprises polypeptides exhibiting fructokinase-6 activity and polypeptides exhibiting 6-phosphofructokinase-1 activity (FruK or phosphofructokinase) to provide a metabolic pathway from internalized fructose through fructose-6-phosphate to fructose-1,6-bisphosphate. In a further and / or alternative embodiment, the genetically modified microbial cell comprises a fructose translocating phosphotransferase (PtsF) system. The fructose translocating phosphotransferase system catalyzes the phosphorylation of incoming fructose simultaneously with its translocation across the cell membrane. In this way, the PtsF system acquires exogenous fructose and provides fructose-1-phosphate to the microbial cell. The PtsF system comprises a membrane-spanning protein, FruA, a 1-phosphofructokinase (FruK), and a diphosphoryl transfer protein, FruB. Fruose is translocated by FruA and FruB to provide fructose-1-phosphate in the cytoplasm. Fructose-1-phosphate can be further phosphorylated by phosphofructokinase (FruK) to produce fructose-1,6-bisphosphate, which can then be used by the microbial cell to generate energy-rich triphosphates in central metabolism. Another appropriate PtsF system comprises LevD, LevE, LevF, and LevG. LevD is the component of the fructose-specific phosphotransferase IIA enzyme. LevE is the component of the fructose-specific phosphotransferase IIB enzyme. LevF is the IIC component of fructose permease, and LevG is the IID component of fructose permease. The corresponding genes levD, levE, levF, and levG are known, for example, from Bacillus subtilis (strain 168). This PtsF system supplies fructose-1-phosphate to the cell. In a further and / or alternative embodiment, the genetically modified microbial cell comprises at least one 1-phosphofructokinase (FruK). In a further and / or alternative embodiment, the genetically modified microbial cell comprises a fructose-1,6-bisphosphatase (GIpX). This fructose-1,6-bisphosphatase dephosphorylates fructose-1,6-bisphosphate to yield fructose-6-phosphate. The fructose-6-phosphate can be used by the microbial cell in the GDP-galactose biosynthesis pathway or in another metabolic pathway, for example, in the biosynthesis of nucleotide-activated saccharides such as GDP-fucose. Preferably, the microbial cell also comprises a deletion or functional inactivation of its phosphofructokinase gene(s). The deletion or functional inactivation of the phosphofructokinase gene(s) leads to a microbial cell that lacks phosphofructokinase activity, thus preventing the conversion of Fru-6-P to Fru-I, 6-bisP. In E. coli, two phosphofructokinase isoforms are present, designated PfkA and PfkB. The corresponding genes are pfkA and pfkB. In a further and / or alternative embodiment, the genetically modified microbial cell possesses a GDP-L-fucose biosynthesis pathway. In a further and / or alternative embodiment, the GDP-L-fucose biosynthesis pathway comprises a mannose-6-phosphate isomerase (ManA), a phosphomannomutase (ManB), a mannose-1-phosphateguanylyltransferase (ManC), a GDP-mannose-4,6-dehydratase (Gmd), and a GDP-L-fucose synthase (WcaG). Preferably, the microbial cell possessing a GDP-L-fucose biosynthesis pathway also possesses a fucosyltransferase. In a further and / or alternative embodiment, the microbial cell comprises an exporter protein or permease that exports the oligosaccharide of interest from the cell, preferably a sugar efflux transporter. In a further and / or alternative embodiment, the genetically modified microbial cell comprises a functional deletion or inactivation of its glucose-6-phosphate isomerase gene, such that the microbial cell lacks glucose-6-phosphate isomerase activity. Glucose-6-phosphate isomerase, designated Pgi in E. coli, converts glucose-6-phosphate to fructose-6-phosphate. By deleting the glucose-6-phosphate gene(s) or by inactivating their expression, any glucose-6-phosphate present in the cytoplasm of the microbial cell can be directed toward lactose production. In an additional and / or alternative embodiment, the microbial cell is a bacterial cell selected from the group comprising bacteria of the genus Escherichia, Lactobacillus, Cor y nebacterium, Bacillus, Streptococcus, Enterococcus, Lactococcus and Clostidium, preferably a bacterial cell selected from the group of bacterial species consisting of Escherichia coli, Cor y nebacterium glutamicum, Clotridium cellulolyticum, Clostridium Ijungdahlii, Clostridium autoethanogenum, Clostridium acetobutylicum, Bacillus subtilis, Bacillus megaterium, Lactobacillus, Lactobacillus, actiophilus Lactobacillus helveticus, Lactobacillus delbrueckii, and Lactococcus lactis. In another embodiment, the microbial cell is an Escherichia coli. One skilled in the art will become familiar with other bacterial strains upon reading the present disclosure. In a further and / or alternative embodiment, the oligosaccharide of interest is a human milk oligosaccharide selected from the group consisting of 2'-fucosillactose, 3-fucosillactose, 2', 3-difucosillactose, 3-sialillactose, 6'-sialillactose, 3-fucosyl-3'-sialillactose, lacto-W-tetraose, lacto-A / -neotetraose, lacto-A / -fucopentaose I, lacto-W-fucopentaose II, lacto-W-fucopentaose III, lacto-W-fucopentaose V, lacto-W-difucosylhexose I, lacto-W-difucosylhexaose II, lacto-W-sialpentaose LSTa, LSTb, LSTc. Preferably, the mixture of glucose and at least one additional monosaccharide is a mixed raw material of glucose and fructose, preferably obtained by hydrolysis of sucrose. In a further and / or alternative embodiment, the microbial cell is cultured without exogenous supply of lactose, particularly when it is cultured for the production of the oligosaccharide of interest. The present invention will be described with regard to particular embodiments and with reference to the drawings, but the invention is not limited to them but only to the claims. Additionally, the terms "first," "second," and "similar" in the description and in the claims are used to distinguish between similar elements and not necessarily to describe a sequence, whether temporal, spatial, in classification, or otherwise. It should be understood that the terms so used are interchangeable in appropriate circumstances and that the embodiments of the invention described herein are capable of functioning in sequences other than those described or illustrated herein. It should be noted that the term "comprising," used in the claims, should not be interpreted as restricted to the means listed below; it does not exclude other elements or steps. Thus, it should be interpreted as specifying the presence of the indicated features, integers, steps, or components to which reference is made, but not excluding the presence or addition of one or more features, integers, steps, or components, or groups thereof. Therefore, the scope of the expression "a device comprising means A and B" should not be limited to devices consisting solely of components A and B. It means that, with respect to the present invention, the only relevant components of the device are A and B. In one embodiment, an E. coli strain possessing the genotype lacY-, lacZ-, fuclK-, wcaJ- is metabolically engineered to efficiently produce 2'-fucosyllactose via total fermentation using a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) as the primary carbon and energy source. Therefore, the expression of the glucokinase gene glk and / or the glucose dehydrogenase gene gcd and / or the glucose permease gene ptsG is reduced and / or suppressed. Furthermore, a glucose permease gene is expressed or overexpressed in this E. coli strain. Additionally, at least one of the E. coli genes manA, manC, manB, gmd, wcaG, pgm, galU and galE, as well as the expression of a heterologous p-1,4-galactosyltransferase, capable of transferring galactose from UDP-galactose to glucose, thereby generating lactose, and an a-1,2-fucosyltransferase, capable of transferring fucose from GDP-fucose to lactose, thereby generating 2'-fucosylactose, are expressed / overexpressed. In a preferred embodiment, this production strain is further modified by reducing and / or decreasing the expression of the phosphofructokinase genes pfkA and / or pfkB and / or the glucose-6-phosphate dehydrogenase gene zwf and / or the glucose-6-phosphate isomerase gene pgi. This further genetic modification allows the cultivation of the thus engineered production strain on a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) as the main source of carbon and energy, while avoiding hindering the strain's metabolism but increasing the supply of precursors (glucose and fructose-6-phosphate and glucose-6-phosphate) for the production of 2'-fucosyllactose by total fermentation. With reference to Figure 1, an example microbial cell of the invention is shown schematically. This microbial cell is capable of producing 2'-FL when cultured on a mixed raw material consisting of glucose (Glu) and fructose (Fru), but this mixed raw material does not contain lactose (Lac). The microbial cell expresses polynucleotides encoding a glucose transporter (Glf) and a fructose transporter for the import of glucose and fructose into the cell, respectively. Since the expression of the glucose kinase Glk has been abolished by the deletion or functional inactivation of the glk gene(s), any glucose imported by the cell becomes available as a substrate for the p1,4-galactosyltransferase GalTpm1141 via the UDP-galactose biosynthesis pathway to generate lactose (Lac) intracellularly. The cell's fructose-6-kinase phosphorylates imported fructose to generate an intracellular pool of Fru-6-P. Part of this Fru-6-P pool is used in the UDP-Gal biosynthesis pathway to synthesize UDP-Gal intracellularly, which serves as a galactose donor for the galactosyltransferase GalTpm1141 to generate lactose. Another part of the Fru-6-P pool is used in the GDP-L-Fuc biosynthesis pathway for the production of GDP-L-fucose. This GDP-L-fucose serves as a fucose donor for the 2'-fucosyltransferase WbgL. However, a third of the intracellular Fru-6-P reserve is used for energy and biomass production, as its fru-6-P is converted into Fru-1, 6-bisP by the cellular phosphofructokinases PfkA and / or PfkB. Figure 2 schematically shows another example microbial cell of the invention that is capable of producing 2'-FL when grown on a mixed feedstock consisting of glucose (Glu) and fructose (Fru), but said mixed feedstock does not contain lactose (Lac). In addition to the example microbial cell shown in Figure 1, the microbial cell further comprises a glucose-specific Pts system (PtsG). Said PtsG system imports and phosphorylates Glu to provide Glu-6-P in the cell cytosol. Said Glu-6-P can be used by the microbial cell to generate UDP-Gal or Fru-6-P. In a variant of the microbial cell (not shown), the pgi gene(s) encoding a glucose-6-phosphate isomerase (Pgi) are deleted from the cell.Along with the deletion of the glk gene, the microbial has been genetically modified so that the free glucose monomer acquired by Glf is available as a substrate for p1,4-galactosyltransferase, while any Glu-6-P acquired by PtsG is available for UDP-Gal biosynthesis. Figure 3 schematically shows another exemplary microbial cell of the invention capable of producing 2'-FL when grown on a mixed feedstock consisting of glucose (Glu) and fructose (Fru), but said mixed feedstock does not contain lactose (Lac). In addition to the exemplary microbial cell shown in Figure 2, the microbial cell further comprises a fructose-specific Pts system (PtsF). This PtsF system imports and phosphorylates Fru to provide Fru-1-P. Fru-1-P is phosphorylated by FruK to provide Fru-1,6-bisP. The microbial cell possesses fructose-1,6-bisphosphatase activity (GlpX). Thus, the genetically modified microbial cell is metabolically modified so that the cell can use fructose and / or fructose-1-P for the biosynthesis of UDP-Gal. Furthermore, the phosphofructokinase gene(s) are deleted or functionally inactivated so that the cell lacks phosphofructokinase activity (PfkA / PfkB). Deletion or functional inactivation of the phosphofructokinase genes impairs the conversion of Fru-6P to Fru-1,6-P, thus preventing the use of Fru-6-P to generate energy-rich triphosphates and enhancing the production of 2'-FL. Examples Example 1 - Preparation of a mixed monosaccharide raw material A 50% (w / v) sucrose solution was prepared by dissolving 500 g of sucrose in water. The final volume of the solution was 1 liter. At a temperature of 30–35 °C, the pH was adjusted using 96% (v / v) sulfuric acid. Subsequently, the solution was sterilized in a vertical autoclave (Systec VX-65, Linden, Germany) at 121 °C for 45 minutes. Samples were taken before and after thermal sterilization and kept frozen until analysis by high-performance liquid chromatography (HPLC). HPLC was performed using a RID-10A refractive index detector (Shimadzu, Germany) and a 3.5 pm (250 × 4.6 mm) Waters XBridge amide column (Eschborn, Germany) connected to a Shimadzu HPLC system. Isocratic elution was carried out with 30% solvent A (50% (v / v) acetonitrile in double-distilled water, 0.1% (v / v) NH4OH) and 70% solvent B (80% (v / v) acetonitrile in double-distilled water, 0.1% (v / v) NH4OH) at 35 °C and a flow rate of 1.4 mL min⁻¹. The samples were clarified by solid-phase extraction on an ion-exchange matrix (Strata ABW, Phenomenex). Ten microliters of the sample (1:5 dilution) were applied to the column. Finally, the relative amount of sugars detected was determined. As shown in Table 1, the conversion of sucrose to the monosaccharides glucose and fructose increased with decreasing pH values ​​of the solutions before heat treatment. Complete cleavage of sucrose was observed at pH values ​​< 3.50 when acidification was carried out with sulfuric acid. Table 1: Relative amount of sugars detected in a 50% (w / v) sucrose solution with adjusted pH before and after thermal sterilization. The pH adjustment was performed using 96% (v / v) sulfuric acid. The percentage of sugars (area under the curve; AUC) detected by HPLC is shown. Example 2: Raw material-dependent growth of various gene deletion strains The growth behavior of an E. coli BL21 (DE3) (wild type) strain was compared with that of the mutated strains E. coli pfkA- (ApfkA), E. coli pfkB- (ApfkB), and E. coli pfkA- pfkB- (ApfkA ApfkA). Genomic deletions were performed according to the method of Datsenko and Wanner (Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)). All strains were cultured at 30 °C in 100 mL flasks with shaking and 20 mL of mineral salt medium containing 7 g L⁻¹ of NH₄H₂PO₄, 7 g L⁻¹ of K₂HPO₄, 2 g L⁻¹ of KOH, 0.3 g L⁻¹ of citric acid, 2 g L⁻¹ of MgSO₄ * 7 H₂O and 0.015 g L⁻¹ of CaCh₄ * 6 H₂O, supplemented with 1 mL L⁻¹ of trace element solution (54.4 g L⁻¹ of ferric ammonium citrate, 9.8 g L⁻¹ of MnCh₄ * 4 H₂O, 1.6 g L⁻¹ of CoCh₄ * 6 H₂O, 1 g L⁻¹ of CuCh₄ * 2 H₂O, 1.9 g L⁻¹ of H₃BO₃, 9 g L⁻¹ of ZnSO₄ * 7 H2O, 1.1 g L-1 of Na2MoO4 * 2 H2O, 1.5 g L-1 of Na2SeO3, 1.5 g L-1 of NiSO4 * 6 H2O) and containing either 2% (w / v) glucose (A) or 1% (w / v) glucose / 1% (w / v) fructose (B) as the carbon source. Cultures were inoculated to OD 0.1 and growth development was monitored for 26 hours by measuring OD600. As shown in Figure 2, E. coli pfkA- pfkB- showed little growth when glucose was provided as the sole carbon and energy source, whereas its growth was indistinguishable from the wild-type strain as well as from single-deletion mutants when the mixed monosaccharide feedstock was available. Example 3 - Total fermentation of 2'-fucosyllactose by an engineered E. coli strain during growth on a mixed monosaccharide feedstock A strain of E. coli BL21 (DE3) exhibiting the genotype. pfkA-, lacZ-, fuclK-, wcaJ-, glk-, gcd-, ptosG-, and were also genetically modified by overexpression of enzymes for de novo GDP-fucose synthesis (ManB, ManC, Gmd, WcaG), the 2'-fucosyltransferase gene wbgL from E. coli:O126, the sugar efflux transporter gene yberc0001_9420 from Yersinia bercovieri ATCC 43970, the glucose facilitator gene glf from Zymomonas mobilis, the p-1,4-galactosyltransferase gene galTpm1141 from Pasteurella multocida (GenBank: AEC04686), as well as the galE and pgm genes from E. coli, which encode a UDP-glucose 4-epimerase and a phosphoglucomutase, respectively. Genomic deletions were performed according to the method of Datsenko and Wanner (Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)). Genomic integration of heterologous genes was performed by transposition. The EZ-Tn5TM transposase (Epicentre, USA) was used.The C9 hyperactive mutant of the mariner transposase Himar1 (Proc. Natl. Acad. Sci. 1999, USA 96:11428-11433) was used for transposition to integrate linear DNA fragments. The genes were codon-optimized for expression in E. coli and synthetically prepared using GenScript. The resulting E. coli strain was cultured at 30 °C in a 3 L fermenter (New Brunswick, Edison, USA) starting with 1000 mL of mineral salts medium containing 7 g L-1 of NH4H2PO4, 7 g L-1 of K2HPO4, 2 g L-1 of KOH, 0.3 g L-1 of citric acid, 2 g L-1 of MgSO4 x 7 H2O and 0.015 g L-1 of CaCh x 6 H2O, supplemented with 1 mL L-1 of trace element solution (54.4 g L-1 of ferric ammonium citrate, 9.8 g L-1 of MnCh x 4 H2O, 1.6 g L-1 of COCl2 x 6 H2O, 1 g L-1 of CuCh x 2 H2O, 1.9 g L-1 of H3BO3, 9 g L-1 of ZnSO4 x 7 H2O, 1.1 g L-1 of Na2MoO4 x 2 H2O, 1.5 g L-1 of Na2SeO3, 1.5 g L-1 of NiSO4 x 6 H2O) and containing 2% (w / v) hydrolyzed sucrose as a carbon source. The culture was initiated by the addition of a 2.5% (v / v) inoculum from a preculture grown in the same medium. The end of the batch phase was characterized by an increase in the dissolved oxygen level.A carbon feed consisting of fully hydrolyzed sucrose, supplemented with 2 g L⁻¹ of MgSO₄ x 7 H₂O, 0.015 g L⁻¹ of CaCh x 6 H₂O, and 1 mL L⁻¹ of trace element solution, was applied immediately after exiting the batch phase. A feed rate of 12.0–15.0 mL L⁻¹ h⁻¹ was applied, based on the initial volume. Aeration was maintained at 3 L min⁻¹. Dissolved oxygen was maintained at 20–30% saturation by controlling the stirring speed. The pH was maintained at 6.7 by adding a 25% ammonia solution. The culture lasted 86 hours and produced substantial amounts of 2'-FL in the culture supernatant.

Claims

1. A method for producing lactose or an oligosaccharide of interest comprising a galactosep1,4-glucose structural unit at its reducing end, the method comprising the steps of: a) providing a genetically modified microbial cell, wherein said microbial cell possesses: - at least one glucose transporter for translocating glucose from the culture medium to the cytoplasm of the microbial cell so that free glucose is available for intracellular lactose biosynthesis, wherein the at least one glucose transporter is selected from the group consisting of glucose-facilitated diffusion proteins and glucose-translocating permeases; - a UDP-galactose biosynthesis pathway; and - at least one p-1,4-galactosyltransferase to galactosylate free glucose to produce lactose intracellularly; b) cultivating the microbial cell in a culture medium and under conditions that are permissive for the production of said lactose or oligosaccharide of interest, wherein the culture medium contains a mixture of glucose and at least one additional compound selected from the group consisting of fructose, galactose, mannose, xylose, rhamnose, glycerol, succinate, pyruvate and malate as the main carbon source; and c) recovering the lactose or oligosaccharide of interest from the culture medium and / or from the microbial cell.

2. The method according to claim 1, wherein the oligosaccharide of interest is a human milk oligosaccharide selected from the group consisting of 2'-fucosillactose, 3-fucosillactose, 2',3-difucosillactose, 3'-sialillactose, 6'-sialillactose, 3-fucosyl-3'-sialillactose, lacto-W-tetraose, lacto-W-neotetraose, lacto-A / -fucopentaose I, lacto-W-fucopentaose II,lacto-W-fucopentaose III, lacto-W-fucopentaose V, lacto-W-difucosylhexose I, lacto-W-difucosylhexaose II, lacto-W-sialylpentaose LSTa, LSTb, LSTc.

3. The method according to claim 1 or 2, wherein the mixture of glucose and at least one additional monosaccharide is a mixed glucose and fructose raw material, preferably obtained by sucrose hydrolysis.

4. The method according to any one of claims 1 to 3, wherein the microbial cell is cultured without exogenous lactose supply.

5. The method according to any one of claims 1 to 4, wherein said microbial cell expresses or overexpresses at least one gene encoding the glucose transporter, preferably at least one gene selected from the group consisting of glf, galP, and functional variants thereof.

6. The method according to any one of claims 1 to 5, wherein said microbial cell possesses a phosphoglucomutase,7. The method according to any one of claims 1 to 6, wherein the p-1,4-galactosyltransferase is encoded by a gene selected from the group consisting of Neisseria meningitidis IgtB, Aggregatibacter aphrophilus lex-1, Pasteurella multocida galTpm1141, and functional variants thereof.

8. The method according to any one of claims 1 to 7, wherein the microbial cell possesses at least one additional glycosyltransferase, preferably a glycosyltransferase selected from the group consisting of fucosyltransferases, sialyltransferases, glucosaminelltransferases, and galactosyltransferases.

9. The method according to any one of claims 1 to 8, wherein the microbial cell comprises a glucose translocating phosphotransferase system.

10. The method according to any one of claims 1 to 9,11. The method according to any one of claims 1 to 10, wherein the microbial cell possesses a fructose-specific phosphotransferase system, and wherein the cell further comprises 1-phosphofructokinase.

12. The method according to claim 10, wherein the microbial cell comprises fructokinase-6 activity and 6-phosphofructokinase-1 activity.

13. The method according to claim 11, wherein the microbial cell comprises a fructose-1,6-bisphosphatase.

14. The method according to claim 11 or 13, wherein the microbial cell comprises a functional deletion or inactivation of its glucose-6-phosphate isomerase.

15. The method according to any one of claims 8 to 14, wherein the additional glycosyltransferase is a fucosyltransferase, and wherein said microbial cell possesses a mannose-6-phosphate isomerase, a phosphomannomutase, a mannose-1-phosphate-guanylyltransferase,a GDP-mannose-4,6-dehydratase, a GDP-L-fucose synthase.

16. The method according to any one of claims 1 to 15, wherein said microbial cell comprises an exporting protein or a permease that exports the oligosaccharide of interest from the cell, preferably a sugar efflux transporter.

17. The method according to any one of claims 1 to 16, wherein said microbial cell is a bacterial cell selected from the group consisting of bacteria of the genera Escherichia, Lactobacillus, Cor y nebacterium, Bacillus, Streptococcus, Enterococcus, Lactococcus and Clostidium, preferably a bacterial cell that is selected from the group of bacterial species consisting of Escherichia coli, Cor y nebacterium glutamicum, Clotridium cellulolyticum, Clostridium Ijungdahlii, Clostridium autoethanogenum, Clostridium acetobutylicum, Bacillus subtilis, Bacillus megaterium, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus helveticus,Lactobacillus delbrueckii, y Lactococcus lactis.,

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