Oligosaccharide production by total fermentation using mixed raw materials

Abstract

Disclosed are genetically engineered microbial cells that produce oligosaccharides containing a galactose-β1,4-glucose moiety at their reducing ends. The microbial cells are capable of producing the oligosaccharides without the addition of exogenous lactose. Also disclosed are methods for producing the oligosaccharides using the microbial cells. [Selected figure] Figure 3

Description

[Technical field]

[0001] The present invention relates to bacterial host cells capable of producing lactose or target oligosaccharides that contain a galactose-β1,4-glucose moiety at their reducing ends, and methods for producing lactose or target oligosaccharides that contain a terminal galactose-β1,4-glucose moiety. [Background technology]

[0002] Human milk consists of a complex mixture of sugars (carbohydrates), fats, proteins, vitamins, minerals and trace elements. The major fraction of human milk consists of carbohydrates. The carbohydrate fraction in human milk is further divided into (i) lactose (milk sugar) and (ii) oligosaccharides (human milk oligosaccharides, HMOs). In infants, lactose (galactose-β1,4-glucose) is used as an energy source, whereas oligosaccharides are not metabolized. The oligosaccharide fraction represents up to one-tenth of the total carbohydrate fraction and is composed of probably more than 150 different oligosaccharides. The presence and concentration of these complex oligosaccharides are unique to humans and therefore cannot be found in significant amounts in the milk of other mammals, including dairy animals.

[0003] The most prominent human milk oligosaccharides are 2'-fucosyllactose and 3'-fucosyllactose, which together may represent up to one-third of the total HMO fraction. Further prominent HMOs present in human milk are lacto-N-tetraose, lacto-N-neotetraose and lacto-N-fucopentaose I. Besides these neutral oligosaccharides, acidic HMOs such as 3'-sialyllactose, 6'-sialyllactose and 3-fucosyl-3'-sialyllactose, sialyl-lacto-N-tetraose and disialyl-lacto-N-tetraose can also be found in human milk. Of note, the majority of HMOs contain a galactose-β1,4-glucose moiety at their reducing end. The structure of HMOs is closely related to Lewis blood group antigens such as Lewis x (LeX), an epitope of epithelial cell surface glycoconjugates. The structural similarity of HMOs to epithelial epitopes explains the protective properties of HMOs against bacterial pathogens.

[0004] The presence of oligosaccharides in human milk has long been known, and the physiological functions of these oligosaccharides have been the subject of medical research for decades. Specific functions have already been identified for some of the most abundant human milk oligosaccharides.

[0005] Besides the local effects in the intestinal tract mentioned above, HMOs have also been shown to exert systemic effects in infants by entering the systemic circulation. Also, their effects on protein-carbohydrate interactions, e.g., selectin-leukocyte binding, can modulate immune responses and suppress inflammatory responses. Moreover, it is increasingly recognized that HMOs are important substrates for the development of the infant microbiota.

[0006] The benefits of prebiotic oligosaccharides, especially HMOs, have been well studied, but due to limited availability from natural sources, efficient commercial, i.e., large-scale, production of HMOs is highly desirable.

[0007] Chemical routes to some of the individual human milk oligosaccharides have been developed with the aim of large-scale production of these oligosaccharides. However, such methods involve the use of several hazardous chemicals, which pose a risk of contaminating the final product. To date, at least, chemical synthesis methods have not been able to provide large-scale quantities, let alone quantities sufficient for food use.

[0008] To avoid the drawbacks associated with chemical synthesis of human milk oligosaccharides, several enzymatic and fermentation methods have been developed for their production. Fermentation production methods have been developed for some HMOs, such as 2'-fucosyllactose, 3'-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, 3'-sialyllactose and 6'-sialyllactose. These production methods typically use genetically engineered bacterial strains, such as recombinant Escherichia coli.

[0009] Today, all fermentation and biocatalytic processes for the production of HMOs are based exclusively on exogenously added lactose as the starting substrate. In these processes, one or more monosaccharides are added to lactose (US Pat. No. 7,521,212 B1; Albermann et al. (2001) Carbohydr. Res. 334(2) p97-103). The addition of monosaccharides to lactose can be catalyzed by glycosyltransferases or glycosidases using appropriate activated monosaccharide substrates. Furthermore, additional monosaccharides can also be added to lactose by transglucosidase reactions.

[0010] In particular, the fermentative production of HMOs has proven to be efficient because the necessary but difficult to synthesize nucleotide-activated monosaccharides are provided by the metabolism of the microbial cells used. However, the use of whole cells for the synthesis of HMOs also has some major disadvantages compared to biocatalytic approaches. These disadvantages are related to transport processes across the cell membrane, metabolic side reactions, and the need to purify the oligosaccharides synthesized by the microbial cells from a complex mixture containing, inter alia, various polyols (e.g. carbohydrates), nucleic acids, polypeptides, inorganic substances, etc.

[0011] A technical problem to be overcome related to the use of lactose in fermentation processes is the rearrangement of lactose (beta-D-galactopyranosyl-(1→4)-D-glucose) to lactulose (beta-D-galactopyranosyl-(1→4)-D-fructofuranose) during heat treatment of lactose, especially if the oligosaccharides produced are to be used for human consumption. This rearrangement can occur to an extensive extent upon heat sterilization of lactose, so that a few percent of the lactose present in the fermentation medium or in the lactose fermentation feedstock is rearranged to lactulose. However, lactulose is a non-digestible sugar for humans and is widely used as a laxative to treat chronic constipation.

[0012] The conversion of lactose to lactulose not only results in the production of undesired lactulose, but also provides microbial cells with undesired substrates for glycosylation reactions, which result in the production of more complex oligosaccharides (e.g., 2'-fucosyl-lactulose) as by-products. Thus, the production of lactulose from lactose results in the contamination of the desired product with closely related oligosaccharides that are difficult or even impossible to separate from the desired product.

[0013] Furthermore, lactose can be converted to another undesirable contaminant, allolactose (beta-D-galactopyranosyl-(1→6)-D-glucopyranose), when fed to beta-galactosidase positive E. coli strains (Huber et al., "Efflux of beta-galactosidase products from Escherichia coli" (1980) J. Bacteriol. 141, 528-533).

[0014] Furthermore, the addition of lactose can also cause a well-documented effect known as “lactose-induced cell death”. This effect appears to be caused by the excessive uptake of lactose by microbial cells and the concomitant disruption of the proton gradient across the microbial membrane. In particular, the combination of overexpression of lactose permease genes (e.g., lacY in E. coli) and exposure of recombinant microbial cells to excess lactose can cause a significant growth retardation and increased synthesis of cellular polysaccharides in the recombinant strain (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).

[0015] Furthermore, all commercially available lactose today is derived from whey, a waste product of the dairy industry. Whey is produced in large quantities in the manufacture of cheese and casein. Its origin from the dairy industry therefore means that concerns remain regarding the possibility of lactose being contaminated with prion proteins, the causative agent of bovine spongiform encephalopathy (BSE), also commonly known as mad cow disease. BSE is a fatal neurodegenerative disease in cattle that causes spongy degeneration of the brain and spinal cord. BSE can also be transmitted to humans, where it is known as variant Creutzfeldt-Jakob disease.

[0016] Moreover, lactose remains one of the most expensive components of the fermentation medium, so its replacement with glucose, glycerol, sucrose, etc. would result in a more cost-efficient production of HMOs.

[0017] In order to overcome the above drawbacks, improved means and methods for producing HMOs have been developed. For example, WO2015 / 150328 A1 discloses a bacterial host cell capable of producing oligosaccharides containing terminal galactose-(1→4)-glucose disaccharides. The bacterial host cell expresses at least one recombinant nucleic acid sequence encoding a β-1,4-galactosyltransferase capable of galactosylating a released glucose monosaccharide to produce lactose intracellularly, and also contains and expresses at least one recombinant nucleic acid sequence encoding a fucosyltransferase, a sialyltransferase, a glucosamyltransferase or a galactosyltransferase. Since the bacterial host cell can produce oligosaccharides without exogenous lactose, the bacterial host cell can be cultured in a medium without exogenous lactose to produce the oligosaccharides. More specifically, WO2015 / 150328 A1 discloses an E. coli strain genetically engineered to produce 2'-fucosyllactose using sucrose or a combination of glucose and sucrose as a carbon source. When utilizing sucrose, the E. coli strain is genetically engineered to express four genes of the csc-gene cluster of E. coli W strain, namely, genes encoding sucrose permease (cscB), fructokinase (cscK), sucrose hydrolase (cscA), and a transcriptional repressor (cscR).

[0018] However, the production of 2'-FL by the engineered E. coli strain using sucrose as the sole carbon and energy source also has the disadvantage that sucrose is difficult to heat sterilize without significant hydrolysis and formation of unwanted by-products. Alternatively, sterile filtration of the sucrose solution may be used, but sterile filtration carries a high risk of contamination with adventitious fermentation growths, especially in large-scale fermentations.

[0019] Furthermore, culturing microbial cells in the presence of sucrose as a carbon source for the production of HMOs, where the microbial cells are genetically engineered to have a split metabolism such that the monomers that make up sucrose are used in completely separate metabolic pathways, results in undesirable growth characteristics of the microbial cell cultures, presumably due to the stoichiometry of the monomers produced from intracellular sucrose hydrolysis (which does not match the quantitative needs of the different monomers in the separate pathways). [Prior art documents] [Patent documents]

[0020] [Patent Document 1] U.S. Patent No. 7,521,212 B1 [Patent Document 2] International Patent Publication No. 2015 / 150328 A1 [Non-patent literature]

[0021] [Non-Patent Document 1] Albermann et al., (2001) Carbohydr.Res.334(2)p97-103 [Non-Patent Document 2] Huber et al., (1980) J. Bacteriol. 141, 528-533 [Non-Patent Document 3] Grube et al.,(2013) Biotechnol.Appl.Biochem.5,31 Summary of the Invention [Problem to be solved by the invention]

[0022] To overcome the above drawbacks, a genetically engineered microbial cell is provided that is capable of producing a target oligosaccharide containing a galactose-β1,4-glucose moiety at its reducing end when cultured on a mixed monosaccharide feedstock as the main carbon and energy source without exogenous lactose addition. [Means for solving the problem]

[0023] In a first aspect, there is provided a microbial cell genetically engineered to produce lactose or a target oligosaccharide comprising a galactose-β1,4-glucose moiety at its reducing end, wherein the microbial cell is capable of producing the lactose de novo or of producing the target oligosaccharide when cultured without exogenous lactose addition.

[0024] In a second aspect, there is provided the use of a genetically engineered microbial host cell to produce a desired oligosaccharide that contains lactose or a galactose-β1,4-glucose moiety at its reducing end.

[0025] In a third aspect, there is provided a method for producing a target oligosaccharide containing lactose or a galactose-β1,4-glucose moiety at its reducing end by culturing genetically engineered microbial cells in the presence of a mixed feed containing glucose and recovering the target oligosaccharide. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 shows a schematic diagram of an exemplary embodiment of a microbial cell genetically engineered to produce 2'-fucosyllactose according to the present invention. [Diagram 2] FIG. 2 shows a schematic diagram of another exemplary embodiment of a microbial cell genetically engineered to produce 2'-fucosyllactose according to the present invention. [Diagram 3] FIG. 3 shows a schematic diagram of a further exemplary embodiment of a microbial cell genetically engineered to produce 2'-fucosyllactose according to the present invention. [Figure 4] FIG. 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 carbon and energy source. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] According to a first aspect, there is provided a microbial cell genetically engineered to produce lactose or a target oligosaccharide comprising a galactose-β1,4-glucose moiety at its reducing end, wherein the microbial cell has at least one glucose transporter for the transfer of glucose from a medium into the cytoplasm of the microbial cell, a UDP-galactose biosynthetic pathway for the intracellular biosynthesis of UDP-galactose, and at least one galactosyltransferase capable of galactosylating intracellular free glucose to produce lactose intracellularly.

[0028] The genetically engineered microbial cells are capable of producing lactose. In certain embodiments, the microbial cells can utilize the lactose produced by themselves to produce a target oligosaccharide having a galactose-β1,4-glucose moiety at its reducing end. There is no need to exogenously supply lactose to the microbial cells for the production of said target oligosaccharide.

[0029] The genetically engineered microbial cells have at least one glucose transporter for the transfer of glucose from the medium in which the microbial cells are cultured into the cytoplasm of the microbial cells, such that free glucose is available for the intracellular biosynthesis of lactose.

[0030] Typically, the genetically engineered microbial cell contains at least one functional gene encoding a glucose transporter capable of translocating glucose (Glu) from the medium into the cytoplasm of the cell.

[0031] The term "functional gene" as used herein refers to a nucleic acid molecule that includes a nucleotide sequence that encodes a protein or polypeptide and also contains a regulatory sequence operably linked to the protein-encoding nucleotide sequence such that the nucleotide sequence encoding the protein or polypeptide can be expressed in / by a microbial cell harboring the functional gene. Thus, when cultured under conditions that allow expression of the functional gene, the functional gene is expressed and a microbial cell expressing the functional gene typically contains the protein or polypeptide encoded by the protein coding region of the functional gene. The terms "nucleic acid" and "polynucleotide" as used herein refer to deoxyribonucleotide or ribonucleotide polymers in either single- or double-stranded form and, unless otherwise limited, also include known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to natural nucleotides. Unless otherwise indicated, a particular nucleic acid sequence includes its complementary sequence.

[0032] The term "operably linked" as used herein is intended to mean a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or a set of transcription factor binding sites) and a second nucleic acid sequence, where the expression control sequence affects the transcription and / or translation of the nucleic acid corresponding to the second sequence. Thus, the term "promoter" refers to a DNA sequence that usually "precedes" a gene in a DNA polymer and provides a site for initiation of transcription into mRNA. "Regulator" DNA sequences are also usually "upstream" (i.e., precede) a gene in a given DNA polymer and bind proteins that determine the frequency (or rate) of transcription initiation. Collectively referred to as "promoter / regulator" or "regulatory" DNA sequences, these sequences that precede a selected gene (or set of genes) in a functional DNA polymer cooperate to determine whether transcription (and ultimately expression) of the gene occurs. DNA sequences that "follow" a gene in a DNA polymer and provide a signal for termination of transcription into mRNA are referred to as transcription "terminator" sequences.

[0033] The term "recombinant" as used herein with reference to bacterial host cells indicates that the bacterial cell replicates heterologous nucleic acid or expresses a peptide or protein encoded by a heterologous nucleic acid (i.e., a sequence "foreign to the cell"). A recombinant cell may contain genes not found in the native (non-recombinant) form of the cell. A recombinant cell may also contain genes found in the native form of the cell that have been modified by artificial means and reintroduced into the cell. The term also includes cells that contain nucleic acid that is endogenous to the cell and has been modified without removing it from the cell. Such modifications include those obtained by gene replacement, site-directed mutagenesis, and related techniques. Thus, a "recombinant polypeptide" is one that has been produced by a recombinant cell. As used herein, a "heterologous sequence" or "heterologous nucleic acid" is one that is from a source foreign to a particular host cell (e.g., from a different species) or, if from the same source, has been modified from its original form. Thus, a heterologous nucleic acid operably linked to a promoter is either from a source different from that from which the promoter is derived, or, if from the same source, is modified from its original form. Heterologous sequences can be stably introduced into the genome of a host microbial cell, for example, by transfection, transformation, conjugation, or transduction. In this case, the technique applied depends on the host cell into which the sequence is to be introduced. Various techniques are known to those skilled in the art and are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

[0034] Thus, a "genetically engineered microbial cell" is understood to be a microbial cell which has been transformed or transfected, or which is capable of being transformed or transfected, by an exogenous polynucleotide sequence.

[0035] Thus, the nucleic acid sequences used in the present invention may, for example, be comprised in a vector that is stably transformed / transfected or otherwise introduced into a host microbial cell. A wide variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, inter alia, chromosomal, episomal and virus-derived vectors, such as those derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses, and combinations thereof, such as vectors derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. Expression system constructs may contain control regions that not only induce but also regulate expression. In general, any system or vector suitable to maintain, amplify or express polynucleotides and synthesize polypeptides in a host can be used for expression in the present context. The appropriate DNA sequence can be inserted into the expression system by any of a variety of well-known and routine techniques, such as those set out in Sambrook et al., supra.

[0036] The art is replete with patents and published literature on "recombinant DNA" methodologies for isolating, synthesizing, purifying and amplifying genetic material for use in transforming a selected host organism. Thus, it is common knowledge to transform a host organism with a "hybrid" viral or circular plasmid DNA containing a selected exogenous (i.e., foreign or "heterologous") DNA sequence. The procedure known in the art involves first creating a transformation vector by enzymatically cleaving a circular viral or plasmid DNA to form a linear DNA strand. The selected foreign DNA strand, which usually contains the sequence encoding the desired protein product, is also made linear using the same / similar enzyme. The linear viral or plasmid DNA is incubated with the foreign DNA in the presence of a ligase capable of carrying out the renaturation process, forming a "hybrid" vector containing the selected foreign DNA segment "spliced" into the viral or circular DNA plasmid.

[0037] The term "nucleotide sequence encoding ..." generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA, and generally represents a portion of a gene that codes for a particular polypeptide or protein. The term includes, but is not limited to, 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 containing DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or may be a mixture of single-stranded and double-stranded regions. The term also encompasses polynucleotides that contain a single contiguous or discontinuous region that codes for a polypeptide (e.g., interrupted by integrated phage or insertion sequence or editing), as well as additional regions that may also contain coding and / or non-coding sequences.

[0038] In embodiments, a suitable glucose transporter is a glucose facilitated diffusion protein. A suitable glucose facilitated fusion protein is encoded by the glf gene of Zymomonas mobilis subsp. mobilis (ATCC strain 31821 / ZM4 / CP4).

[0039] In additional and / or alternative embodiments, another suitable glucose transporter is a glucose translocation permease. A suitable glucose translocation permease is encoded by the galP gene of E. coli K-12. Glucose translocation permease, also known as galactose-proton symporter or galactose permease, also imports glucose through the cell membrane.

[0040] Thus, in further and / or alternative embodiments, the genetically engineered microbial cell contains and expresses at least one gene comprising the protein coding region of the glf gene of Zymomonas mobilis subsp. mobilis (ATCC strain 31821 / ZM4 / CP4), the galP gene of an Escherichia coli K-12 strain, or a functional variant thereof.

[0041] The term "variant" as used herein refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains essential (enzymatic) properties of the reference polynucleotide or polypeptide, respectively. A typical variant of a polynucleotide differs in nucleotide sequence from another reference polynucleotide. A change in the nucleotide sequence of a variant may or may not change the amino acid sequence of a polypeptide encoded by a reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations of the polypeptide encoded by a reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, the differences are limited, so that the sequences of a reference polypeptide and a variant are generally closely similar, and in many regions identical. A variant and a reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, in any combination. The substituted or inserted amino acid residues may or may not be those 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 a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art.

[0042] Within the scope of the present invention, the terms also include polymorphic variants, alleles, mutants, and interspecies homologs of nucleic acids / polynucleotides and polypeptides having an amino acid sequence with greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more amino acid sequence identity to a polypeptide encoded by a wild-type protein, preferably over a region of at least about 25, 50, 100, 200, 500, 1000 or more amino acids.

[0043] Thus, a "functional variant" of any gene / protein disclosed herein is intended to refer to a sequence variant of the gene / protein that still retains the same activity or slightly less activity as the original gene or protein from which the respective fragment is derived.

[0044] The genetically engineered microbial cells possess a UDP-galactose biosynthetic pathway for the intracellular formation of GDP-galactose (GDP-Gal), since an efficient supply of UDP-galactose is required for the intracellular biosynthesis of lactose.

[0045] In additional and / or alternative embodiments, UDP-galactose can be obtained from the microbial cell's own metabolism, namely, the activity of phosphoglucomutase, UTP-glucose-1-phosphate-uridyltransferase and UDP-glucose-4-epimerase.

[0046] The intracellular supply of GDP-galactose can be improved by genetic modification, such as expression or overexpression of one or more of the genes encoding polypeptides exhibiting phosphoglucomutase activity, UDP-glucose-1-phosphate-uridyltransferase activity and UDP-glucose-4-epimerase activity, respectively.

[0047] As used herein, the terms "overexpression" or "overexpressed" refer to levels of enzyme or polypeptide expression that are greater than that measured in a wild-type cell of the same species as the host cell that has not been genetically modified.

[0048] Phosphoglucomutase is an enzyme that promotes the interconversion of glucose-1-phosphate to glucose-6-phosphate from the 1' to 6' or 6' to 1' positions on α-D-glucose monomers. An example of a suitable phosphoglucomutase-encoding gene is the pgm gene of E. coli K-12 strain (GenBank: U08369.1). Thus, in further and / or alternative embodiments, the genetically engineered microbial cell comprises and expresses / overexpresses a gene encoding phosphoglucomutase, which preferably comprises the protein coding region of the E. coli pgm gene or a variant thereof.

[0049] A 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 utilizing UTP. An example of a gene encoding a suitable UTP-glucose-1-phosphate uridyltransferase is the galU gene of E. coli K-12 strain (GenBank: M98830.1). Thus, in additional and / or alternative embodiments, the genetically engineered microbial cell comprises and expresses / overexpresses a gene encoding a UTP-glucose-1-phosphate uridyltransferase, which preferably comprises the protein coding region of the E. coli galU gene or a variant thereof.

[0050] UDP-glucose-4-epimerase, such as GalE or a functional variant thereof, catalyzes the epimerization of UDP-glucose to UDP-galactose. An example of a gene encoding a UDP-glucose-4-epimerase is the galE gene of E. coli K-12 strain. Thus, in additional and / or alternative embodiments, the genetically engineered microbial cell comprises and expresses / overexpresses a gene encoding a UDP-glucose-4-epimerase, which preferably comprises the protein coding region of the E. coli galE gene or a variant thereof.

[0051] In further and / or alternative embodiments, the UDP-galactose biosynthetic pathway further comprises the enzyme activity of glucose-6-phosphate isomerase, which converts fructose-6-phosphate to glucose-6-phosphate and vice versa. An example of a gene encoding glucose-6-phosphate isomerase is the pgi gene of E. coli K-12 strain. Thus, in further and / or alternative embodiments, the genetically engineered microbial cell comprises and expresses / overexpresses a gene encoding glucose-6-phosphate isomerase, which preferably comprises the protein coding region of the E. coli pgi gene or a variant thereof.

[0052] Alternatively, UDP-galactose can be obtained by feeding galactose to the microbial cells from the medium. Galactose is taken up by the cells and phosphorylated to galactose-1-phosphate, which is then converted to UDP-galactose. Genes encoding enzymes with the required enzymatic activity 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).

[0053] The genetically engineered microbial cell comprises a β-1,4-galactosyltransferase capable of galactosylating free glucose monosaccharides. In additional and / or alternative embodiments, the suitable β-1,4-galactosyltransferase is derived from Neisseria menningitidis, Aggregatibacter aphrophilus or Pasteurella multocida, preferably encoded by the lgtB gene of Neisseria meningitidis, by the lex-1 gene of Aggregatibacter aphrophilus or by the β-1,4-galactosyltransferase gene galTpm1141 of Pasteurella multocida (GenBank: AEC04686). Thus, in further and / or alternative embodiments, the genetically engineered microbial cell comprises and expresses / overexpresses a gene encoding a β-1,4-galactosyltransferase, preferably comprising the protein coding region of the lgtB gene of Neisseria meningitidis, the lex-1 gene of Agregatibacter aphrophyllus, the galTpm1141 gene of Pasteurella multocida or a variant thereof.

[0054] β-1,4-galactosyltransferase synthesizes the galactose-β1,4-glucose disaccharide, i.e., lactose, by using UDP-galactose as a substrate to transfer a galactose moiety to a free glucose monosaccharide.

[0055] In further and / or alternative embodiments, the genetically engineered microbial cell comprises, in addition to the β-1,4-galactosyltransferase, at least one additional glycosyltransferase.

[0056] Generally, and throughout this disclosure, the term "glycosyltransferase activity" or "glycosyltransferase" refers to and includes enzymes responsible for the biosynthesis of di-, oligo-, and polysaccharides, which catalyze the transfer of a monosaccharide moiety from an activated nucleotide monosaccharide / sugar ("glycosyl donor") to a glycosyl acceptor molecule.

[0057] In a preferred embodiment, the at least one additional glycosyltransferase is a fucosyltransferase, a sialyltransferase, a glucosaminyltransferase or a galactosyltransferase, and 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-acetylglucosaminyltransferase, beta-1,3-galactosyltransferase, alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase, beta-1,4-galactosyltransferase or beta-1,6-galactosyltransferase.

[0058] The enzymatic activity of at least one additional glycosyltransferase allows for the production of target oligosaccharides containing a galactose-β-1,4-glucose moiety at their reducing end by using lactose as an acceptor for the activity of the additional glycosyltransferase. Table 1 shows the most abundant HMOs that can be produced as target oligosaccharides by the microbial cells and methods disclosed herein.

[0059] [Table 1-1]

[0060] [Table 1-2]

[0061] [Table 1-3]

[0062] Table 1: List of oligosaccharides of interest that can be produced using the engineered microbial cells and / or methods described herein. In additional and / or alternative embodiments, the microbial cell comprises a glucose-translocating phosphotransferase system (PtsG) that catalyzes the phosphorylation of incoming glucose as well as its transfer across the cell membrane.

[0063] The general mechanism of the Pts system is as follows: the phosphoryl group of phosphoenolpyruvate (PEP) is transferred via a signal transduction pathway to enzyme I (EI), which then transfers it to the phosphoryl carrier histidine protein (HPr). Phospho-HPr then transfers the phosphoryl group to a membrane-bound complex known as the sugar-specific permease enzyme 2 (EII), which transports the sugar into the cell. EII consists of at least three structurally distinct domains, IIA, IIB, and IIC, which may be fused together in a single polypeptide chain or may exist as two or three interacting chains, previously termed enzymes II (EII) and III (EIII).

[0064] The first domain (IIA or EIIA) contains the first permease-specific phosphorylation site, a histidine, which is phosphorylated by phospho-HPr. The second domain (IIB or EIIB) is phosphorylated by phospho-IIA on a cysteinyl or histidyl residue depending on the sugar to be transported. Finally, a phosphoryl group (phosphate group) is transferred from the IIB domain to the sugar substrate while the sugar uptake is processed by the IIC domain. This third domain (IIC or EIIC) forms the translocation channel and the specific substrate binding site.

[0065] Thus, the PtsG system obtains exogenous glucose and provides glucose-6-phosphate to the microbial cell, which can either be utilized in the UDP-galactose biosynthetic pathway and / or converted to fructose-6-phosphate, which can then be used to generate the energy-rich triphosphate in central metabolism and / or can be used for the biosynthesis of nucleotide-activated sugars, such as GDP-fucose.

[0066] In additional and / or alternative embodiments, the glucokinase gene(s) of the microbial cell are deleted or functionally inactivated such that the microbial cell does not have any polypeptides with glucokinase activity. Glucokinase (Glk)b phosphorylates free glucose at its carbon atom 6 to produce glucose-6-phosphate. In the absence of glucokinase activity, free glucose transferred into the cytoplasm of the microbial cell becomes available as a substrate for β1,4-galactosyltransferase to produce lactose, while glucose-6-phosphate obtained from PtsG activity is available for UDP-galactose formation or other metabolic pathways.

[0067] In additional and / or alternative embodiments, the genetically engineered microbial cell comprises a fructose transporter for transferring fructose (Fru) from the medium into the cytoplasm of the microbial cell. A suitable fructose transporter for importing free fructose is the isoform (PtsG-F) as described by Kornberg et al. in PNAS 97:1808-1812 (2000).

[0068] Fructose taken up into the cell can then be phosphorylated by fructokinase (FrK) to provide fructose-6-phosphate (Fru-6-P), which can then be utilized in other metabolic pathways, such as the UDP-galactose biosynthetic pathway and / or central metabolism to generate energy-rich triphosphates, and / or for the biosynthesis of nucleotide-activated sugars, e.g., GDP-fucose.

[0069] In further and / or alternative embodiments, the genetically engineered microbial cells contain a polypeptide exhibiting fructokinase-6 activity and a polypeptide exhibiting 6-phosphofructokinase-1 activity (FruK or phosphofructokinase), providing a metabolic pathway from intracellularly taken-up fructose to fructose-1,6-bisphosphate via fructose-6-phosphate.

[0070] In additional and / or alternative embodiments, the genetically engineered microbial cell comprises a fructose-transferring phosphotransferase system (PtsF) that catalyzes the phosphorylation of incoming fructose as well as its transfer across the cell membrane.

[0071] Thus, the PtsF system acquires exogenous fructose and provides fructose-1-phosphate in the microbial cell. The PtsF system includes the transmembrane proteins FruA, 1-phosphofructose kinase (FruK) and diphosphoryl transfer protein FruB. Fructose is transferred by FruA and FruB to provide fructose-1-phosphate in the cytoplasm. Fructose-1-phosphate is further phosphorylated by phosphofructokinase (FruK) to fructose-1,6-diphosphate, which the microbial cell can then use to generate energy-rich triphosphate in the central metabolism.

[0072] Another suitable PtsF system comprises LevD, LevE, LevF and LevG. LevD is a component of the fructose-specific phosphotransferase enzyme IIA. LevE is a component of the fructose-specific phosphotransferase enzyme IIB. LevF is a component of the fructose permease IIC and LevG is a component of the fructose permease IID. The corresponding genes levD, levE, levF and levG are known, for example, from Bacillus subtilis (strain 168). The PtsF system provides fructose-1-phosphate in the cell.

[0073] In further and / or alternative embodiments, the genetically engineered microbial cell contains at least one 1-phosphofructokinase (FruK). In additional and / or alternative embodiments, the engineered microbial cell comprises a fructose-1,6-bisphosphatase (GlpX) that dephosphorylates fructose-1,6-bisphosphate to provide fructose-6-phosphate that can be used by the microbial cell in the GDP-galactose biosynthetic pathway or another metabolic pathway, such as the biosynthesis of a nucleotide-activated sugar, such as GDP-fucose.

[0074] Preferably, the microbial cell also comprises a deletion or functional inactivation of its phosphofructokinase gene(s). Deletion or functional inactivation of the phosphofructokinase gene(s) results in a microbial cell lacking phosphofructokinase activity, preventing the conversion of Fru-6-P to Fru-1,6-bisP. Two isoforms of phosphofructokinase exist in E. coli, called PfkA and PfkB. The corresponding genes are pfkA and pfkB.

[0075] In additional and / or alternative embodiments, the engineered microbial cell has a GDP-L-fucose biosynthetic pathway. In additional and / or alternative embodiments, the GDP-fucose biosynthetic pathway includes mannose-6-phosphate isomerase (ManA), phosphomannomutase (ManB), mannose-1-phosphate-guanylyltransferase (ManC), GDP-mannose-4,6-dehydratase (Gmd), and GDP-L-fucose synthase (WcaG). Preferably, the microbial cell having a GDP-L-fucose biosynthetic pathway also includes a fucosyltransferase.

[0076] In additional and / or alternative embodiments, the microbial cell comprises an exporter protein or permease, preferably a sugar efflux transporter, that transports the oligosaccharide of interest out of the cell.

[0077] In additional and / or alternative embodiments, the genetically engineered microbial cell comprises a deletion or functional inactivation of its glucose-6-phosphate isomerase gene such that the microbial cell does not have glucose-6-phosphate isomerase activity. Glucose-6-phosphate isomerase (called 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 to the production of lactose.

[0078] In further and / or alternative embodiments, the microbial cell is a bacterial cell selected from the group consisting of bacteria of the genera Escherichia, Lactobacillus, Corynebacterium, Bacillus, Streptococcus, Enterococcus, Lactococcus and Clostridium, preferably Escherichia coli, Corynebacterium glutamicum, Clostridium cellulolyticum, Clostridium ljungdahlii, Clostridium autoethanogenum, or the like. The bacterial cell is selected from the group of bacterial species consisting of Bacillus autoethanogenum, Clostridium acetobutylicum, Bacillus subtilis, Bacillus megaterium, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii, and Lactococcus lactis. In another embodiment, the microbial cell is Escherichia coli. Other bacterial strains will be apparent to those of skill in the art upon reading this disclosure.

[0079] According to a second aspect, there is provided the use of a genetically engineered microbial cell as described herein for producing lactose or oligosaccharides comprising a galactose-β1,4-glucose moiety at its reducing end. In a further and / or alternative embodiment, the genetically engineered microbial cell is used for producing lactose or oligosaccharides comprising a galactose-β1,4-glucose moiety at its reducing end, wherein the microbial cell is cultured in the presence of a mixed feedstock comprising glucose and at least one additional carbon source. The additional carbon source may be selected from the group consisting of fructose, galactose, mannose, xylose, rhamnose, glycerol, succinic acid, pyruvic acid and malic acid. Preferably, the mixed feedstock comprises or consists of a mixture of glucose and fructose, more preferably an equimolar mixture of glucose and fructose, most preferably hydrolyzed sucrose.

[0080] According to a third aspect, there is provided a method for producing a target oligosaccharide containing lactose or a galactose-β1,4-glucose moiety at its reducing end, the method comprising the steps of: a) providing a genetically engineered microbial cell as described herein; b) culturing the microbial cells in a medium and under conditions allowing the production of the lactose or oligosaccharide of interest, wherein the medium contains as the main carbon source a mixture of glucose and at least one additional compound selected from fructose, galactose, mannose, xylose, rhamnose, glycerol, succinic acid, pyruvic acid and malic acid; and c) Recovering lactose or the oligosaccharide of interest from the medium and / or the microbial cells. Each step is included.

[0081] In further and / or alternative embodiments, the oligosaccharide of interest is a human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, 3'-sialyllactose, 6'-sialyllactose, 3-fucosyl-3'-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-difucosylhexose I, lacto-N-difucosylhexose II, lacto-N-sialylpentaose LSTa, LSTb, LSTc.

[0082] Preferably, the mixture of glucose and at least one additional monosaccharide is a mixed feedstock of glucose and fructose, preferably obtained by hydrolysis of sucrose. In additional and / or alternative embodiments, the microbial cells, particularly when cultured for the production of an oligosaccharide of interest, are cultured without an exogenous supply of lactose.

[0083] Aspects of the invention also include the following. Aspect 1: A microbial cell genetically engineered to produce lactose or a target oligosaccharide containing a galactose-β1,4-glucose moiety at its reducing end, the microbial cell comprising: - at least one glucose transporter for transferring glucose from the medium into the cytoplasm of the microbial cell; - UDP-galactose biosynthetic pathway; and - at least one β-1,4-galactosyltransferase capable of galactosylating free glucose to produce lactose intracellularly; A genetically engineered microbial cell comprising: 2. The genetically engineered microbial cell of claim 1, wherein the at least one glucose transporter is selected from the group consisting of glucose facilitated diffusion proteins and glucose translocating permeases. 3. The genetically engineered microbial cell according to claim 1 or 2, wherein the microbial cell expresses or overexpresses at least one gene encoding a glucose transporter, preferably at least one gene selected from the group consisting of glf, galP and functional mutants thereof. Aspect 4. The genetically engineered microbial cell according to any one of aspects 1 to 3, wherein the microbial cell comprises phosphoglucomutase, UTP-glucose-1-phosphate-uridyltransferase, and UDP-glucose 4-epimerase. Aspect 5. The genetically engineered microbial cell of any one of aspects 1 to 4, wherein the β-1,4-galactosyltransferase is encoded by a gene selected from the group consisting of lgtB of Neisseria meningitidis, lex-1 of Agregatibacter aphrofilus, galTpm1141 of Pasteurella multocida, and functional mutants thereof. Aspect 6. The genetically engineered microbial cell according to any one of aspects 1 to 5, wherein the microbial cell comprises at least one additional glycosyltransferase, preferably a glycosyltransferase selected from the group consisting of a fucosyltransferase, a sialyltransferase, a glucosaminyltransferase and a galactosyltransferase.

[0023] Aspect 7. The genetically engineered microbial cell of any of Aspects 1 to 6, wherein said microbial cell comprises a glucosyltransferase system. Aspect 8. The genetically engineered microbial cell according to any of aspects 1 to 7, wherein the microbial cell comprises a fructose transporter. Embodiment 9. The genetically engineered microbial cell according to any of embodiments 1 to 8, wherein said microbial cell has a fructose-specific phosphotransferase system and further comprises 1-phosphofructokinase.

[0023] Aspect 10. The genetically engineered microbial cell of aspect 8, wherein the microbial cell comprises fructokinase-6 activity and 6-phosphofructokinase-1 activity.

[0023] Aspect 11. The genetically engineered microbial cell of aspect 9, wherein the microbial cell comprises fructose-1,6-bisphosphatase.

[0023] Aspect 12. The genetically engineered microbial cell of aspect 9 or 11, wherein said microbial cell comprises a deletion or functional inactivation of its glucose-6-phosphate isomerase. Aspect 13. The genetically engineered microbial cell of any of aspects 6 to 12, wherein the additional glycosyltransferase is a fucosyltransferase and the microbial cell comprises mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-phosphate guanylyltransferase, GDP-mannose-4,6-dehydratase, and GDP-L-fucose synthase. 14. The genetically engineered microbial cell according to any of the preceding claims, wherein said microbial cell comprises an exporter protein or permease, preferably a sugar exporter, that exports the oligosaccharide of interest out of the cell. Aspect 15. The genetically engineered microbial cell according to any one of aspects 1 to 14, wherein the microbial cell is a bacterial cell selected from the group consisting of bacteria of the genera Escherichia, Lactobacillus, Corynebacterium, Bacillus, Streptococcus, Enterococcus, Lactococcus and Clostridium, preferably a bacterial cell selected from the group of bacterial species consisting of Escherichia coli, Corynebacterium glutamicum, Clostridium celluloticum, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium acetobutylicum, Bacillus subtilis, Bacillus megaterium, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii, and Lactococcus lactis. Aspect 16. Use of the genetically engineered microbial cell according to any of aspects 1 to 15 for producing lactose or oligosaccharides comprising a galactose-β1,4-glucose moiety at its reducing end. A method for producing a target oligosaccharide containing lactose or a galactose-β1,4-glucose moiety at its reducing end, the method comprising the steps of: a) providing a genetically engineered microbial cell according to any one of aspects 1 to 15; b) culturing the microbial cells in a medium and under conditions allowing the production of the lactose or oligosaccharide of interest, wherein the medium contains as the main carbon source a mixture of glucose and at least one additional compound selected from fructose, galactose, mannose, xylose, rhamnose, glycerol, succinic acid, pyruvic acid and malic acid; and c) Recovering lactose or the oligosaccharide of interest from the medium and / or the microbial cells. A method including each step. A method according to embodiment 17, wherein the oligosaccharide of interest is a human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, 3'-sialyllactose, 6'-sialyllactose, 3-fucosyl-3'-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-difucosylhexose I, lacto-N-difucosylhexose II, lacto-N-sialylpentaose LSTa, LSTb, LSTc. A method according to any one of claims 17 to 18, wherein the mixture of glucose and at least one additional monosaccharide is a mixed feedstock of glucose and fructose, preferably a mixed feedstock of glucose and fructose obtained by hydrolysis of sucrose.

[0046] Aspect 20. The method according to any of aspects 17 to 19, wherein the microbial cells are cultured without exogenous lactose supply. The present invention will be described with respect to certain embodiments and with reference to the drawings, but the present invention is not limited thereto, but only by the claims. Moreover, terms such as first, second, etc. in the description and claims are used to distinguish similar elements, and do not necessarily describe an order in time, space, ranking, or any other manner. It should be understood that the terms so used are interchangeable under appropriate circumstances, and that the aspects of the invention described herein can operate in other orders than those described or illustrated herein.

[0084] It should be noted that the term "comprising" used in the claims should not be interpreted as being limited to the means listed thereafter; it does not exclude other elements or steps. Thus, it should be interpreted as embodying the presence of stated features, integers, steps or elements, but without excluding the presence or addition of one or more other features, integers, steps or elements, or groups thereof. Thus, the scope of the expression "a device comprising means A and B" should not be limited to a device consisting of only elements A and B, but means that in the context of the present invention, the only relevant elements of the device are A and B.

[0085] Throughout this specification, a reference to "one embodiment" or "an embodiment" means that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as will be apparent to one of ordinary skill in the art from this disclosure.

[0086] Similarly, in describing exemplary embodiments of the invention, it should be understood that various features of the invention may be grouped together in a single embodiment, figure, or description thereof to streamline the disclosure and to aid in understanding one or more of the various inventive aspects. This method of disclosure, however, should not be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as reflected in the following claims, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of the invention.

[0087] Furthermore, although some embodiments described herein include some features (but not other features) that are included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention and form different embodiments, as will be understood by those skilled in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0088] Additionally, some of the embodiments are described herein as methods or combinations of method elements that may be implemented by a processor of a computer system or by other means for implementing that functionality. Thus, the processor, together with the necessary instructions for implementing such method or method elements, forms a means for implementing the method or method elements. Additionally, the elements described herein of the apparatus aspects are examples of means for performing the functions implemented by the elements to implement the invention.

[0089] Numerous specific details are set forth in the description and drawings provided herein. However, it should be understood that aspects of the invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.

[0090] The present invention will now be described by describing in detail some embodiments of the present invention. Other embodiments of the present invention can be constructed according to the knowledge of those skilled in the art without departing from the true spirit or technical teaching of the present invention, and it is apparent that the present invention is limited only by the provisions of the appended claims.

[0091] In an embodiment, the genotype is lacY - , lacZ - , fucIK - , wcaJ - The E. coli strain having the above structure is metabolically engineered to efficiently produce 2'-fucosyllactose by total fermentation using mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) as the main carbon and energy source. Thus, the expression of the glucokinase gene glk and / or the glucose dehydrogenase gene gcd and / or the glucose PTS permease gene ptsG is reduced and / or silenced. Furthermore, the glucose permease gene is expressed or overexpressed in the E. coli strain.

[0092] Additionally, at least one of the E. coli genes manA, manC, manB, gmd, wcaG, pgm, galU and galE are also expressed / overexpressed, as well as expression of a heterologous β-1,4-galactosyltransferase capable of transferring galactose from UDP-galactose to glucose to produce lactose, and an α-1,2-fucosyltransferase capable of transferring fucose from GDP-fucose to lactose to produce 2'-fucosyllactose.

[0093] In a preferred embodiment, the production strain is further modified by reducing and / or attenuating 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 additional genetic modification allows the production strain modified in this way to be cultivated on mixed monosaccharide feedstocks (e.g., hydrolyzed sucrose) as the main carbon and energy source, with an increased precursor supply (glucose and fructose-6-phosphate and glucose-6-phosphate) for the production of 2'-fucosyllactose by total fermentation, without interfering with the metabolism of the strain.

[0094] Referring to FIG. 1, an exemplary microbial cell of the present invention is shown diagrammatically. The microbial cell is capable of producing 2'-FL when cultured on a mixed feed consisting of glucose (Glu) and fructose (Fru), but the mixed feed 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. Expression of glucose kinase Glk is stopped by deletion or functional inactivation of the glk gene(s), so that all glucose imported by the cell is made available as a substrate for β1,4-galactosyltransferase GalTpm1141 via the UDP-galactose biosynthetic pathway for the intracellular production of lactose (Lac).

[0095] The imported fructose is phosphorylated by cellular fructose-6 kinase to generate an intracellular Fru-6-P pool. A portion of the Fru-6-P pool is used in the "UDP-Gal biosynthetic pathway" to synthesize UDP-Gal, which serves as a Gal donor for the GalTpm1141 galactosyltransferase to generate Lac. Another portion of the fru-6-P pool is used in the "GDP-L-Fuc biosynthetic pathway" to produce GDP-L-fucose, which serves as a fucose donor for the 2'-fucosyltransferase WbgL. A third portion of the Fru-6-P pool is further used for energy and biomass production, in that the fru-6-P is converted to Fru-1,6-bisP by cellular phosphofructokinases PfkA and / or PfkB.

[0096] FIG. 2 shows a schematic of another exemplary microbial cell of the present invention capable of producing 2'-FL when cultured on a mixed feedstock consisting of glucose (Glu) and fructose (Fru). Again, the mixed feedstock does not contain lactose (Lac). In addition to the exemplary microbial cell shown in FIG. 1, the microbial cell further comprises a glucose-specific Pts system (PtsG). The PtsG system imports and phosphorylates Glu to provide Glu-6-P in the cytosol of the cell. The Glu-6-P can be utilized by the microbial cell to produce UDP-Gal or Fru-6-P. In a mutant of the microbial cell (not shown), the cellular pgi gene(s) encoding glucose-6-phosphate isomerase (Pgi) have been deleted. In conjunction with the deletion of the glk gene, the microbial cell has been engineered so that free glucose monomers acquired by Glf can be utilized as a substrate for β1,4-galactosyltransferase, but all Glu-6-P acquired by PtsG is also available for UDP-Gal biosynthesis.

[0097] FIG. 3 shows a schematic diagram of another exemplary microbial cell of the present invention capable of producing 2'-FL when cultured on a mixed feedstock consisting of glucose (Glu) and fructose (Fru). Again, the mixed feedstock does not contain lactose (Lac). In addition to the exemplary microbial cell shown in FIG. 2, the microbial cell further comprises a fructose-specific Pts system (PtsF). The 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 has fructose-1,6-bisphosphatase (GlpX) activity. The genetically engineered microbial cell is thereby metabolically engineered to allow fructose and / or fructose-1-P to be utilized by the cell for UDP-Gal biosynthesis.

[0098] In addition, the phosphofructokinase gene(s) is deleted or functionally inactivated such that the cell has no phosphofructokinase (PfkA / PfkB) activity. Deletion or functional inactivation of the phosphofructokinase gene impairs the conversion of Fru-6-P to Fru-1,6-P, thereby preventing the utilization of Fru-6-P for the production of the energy-rich triphosphate and enhancing the production of 2'-FL. EXAMPLES

[0099] Example 1 - Preparation of mixed monosaccharide feedstock 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. The pH was adjusted using 96% (v / v) sulfuric acid at a temperature of 30°C–35°C. The solution was then sterilized at 121°C for 45 min in a vertical autoclave (Systec VX-65, Linden, Germany). Samples were taken before and after heat sterilization and kept frozen until analysis by high-performance liquid chromatography (HPLC). HPLC was performed using a RID-10A refractive index detector (Shimadzu, Germany) connected to a Shimadzu HPLC system and a Waters XBridge amide column 3.5 μm (250 × 4.6 mm) (Eschborn, Germany). Isocratic elution 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) was performed at 35 °C and a flow rate of 1.4 mL / min. The samples were clarified by solid phase extraction on an ion exchange matrix (Strata ABW, Phenomenex). Ten microliters of sample (diluted 1:5) were applied to the column. Finally, the relative amounts of detected sugars were determined. As shown in Table 1, the conversion of sucrose to the monosaccharides glucose and fructose increased as the pH value of the solution before heat treatment decreased. Complete sucrose cleavage was observed at pH values ​​≦3.50 when acidification was performed with sulfuric acid.

[0100] [ka]

[0101] Table 1: Relative amounts of sugars detected in pH-adjusted 50% (w / v) sucrose solutions before and after heat sterilization. pH adjustment was performed with 96% (v / v) sulfuric acid. Shown are the percentage amounts of sugars detected by HPLC (area under the curve, AUC).

[0102] Example 2 - Feedstock-dependent growth of various gene deletion strains In addition to the wild-type E. coli BL21(DE3) strain, the mutant E. coli pfkA - (ΔpfkA), E. coli pfkB - (ΔpfkB), E. coli pfkA - pfkB - The growth behavior of the two strains, ΔpfkA, ΔpfkA, and ΔpfkA, was compared. Genomic deletions were performed according to the method of Datsenko and Wanner (Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)). All strains were grown at 30°C in 100-mL shake flasks containing 20 mL of mineral salts medium. The medium was diluted with 7 g L -1 NH4H2PO4, 7 g L -1 K2HPO4, 2 g L -1 KOH, 0.3 g L -1 of citric acid, 2 g L -1 of MgSO4 × 7 H2O and 0.015 g L -1 CaCl2×6·H2O, 1 mL L -1 of trace element solution (54.4 g L -1 of ferric ammonium citrate, 9.8 g L -1 MnCl2 × 4 H2O, 1.6 g L -1 CoCl2×6 H2O, 1 g L -1 CuCl2×2 H2O, 1.9 g L -1 H3BO3, 9 g L -1 ZnSO4 × 7 H2O, 1.1 g L -1 Na2MoO4×2 H2O, 1.5 g L -1 Na2SeO3, 1.5 g L -1 The cultures were supplemented with 1000 ml of NiSO4 × 6·H2O) and contained either 2% (m / v) glucose (A) or 1% (w / v) glucose / 1% (w / v) fructose (B) as carbon source. Cultures were inoculated to an OD of 0.1 and growth progression was monitored by measuring the OD over a period of 26 h. 600 As shown in Figure 2, E. coli pfkA - pfkB -showed little growth when glucose was supplied as the sole carbon and energy source, but when a mixed monosaccharide feedstock was available, its growth was indistinguishable from that of the wild-type strain and single deletion mutants.

[0103] Example 3 - Total fermentation of 2'-fucosyllactose by engineered E. coli strains growing on mixed monosaccharide feedstocks Genotype pfkA - , lacZ - , fucIK - , wcaJ - , glk - , gcd - , ptsG -The E. coli BL21(DE3) strain was further engineered by overexpression of enzymes for de novo synthesis of GDP-fucose (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 transport facilitator gene glf (from Zymomonas mobilis), the β-1,4-galactosyltransferase gene galTpm1141 (from Pasteurella multocida (GenBank: AEC04686)), and the E. coli genes galE and pgm encoding UDP-glucose 4-epimerase and 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. Linear DNA fragments were integrated using EZ-Tn5TM transposase (Epicentre, USA) or transposition was performed using the hyperactive C9-mutant of the mariner transposase Himar1 (Proc. Natl. Acad. Sci. 1999, USA 96:11428-11433). Genes were codon-optimized for expression in E. coli and synthetically prepared in collaboration with GenScript.

[0104] The resulting E. coli strain was cultivated in a 3 L fermenter (New Brunswick, Edison, USA) at 30 °C. The starting 1000 mL mineral salts medium contained 7 g L -1 NH4H2PO4, 7 g L -1 K2HPO4, 2 g L -1 KOH, 0.3 g L -1 of citric acid, 2 g L -1 of MgSO4 × 7 H2O and 0.015 g L -1 CaCl2×6·H2O, 1 mL L -1 of trace element solution (54.4 g L-1 of ferric ammonium citrate, 9.8 g L -1 MnCl2 × 4 H2O, 1.6 g L -1 CoCl2×6 H2O, 1 g L -1 CuCl2×2 H2O, 1.9 g L -1 H3BO3, 9 g L -1 ZnSO4 × 7 H2O, 1.1 g L -1 Na2MoO4×2 H2O, 1.5 g L -1 Na2SeO3, 1.5 g L -1 The medium was supplemented with 2 g L NiSO4 × 6 H2O) and contained 2% (m / v) hydrolyzed sucrose as carbon source. Cultivation 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 marked by an increase in the dissolved oxygen concentration. Immediately after leaving the batch phase, the concentration of dissolved oxygen increased by 2 g L -1 MgSO4 × 7 H2O, 0.015 g L -1 of CaCl2 × 6 HO and 1 mL L -1 The carbon source consisted of fully hydrolyzed sucrose supplemented with a trace element solution of 12.0–15.0 mL L based on the starting volume. -1 h -1 A feed rate of 1000 ml was applied. Aeration was maintained at 3 L / min. Dissolved oxygen was maintained at 20-30% saturation by controlling the agitation speed. pH was maintained at 6.7 by adding 25% ammonia solution. The cultivation continued for 86 h, and a significant amount of 2'-FL was obtained in the culture supernatant.

Claims

1. A method for producing lactose or a target oligosaccharide containing a galactose-β1,4-glucose moiety at its reducing end, the method comprising the steps of: a) Below: - at least one glucose transporter for transferring glucose from the medium into the cytoplasm of the microbial cell so that free glucose is available for the intracellular biosynthesis of lactose, the at least one glucose transporter being selected from the group consisting of glucose facilitated diffusion proteins and glucose transfer permeases; - UDP-galactose biosynthetic pathway; - at least one β-1,4-galactosyltransferase capable of galactosylating free glucose to produce lactose intracellularly; and - one or more of the following i) and ii) : i) fructose transporter, fructokinase-6 activity and 6-phosphofructokinase-1 activity; and ii) Fructose-specific phosphotransferase system and 1-phosphofructokinase providing a genetically engineered microbial cell, the genetically engineered microbial cell comprising: b) culturing the microbial cells in a medium and under conditions allowing the production of the lactose or oligosaccharide of interest, wherein the medium contains a mixture of glucose and fructose as the main carbon source; and c) Recovering lactose or the oligosaccharide of interest from the medium and / or the microbial cells. A method including each step.

2. 2. The method of claim 1, wherein the target oligosaccharide is a human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, 3'-sialyllactose, 6'-sialyllactose, 3-fucosyl-3'-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-difucosylhexose I, lacto-N-difucosylhexose II, lacto-N-sialylpentaose LSTa, LSTb, and LSTc.

3. The method according to claim 1, wherein the mixture of glucose and fructose is obtained by hydrolysis of sucrose.

4. The method of claim 1, wherein the microbial cells are cultured without exogenous lactose supply.

5. 2. The method of claim 1, wherein the microbial cell expresses or overexpresses at least one gene encoding a glucose transporter, the at least one gene being selected from the group consisting of glf, galP and functional mutants thereof.

6. 2. The method of claim 1, wherein the microbial cells have phosphoglucomutase, UTP-glucose-1-phosphate-uridyltransferase, and UDP-glucose 4-epimerase.

7. 2. The method of claim 1, wherein the β-1,4-galactosyltransferase is encoded by a gene selected from the group consisting of lgtB of Neisseria meningitidis, lex-1 of Agregatibacter aphrofilus, galTpm1141 of Pasteurella multocida, and functional mutants thereof.

8. The method of claim 1 , wherein the microbial cell comprises at least one additional glycosyltransferase.

9. The method of claim 8, wherein the glycosyltransferase is selected from the group consisting of fucosyltransferase, sialyltransferase, glucosaminyltransferase and galactosyltransferase.

10. The method of claim 1 , wherein the microbial cell comprises a glucose transferring phosphotransferase system.

11. The method of claim 1, wherein the microbial cells contain fructose-1,6-bisphosphatase.

12. The method of claim 1, wherein the microbial cell comprises a deletion or functional inactivation of its glucose-6-phosphate isomerase.

13. 9. The method of claim 8, wherein the additional glycosyltransferase is a fucosyltransferase and the microbial cell has mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-phosphate guanylyltransferase, GDP-mannose-4,6-dehydratase, and GDP-L-fucose synthase.

14. The method of claim 1 , wherein the microbial cell comprises an exporter protein or permease that transports the target oligosaccharide out of the cell.

15. The method of claim 14, wherein the exporter protein or permease that transports the target oligosaccharide out of the cell is a sugar exporter.

16. 2. The method of claim 1, wherein the microbial cells are bacterial cells selected from the group consisting of bacteria of the genera Escherichia, Lactobacillus, Corynebacterium, Bacillus, Streptococcus, Enterococcus, Lactococcus and Clostridium.

17. The method of claim 16, wherein the microbial cells are bacterial cells selected from the group of bacterial species consisting of Escherichia coli, Corynebacterium glutamicum, Clostridium celluloticum, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium acetobutylicum, Bacillus subtilis, Bacillus megaterium, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii, and Lactococcus lactis.

Images