Fermentative production of oligosaccharides by full fermentation using mixed feedstocks

Abstract

The present invention discloses genetically engineered microbial cells for producing oligosaccharides containing a galactose-β1,4-glucose moiety at their reducing end, wherein the microbial cells are capable of producing the oligosaccharides in the absence of exogenously added lactose, and methods for producing the oligosaccharides using the microbial cells.

Description

[0001] The present invention relates to a bacterial host cell capable of producing lactose or an oligosaccharide of interest comprising a galactose-β1,4-glucose moiety at its reducing end, and to a method for producing lactose or an oligosaccharide of interest comprising a terminal galactose-β1,4-glucose moiety. Background Art

[0002] Human milk contains a complex mixture of carbohydrates, fats, proteins, vitamins, minerals, and trace elements. The largest fraction of human milk is composed of carbohydrates. The carbohydrate fraction in human milk can be further divided into (i) lactose and (ii) oligosaccharides (human milk oligosaccharides, HMOs). While lactose (galactose-β1,4-glucose) is used as an energy source, oligosaccharides are not metabolized by infants. The oligosaccharide fraction accounts for up to one-tenth of the total carbohydrate fraction and may be composed of more than 150 different oligosaccharides. The presence and concentration of these complex oligosaccharides are unique to humans and are therefore not found in significant quantities in the milk of other mammals, including dairy animals.

[0003] The most important human milk oligosaccharides are 2'-fucosyllactose and 3'-fucosyllactose, which together account for up to one-third of the total HMO fraction. Other important HMOs present in human milk are lacto-N-tetraose, lacto-N-neotetraose, and lacto-N-fucopentose I. In addition to these neutral oligosaccharides, acidic HMOs such as 3'-sialyllactose, 6'-sialyllactose, 3-fucosyl-3'-sialyllactose, sialyllacto-N-tetraose, and disialyllacto-N-tetraose are also found in human milk. Notably, the vast majority of HMOs contain a galactose-β1,4-glucose moiety at their reducing end. The structure of HMOs is closely related to epitopes of glycoconjugates on the surface of epithelial cells (Lewis tissue 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.

[0004] The presence of oligosaccharides in human milk has long been known, and the physiological functions of these oligosaccharides have been studied medically for decades. Specific functions have been identified for some of the more abundant human milk oligosaccharides.

[0005] In addition to the local effects in the gut mentioned earlier in this article, HMOs have been shown to induce systemic effects by entering the infant's systemic circulation. Furthermore, the effects of HMOs on protein-carbohydrate interactions (e.g., selectin-leukocyte binding) may modulate immune responses and reduce inflammatory responses. Furthermore, HMOs are increasingly recognized as key substrates for the development of the infant microbiome.

[0006] Since the beneficial properties of prebiotic oligosaccharides, particularly HMOs, are well studied, but the availability of natural sources is limited, efficient commercial (ie, large-scale) production of HMOs is highly desirable.

[0007] In an attempt to produce individual human milk oligosaccharides on a large scale, chemical routes to some of these oligosaccharides have been developed. However, these methods involve the use of several toxic chemicals, which increases the risk of contaminating the final product. To date, chemical synthesis has not been able to provide large-scale quantities and qualities that are at least sufficient for food applications.

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

[0009] Currently, all fermentation production methods and biocatalytic reactions for HMO production are based entirely on exogenously added lactose as the starting substrate. In these methods, one or more monosaccharides are added to lactose (US Pat. No. 7,521,212 B1; Albermann et al., (2001) Carbohydr. Res. 334(2) pp. 97-103). The addition of monosaccharides to lactose can be catalyzed by glycosyltransferases or glycosidases using a suitable activated monosaccharide substrate. Furthermore, additional monosaccharides can be added to lactose via transglucosidase reactions.

[0010] In particular, fermentative production of HMOs has proven effective because the metabolism of the microbial cells used provides the necessary, but difficult-to-synthesize, nucleotide-activated monosaccharides. However, compared to biocatalytic methods, the use of whole-cell HMO synthesis also has several major disadvantages. These disadvantages are related to transport processes across cell membranes, metabolic side reactions, and the need to purify the oligosaccharides synthesized by microbial cells from complex mixtures containing, among other things, various polyols (e.g., carbohydrates), nucleic acids, polypeptides, inorganic materials, etc.

[0011] One technical problem that needs to be overcome with the use of lactose in fermentation processes, particularly when the oligosaccharides being produced are intended for human consumption, is that upon heat treatment of lactose, lactose (β-D-galactopyranosyl-(1->4)-D-glucose) rearranges to lactulose (β-D-galactopyranosyl-(1->4)-D-fructofuranosyl). This rearrangement can occur extensively through heat sterilization of lactose, resulting in several percent of the lactose present in the fermentation medium or lactose fermentation feed being rearranged to lactulose. However, lactulose is a sugar that is indigestible to 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 unwanted lactulose, but also provides an unwanted substrate for glycosylation reactions in microbial cells. Consequently, more complex oligosaccharides (e.g., 2'-fucosyl-lactulose) are produced as by-products. Thus, the production of lactulose from lactose results in contamination of the desired product with closely related oligosaccharides that are difficult or even impossible to separate from the desired product.

[0013] Furthermore, if lactose is provided to a β-galactosidase-positive E. coli strain, it can be converted to allolactose (β-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).

[0014] Furthermore, the addition of lactose may induce a well-documented effect known as "lactose-induced cell killing". This effect is most likely due to excessive lactose uptake by microbial cells and the associated collapse of the proton gradient across the bacterial membrane. In particular, overexpression of a lactose permease gene (e.g., lacY in Escherichia coli) combined with exposure of recombinant microbial cells to excess lactose can cause a considerable growth delay in the recombinant strain and an increase in cellular polysaccharide synthesis (Grube et al., "Hydrogen-producing Escherichia coli strains overexpressing lactosepermease: FT-IR analysis of the lactose-induced stress" (2013) Biotechnol. Appl. Biochem. 5, 31).

[0015] Furthermore, any commercially available lactose today is derived from whey, a waste product from the dairy industry. Whey is produced in large quantities during cheese and casein production. Therefore, because lactose originates from the dairy industry, concerns remain about potential contamination with prions, the causative agent of bovine spongiform encephalopathy (BSE), also widely known as mad cow disease. BSE is a fatal neurodegenerative disease in cattle that causes spongiform degeneration of the brain and spinal cord. BSE can be transmitted to humans and is known as a variant of Creutzfeldt-Jakob disease.

[0016] Most importantly, lactose remains one of the most expensive components in fermentation media, and replacing lactose with glucose, glycerol, sucrose, etc. will lead to more cost-effective HMO production.

[0017] To overcome these shortcomings, improved methods and approaches for HMO production have been developed. For example, WO 2015 / 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, which is capable of galactosylating free glucose monosaccharides to produce lactose intracellularly. The bacterial host cell also contains and expresses at least one recombinant nucleic acid sequence encoding a fucosyltransferase, a sialyltransferase, a glucosaminyltransferase, or a galactosyltransferase. The bacterial host cell is capable of producing the oligosaccharide in the absence of exogenously added lactose, thereby allowing the bacterial host cell to be cultured in a culture medium devoid of exogenously added lactose to produce the oligosaccharide. More specifically, WO 2015 / 150328A1 discloses a genetically engineered Escherichia coli strain for producing 2'-fucosyllactose, which utilizes sucrose or a combination of glucose and sucrose as a carbon source. To utilize sucrose, the E. coli strain is genetically engineered to express four genes from the csc-gene cluster of E. coli W, encoding sucrose permease (cscB), fructokinase (cscK), sucrose hydrolase (cscA), and transcription repressor (cscR).

[0018] However, the use of sucrose as the sole carbon and energy source for 2'-FL production in genetically engineered E. coli strains also has its drawbacks: it is difficult to heat-sterilize the sucrose without significant hydrolysis and the formation of unwanted byproducts. Sterile filtration of the sucrose solution can be an alternative, but carries a high risk of contamination of the fermentation with adventitious growth, particularly in industrial-scale fermentations.

[0019] Furthermore, culturing a microbial cell for producing HMOs in the presence of sucrose as a carbon source, wherein the microbial cell has been genetically engineered to have a split metabolism, such that the monomers that make up sucrose are utilized in different metabolic pathways, results in undesirable growth characteristics of the bacterial cell culture, likely due to a mismatch between the stoichiometry of monomers produced by intracellular sucrose hydrolysis and the quantitative requirements of the different monomers in the different pathways.

[0020] In order to overcome the above-mentioned shortcomings, a genetically engineered microbial cell is provided, which is capable of producing a target oligosaccharide containing a galactose-β1,4-glucose moiety at its reducing end when cultured in a mixed monosaccharide feedstock as the main carbon source and energy source, but without exogenously added lactose. Summary of the Invention

[0021] In a first aspect, the present invention provides a genetically engineered microbial cell for producing lactose or an oligosaccharide of interest comprising a galactose-β1,4-glucose moiety at its reducing end, wherein the microbial cell is capable of producing the lactose in a de novo synthetic pathway or producing the oligosaccharide of interest when cultured in the absence of exogenously added lactose.

[0022] In a second aspect, the present invention provides the use of a genetically engineered microbial host cell for producing lactose or an oligosaccharide of interest comprising a galactose-β1,4-glucose moiety at its reducing end.

[0023] In a third aspect, the present invention provides a method for producing lactose or a target oligosaccharide comprising a galactose-β1,4-glucose moiety at its reducing end by culturing genetically engineered microbial cells in the presence of a mixed feedstock containing glucose and recovering the target oligosaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A schematic diagram showing an exemplary embodiment of a genetically engineered microbial cell for producing 2'-fucosyllactose according to the present invention.

[0025] Figure 2 A schematic diagram showing another exemplary embodiment of a genetically engineered microbial cell for producing 2'-fucosyllactose according to the present invention is shown.

[0026] Figure 3 A schematic diagram showing another exemplary embodiment of a genetically engineered microbial cell for producing 2'-fucosyllactose according to the present invention is shown.

[0027] Figure 4 Shown are the growth characteristics of E. coli strains during cultivation on glucose

[0028] (A) or a mixed monosaccharide raw material consisting of glucose and fructose;

[0029] (B) Carried out as the sole carbon and energy source. DETAILED DESCRIPTION

[0030] According to a first aspect, the present invention provides a genetically engineered microbial cell for producing lactose or an oligosaccharide of interest comprising a galactose-β1,4-glucose moiety at its reducing end, wherein the microbial cell has at least one glucose transporter for transporting glucose from a culture medium into the cytoplasm of the microbial cell; a UDP-galactose biosynthetic pathway for biosynthesizing UDP-galactose within the cell; and at least one galactosyltransferase capable of galactosylating free intracellular glucose to produce lactose within the cell.

[0031] Genetically engineered microbial cells are capable of producing lactose. In certain embodiments, the microbial cells can utilize their own lactose production to produce oligosaccharides of interest having a galactose-β1,4-glucose moiety at their reducing end. For the production of the oligosaccharides of interest, it is not necessary to provide an exogenous lactose supply to the microbial cells.

[0032] The genetically engineered microbial cells have at least one glucose transporter for transporting glucose from a culture medium in which the microbial cells are cultured into the cytoplasm of the microbial cells, thereby making the free glucose available for intracellular biosynthesis of lactose.

[0033] Typically, the genetically engineered microbial cell comprises at least one functional gene encoding the glucose transporter, which is capable of transporting glucose (Glu) from the culture medium into the cytoplasm of the cell.

[0034] As used herein, the term "functional gene" refers to a nucleic acid molecule comprising a nucleotide sequence encoding a protein or polypeptide, and further comprising a regulatory sequence operably linked to the nucleotide sequence encoding the protein, such that the nucleotide sequence encoding the protein or polypeptide can be expressed in / by a microbial cell having the functional gene. Thus, when cultured under conditions that allow expression of the functional gene, the functional gene is expressed, and the microbial cell expressing the functional gene typically contains the protein or polypeptide encoded by the protein coding region of the functional gene. As used herein, the terms "nucleic acid" and "polynucleotide" refer to deoxyribonucleotide or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, include known analogs of natural nucleotides that hybridize with nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence includes its complementary sequence.

[0035] As used herein, the term "operably connected" should mean the functional connection between a nucleic acid expression control sequence (such as a promoter, a signal sequence or a series of transcription factor binding sites) and the 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" refers to a DNA sequence that is "before" a gene usually in a DNA polymer and provides a DNA sequence that initiates transcription to mRNA. A "regulator" DNA sequence is also usually "upstream" (i.e., before) of a gene in a given DNA polymer, in conjunction with the protein that determines transcription initiation frequency (or rate). These sequences before the selected gene (or a series of genes) in a functional DNA polymer are collectively referred to as "promoter / regulator" or "regulation" DNA sequences, and they work together to determine whether the transcription (and final expression) of a gene occurs. A DNA sequence that is "after" a gene in a DNA polymer and provides a DNA sequence that terminates transcription to mRNA is referred to as a transcription "terminator" sequence.

[0036] The term "recombinant" when used herein for bacterial host cells means that the bacterial cell replicates heterologous nucleic acids, or expresses peptides or proteins encoded by heterologous nucleic acids (i.e., sequences that are "foreign to the cell"). Recombinant cells may contain genes that are not present in the natural (non-recombinant) form of the cell. Recombinant cells may also contain genes that are present in the natural form of the cell, wherein the genes are modified and reintroduced into the cell by artificial means. The term also encompasses cells that contain nucleic acids that are endogenous to the cell and have been modified, without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, site-specific mutations, and related techniques. Thus, a "recombinant polypeptide" is a polypeptide produced by a recombinant cell. "Heterologous sequence" or "heterologous nucleic acid" as used herein is a nucleic acid derived from a source other than a specific host cell (e.g., from a different species), or, if from the same source, is modified from its natural form. Thus, a heterologous nucleic acid operably linked to a promoter is from a source different from that of the promoter, or, if from the same source, is modified from its natural form. Heterologous sequences can be stably introduced (e.g., by transfection, transformation, conjugation, or transduction) into the genome of a host microbial cell, where the applicable technique 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).

[0037] Thus, "genetically engineered microbial cells" are understood to be bacterial cells that have been transformed or transfected, or are capable of being transformed or transfected with exogenous polynucleotide sequences.

[0038] Thus, the nucleic acid sequences used in the present invention may, for example, be contained in a vector to be stably transformed / transfected or otherwise introduced into a host microbial cell.

[0039] Various expression systems can be used for producing polypeptide of the present invention.This type of vector especially comprises chromosome-derived vector, episome-derived vector and virus-derived vector, for example, derived from bacterial plasmid, derived from phage, derived from transposon, derived from yeast episome, derived from insertion element, derived from yeast chromosome element, derived from virus vector, and the vector derived from its combination, for example, those derived from plasmid and phage genetic elements (for example clay and phagemid).The expression system construct can comprise regulatory region, and it regulates and causes expression.Usually, any system or the vector that is suitable for maintaining, breeding or expressing polynucleotide and is suitable for synthetic polypeptide in host can be used for expression in this respect.By any technology in multiple known and conventional techniques (for example described in those of Sambrook et al. above), suitable dna sequence dna can be inserted into expression system.

[0040] There are many patents and literature publications related to "recombinant DNA" methods in this area, which are used to separate, synthesize, purify and amplify genetic material for transforming selected host organisms. Therefore, it is common knowledge to use "hybrid" viruses or circular plasmid DNA containing selected exogenous (i.e., exogenous or "heterologous") DNA sequences to transform host organisms. Procedures known in the art first include producing transformation vectors by enzyme-digesting circular viruses or plasmid DNA to form linear DNA chains. Selected exogenous DNA chains are prepared in linear form using identical / similar enzymes, which typically contain sequences encoding the desired protein product. Linear viruses or plasmid DNA are incubated with exogenous DNA in the presence of a ligase capable of performing the recovery process, and form a "hybrid" vector comprising the selected exogenous DNA fragments "spliced" into the virus or circular DNA plasmid.

[0041] 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 part of a gene encoding a 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 a mixture of single-stranded, double-stranded and triple-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules comprising DNA and RNA (the DNA and RNA may be single-stranded or, more typically, double-stranded or triple-stranded regions, or a mixture of single-stranded and double-stranded regions). The term also encompasses polynucleotides that comprise a single continuous region or discontinuous regions (e.g., interrupted by integrated phage or insertion sequences or editing) that encode a polypeptide, as well as other regions that may also comprise coding and / or non-coding sequences.

[0042] In one embodiment, 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 (strain ATCC 31821 / ZM4 / CP4).

[0043] In an additional and / or alternative embodiment, another suitable glucose transporter is a glucose transport permease. A suitable glucose transport permease is encoded by the Escherichia coli K-12 galP gene. The glucose transport permease is also referred to as galactose-proton symporter (symporter) or galactose permease, but also crosses the cell membrane to import glucose.

[0044] Thus, in an additional and / or alternative embodiment, the genetically engineered microbial cell comprises and expresses at least one gene comprising a protein coding region of the glf gene of Zymomonas mobilis subsp. mobilis (strain ATCC 31821 / ZM4 / CP4), the Escherichia coli K-12 galP gene, or a functional variant thereof.

[0045] The term "variant" as used herein refers to a polynucleotide or polypeptide that is different from a reference polynucleotide or polypeptide, respectively, but retains the essential (enzyme) properties of the reference polynucleotide or polypeptide. The nucleotide sequence of a typical polynucleotide variant is different from another reference polynucleotide. Changes 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 may result in amino acid substitutions, additions, deletions, fusions, and truncations in a polypeptide encoded by a reference sequence, as discussed below. The amino acid sequence of a typical polypeptide variant is different from another reference polypeptide. Typically, the differences are limited so that the sequences of the reference polypeptide and the variant are very similar overall and identical in many regions. The differences in the amino acid sequences of a variant and a reference polypeptide may be one or more substitutions, additions, or deletions in any combination. The amino acid residues that are replaced or inserted may or may not be amino acid residues encoded by the genetic code. Variants of polynucleotides or polypeptides may be naturally occurring, such as allelic variants, or may be known non-naturally occurring variants. Non-naturally occurring variants of polynucleotides and polypeptides may be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art.

[0046] Within the scope of the present invention, those terms also include nucleic acid / polynucleotide and polypeptide polymorphic variants, alleles, mutants and interspecies homologs whose amino acid sequences have 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 greater amino acid sequence identity to the polypeptide encoded by the wild-type protein, preferably over a region of at least about 25, 50, 100, 200, 500, 1000 or more amino acids.

[0047] Therefore, 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 or slightly less activity than the gene or protein from which the respective fragment was derived.

[0048] Because the intracellular biosynthesis of lactose requires an efficient supply of UDP-galactose, genetically engineered microbial cells possess a UDP-galactose biosynthetic pathway for the intracellular formation of GDP-galactose (GDP-Gal).

[0049] In an additional and / or alternative embodiment, UDP-galactose can be obtained from the metabolism of the microbial cells themselves, ie, the activities of phosphoglucomutase, UTP-glucose-1-phosphate-uridyl transferase, and UDP-glucose-4-epimerase.

[0050] The intracellular supply of GDP-galactose can be improved by genetic modification (e.g., expression or overexpression of one or more genes encoding polypeptides that exhibit phosphoglucomutase activity, UDP-glucose-1-phosphate-uridyl acyltransferase activity, and UDP-glucose-4-epimerase activity, respectively).

[0051] As used herein, the term "overexpression" or "overexpressed" refers to an enzyme or polypeptide being expressed at a level greater than that measured in a wild-type cell of the same species (as the host cell has not been genetically altered).

[0052] Phosphoglucomutase is an enzyme that promotes the interconversion of glucose-1-phosphate to glucose-6-phosphate, i.e., it is on an α-D-glucose monomer from the 1' to 6' position or the 6' to 1' position. An exemplary gene encoding a suitable phosphoglucomutase is the Escherichia coli K-12 pgm gene (GenBank: U08369.1). Therefore, in an additional and / or alternative embodiment, the genetically engineered microbial cell comprises and expresses / overexpresses a gene encoding phosphoglucomutase, preferably comprising the protein coding region of the Escherichia coli pgm gene or a variant thereof.

[0053] UTP-glucose-1-phosphate-uridyl acyltransferase, such as GalU or its functional variants, utilizes UTP to catalyze the conversion of α-D-glucose-1-phosphate into UDP-glucose. An exemplary gene encoding a suitable UTP-glucose-1-phosphate-uridyl acyltransferase is the Escherichia coli K-12 galU gene (GenBank: M98830.1). Therefore, in an additional and / or alternative embodiment, the genetically engineered microbial cell comprises and expresses / overexpresses a gene encoding UTP-glucose-1-phosphate-uridyl acyltransferase, preferably comprising the protein coding region of the Escherichia coli galU gene or its variant.

[0054] UDP-glucose-4-epimerase, such as GalE or its functional variants, catalyzes the epimerization of UDP-glucose to UDP-galactose. An exemplary gene encoding UDP-glucose-4-epimerase is the E. coli K-12 galE gene. Therefore, in an additional and / or alternative embodiment, the genetically engineered microbial cell comprises and expresses / overexpresses a gene encoding UDP-glucose-4-epimerase, preferably comprising the protein coding region of the E. coli galE gene or its variant.

[0055] In an additional and / or alternative embodiment, the UDP-galactose biosynthetic pathway further comprises the enzymatic activity of glucose-6-phosphate isomerase, which converts fructose-6-phosphate into glucose-6-phosphate, and vice versa. An exemplary gene encoding glucose-6-phosphate isomerase is the E. coli K-12 pgi gene. Thus, in an additional and / or alternative embodiment, the genetically engineered microbial cell comprises and expresses / overexpresses a gene encoding glucose-6-phosphate isomerase, preferably comprising a protein coding region of the E. coli pgi gene or a variant thereof.

[0056] Alternatively, UDP-galactose can be obtained by feeding galactose to microbial cells via the culture 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 desired enzyme 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).

[0057] The genetically engineered microbial cell comprises a β-1,4-galactosyltransferase capable of galactosylating free glucose monosaccharides. In an additional and / or alternative embodiment, a suitable β-1,4-galactosyltransferase is derived from Neisseria meningitidis, from Aggregatibacteraphrophilus or from Pasteurella multocida, preferably a β-1,4-galactosyltransferase gene galTpm1141 (GenBank: AEC04686) encoded by Neisseria meningitidis lgtB gene, Aggregatibacteraphrophilus lex-1 gene or from Pasteurella multocida. Thus, in an additional and / or alternative embodiment, 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 Neisseria meningitidis lgtB gene, the Aggregatibacterium foamophilum lex-1 gene, the Pasteurella multocida galTpm1141 gene, or a variant thereof.

[0058] β-1,4-Galactosyltransferase uses UDP-galactose as a substrate to transfer the galactose moiety to free glucose monosaccharide, thereby synthesizing galactose-β1,4-glucose disaccharide, namely lactose.

[0059] In an additional and / or alternative embodiment, the genetically engineered microbial cell comprises at least one additional glycosyltransferase, ie, in addition to the β-1,4-galactosyltransferase.

[0060] In general, throughout this disclosure, the term "glycosyltransferase activity" or "glycosyltransferase" means and encompasses enzymes responsible for the biosynthesis of disaccharides, oligosaccharides, and polysaccharides that catalyze the transfer of a monosaccharide moiety from an activated nucleotide monosaccharide / sugar ("glycosyl donor") to a glycosyl acceptor molecule.

[0061] In a preferred embodiment, the at least one additional glycosyltransferase is a fucosyltransferase, a sialyltransferase, a glucosaminyltransferase or a galactosyltransferase, more preferably, the at least one additional glycosyltransferase is selected from at least one of the following: α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, β-1,3-N-acetylglucosaminyltransferase, β-1,3-galactosyltransferase, α-2,3-sialyltransferase, α-2,6-sialyltransferase, β-1,4-galactosyltransferase or β-1,6-galactosyltransferase.

[0062] The enzymatic activity of at least one additional glycosyltransferase allows for the production of oligosaccharides of interest comprising a galactose-β-1,4-glucose moiety at its reducing end by using lactose as an acceptor for the activity of the additional glycosyltransferase. Table 1 identifies the most abundant HMOs that can be produced as oligosaccharides of interest by the microbial cells and methods disclosed herein.

[0063]

[0064]

[0065]

[0066] Table 1: List of oligosaccharides of interest that can be produced using the genetically modified microbial cells and / or methods described herein.

[0067] In an additional and / or alternative embodiment, the microbial cell comprises a glucose transporting phosphotransferase system (PtsG).The glucose transporting phosphotransferase system catalyzes the phosphorylation of incoming glucose, concomitant with its transport across the cell membrane.

[0068] 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 in turn transfers it to the phosphoryl carrier histidine protein (HPr). Phospho-HPr then transfers the phosphoryl group to a sugar-specific permease, a membrane-bound complex called enzyme II (EII), which transports sugars into the cell. EII is composed of at least three structurally distinct domains, IIA, IIB, and IIC. These can be fused together in a single polypeptide chain or exist as two or three interacting chains, previously referred to as enzymes II (EII) and III (EIII).

[0069] The first domain (IIA or EIIA) carries the first permease-specific phosphorylation site—a histidine phosphorylated by phospho-HPr. The second domain (IIB or EIIB) is phosphorylated by phospho-IIA on either a cysteinyl or histidyl residue, depending on the sugar being transported. Finally, the phosphoryl group is transferred from the IIB domain to the sugar substrate, accompanied by sugar uptake processed by the IIC domain. The third domain (IIC or EIIC) forms the transport channel and specific substrate binding site.

[0070] Thus, the PtsG system obtains exogenous glucose and provides glucose-6-phosphate in microbial cells. Glucose-6-phosphate can be used in the UDP-galactose biosynthetic pathway and / or converted to fructose-6-phosphate, which in turn can be used to produce energy-rich triphosphates in central metabolism and / or in the biosynthesis of nucleotide-activated sugars such as GDP-fucose.

[0071] In an additional and / or alternative embodiment, the glucokinase gene of the microbial cell has been deleted or functionally inactivated, so that the microbial cell does not have any polypeptides having glucokinase activity. Glucokinase (Glk) b phosphorylates free glucose at carbon atom 6 to produce glucose-6-phosphate. In the absence of glucokinase activity, free glucose transported into the cytoplasm of the microbial cell can be used as a substrate for β1,4-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.

[0072] In an additional and / or alternative embodiment, the genetically engineered microbial cell comprises a fructose transporter for transporting fructose (Fru) from the culture medium into the cytoplasm of the microbial cell. A suitable fructose transporter for uptake of free fructose is the isoform (PtsG-F) described by Kornberg et al. PNAS 97: 1808-1812 (2000).

[0073] The 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 biosynthetic pathway and / or other metabolic pathways, such as in central metabolism and / or in the biosynthesis of nucleotide-activated sugars (such as GDP-fucose) to produce high-energy triphosphates.

[0074] In an additional and / or alternative embodiment, the genetically engineered microbial cell comprises a polypeptide exhibiting fructokinase-6 activity and a polypeptide exhibiting 6-phosphofructokinase-1 activity (FruK or phosphofructokinase) to provide a metabolic pathway from internalized fructose via fructose-6-phosphate to fructose-1,6-bisphosphate.

[0075] In an additional and / or alternative embodiment, the genetically engineered microbial cell comprises a fructose transporting phosphotransferase system (PtsF).The fructose transporting phosphotransferase system catalyzes the phosphorylation of incoming fructose, concomitant with its transport across the cell membrane.

[0076] Thus, the PtsF system acquires exogenous fructose and provides fructose-1-phosphate within microbial cells. The PtsF system comprises the transmembrane protein FruA, 1-phosphofructokinase (FruK), and the diphosphotransfer protein FruB. FruA and FruB transport fructose 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 is then used by microbial cells to generate high-energy triphosphates in central metabolism.

[0077] Another suitable PtsF system comprises LevD, LevE, LevF, and LevG. LevD is the fructose-specific phosphotransferase component IIA. LevE is the fructose-specific phosphotransferase component IIB. LevF is the fructose permease component IIC, and LevG is the fructose permease component 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.

[0078] In an additional and / or alternative embodiment, the genetically engineered microbial cell comprises at least one 1-phosphofructokinase (FruK).

[0079] In an additional and / or alternative embodiment, the genetically engineered microbial cell comprises fructose-1,6-bisphosphatase (GlpX). The fructose-1,6-bisphosphatase dephosphorylates fructose-1,6-bisphosphate to provide fructose-6-phosphate. Fructose-6-phosphate can be used by the microbial cell in the GDP-galactose biosynthetic pathway, or in another metabolic pathway, such as the biosynthesis of nucleotide-activated sugars (e.g., GDP-fucose).

[0080] Preferably, the microbial cell also contains a deletion or functional inactivation of its phosphofructokinase gene. Deletion or functional inactivation of the phosphofructokinase gene results in a lack of phosphofructokinase activity in the microbial cell, thereby preventing the conversion of Fru-6-P to Fru-1,6-diP. In Escherichia coli, there are two isoforms of phosphofructokinase, designated PfkA and PfkB. The corresponding genes are pfkA and pfkB.

[0081] In an additional and / or alternative embodiment, the genetically engineered microbial cell has a GDP-L-fucose biosynthetic pathway. In an additional and / or alternative embodiment, the GPD-fucose biosynthetic pathway comprises mannose-6-phosphate isomerase (ManA), phosphomannose mutase (ManB), mannose-1-phosphate-guanylyltransferase (ManC), GDP-mannose-4,6-dehydratase (Gmd), GDP-L-fucose synthase (WcaG). Preferably, the microbial cell having the GDP-L-fucose biosynthetic pathway also has a fucosyltransferase.

[0082] In an additional and / or alternative embodiment, the microbial cell comprises an exporter or permease, preferably a sugar efflux transporter, that exports the oligosaccharide of interest from the cell.

[0083] In an additional and / or alternative embodiment, the genetically engineered microbial cell comprises a deletion or functional inactivation of its glucose-6-phosphate isomerase gene such that the microbial cell lacks glucose-6-phosphate isomerase activity. Glucose-6-phosphate isomerase (called after Pgi in E. coli) converts glucose-6-phosphate into fructose-6-phosphate. By deleting the glucose-6-phosphate gene or by inactivating their expression, any glucose-6-phosphate present in the cytoplasm of the microbial cell can be directed to lactose production.

[0084] In an additional and / or alternative embodiment, the microbial cell is a bacterial cell selected from the group consisting of Escherichia, Lactobacillus, Corynebacterium, Bacillus, Streptococcus, Enterococcus, Lactococcus and Clostridium, preferably a bacterial cell selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Clotridium cellulolyticum, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium acetobutylicum, Bacillus subtilis, Bacillus megaterium, Lactobacillus casei, In one embodiment, the microbial cell is an intestinal bacteria. In another embodiment, the microbial cell is an intestinal bacteria. Those skilled in the art will recognize other bacterial strains upon reading this disclosure.

[0085] According to a second aspect, the present invention provides the use of genetically engineered microbial cells as described herein for producing lactose or oligosaccharides comprising a galactose-β1,4-glucose moiety at its reducing end. In an additional and / or alternative embodiment, genetically engineered microbial cells are used to produce lactose or a method for oligosaccharides comprising a galactose-β1,4-glucose moiety at its reducing end, wherein the microbial cells are 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 fructose, galactose, mannose, xylose, rhamnose, glycerol, succinic acid, pyruvic acid and malic acid. Preferably, the mixed feedstock is a mixture of glucose and fructose, more preferably an equimolar mixture of glucose and fructose, most preferably comprising or consisting of hydrolyzed sucrose.

[0086] According to a third aspect, the present invention provides a method for producing lactose or an oligosaccharide of interest containing a galactose-β1,4-glucose moiety at its reducing end, the method comprising the following steps:

[0087] a) providing a genetically engineered microbial cell as described herein;

[0088] b) cultivating the microbial cells in a culture medium under conditions that allow production of the lactose or oligosaccharide of interest, wherein the culture medium contains as a main carbon source 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; and

[0089] c) recovering lactose or target oligosaccharides from the culture medium and / or microbial cells.

[0090] In an additional and / or alternative embodiment, 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-fucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-difucosylhexose I, lacto-N-difucosylhexose II, lacto-N-sialylpentose LSTa, LSTb, LSTc.

[0091] 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.

[0092] In an additional and / or alternative embodiment, the microbial cells are cultured in the absence of an exogenous supply of lactose, particularly when cultured for the production of oligosaccharides of interest.

[0093] The present invention will be described using specific embodiments and accompanying drawings, but the invention is not limited thereto but only by the claims. In addition, the terms first, second, etc. in the specification and claims are used to distinguish similar elements and are not necessarily used to describe a sequence in time, space, in a sequenced manner, or in any other manner. It should be understood that the terms so used are interchangeable where appropriate, and that the embodiments of the invention described herein can be operated in other sequences than those described or illustrated herein.

[0094] 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 indicating the presence of the stated features, integers, steps, or components, but not excluding the presence or addition of one or more other features, integers, steps, or components, or groups thereof. Thus, 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. This means that, for the purposes of the present invention, the only relevant components of the device are A and B.

[0095] Throughout this specification, reference to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" throughout this specification are not necessarily all referring to the same embodiment, but may refer to the same embodiment. 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 skilled in the art based on this disclosure.

[0096] Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof in order to simplify the disclosure and facilitate understanding of one or more aspects of the invention. However, this method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. On the contrary, as reflected in the following claims, aspects of the invention lie in less than all the features of a single aforementioned disclosed embodiment. Accordingly, the claims following a specific description are expressly incorporated into that specific description, with each claim standing on its own as a separate embodiment of the invention.

[0097] Furthermore, while some embodiments described herein include some features included in other embodiments but not others, combinations of features from different embodiments are 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.

[0098] Furthermore, some embodiments are described herein as methods or combinations of method elements that can be implemented by a processor of a computer system or by other means for performing the functions. Thus, a processor having the necessary instructions to perform such methods or method elements constitutes a means for performing the methods or method elements. Furthermore, the elements of the apparatus embodiments described herein are examples of means for performing the functions performed by the elements in order to implement the invention.

[0099] In the description and drawings provided herein, numerous specific details are provided. However, it should be understood that embodiments of the present invention may be implemented without these specific details. In other cases, well-known methods, structures, and techniques are not shown in detail to avoid obscuring an understanding of this specification.

[0100] The present invention will now be described by the specific description of several embodiments of the present invention. Obviously, other embodiments of the present invention can be provided according to the knowledge of those skilled in the art without departing from the true spirit or technical teachings of the present invention, and the present invention is only limited by the terms of the appended claims.

[0101] In one embodiment, a cell having the genotype lacY - , lacZ - 、fuclK - 、wcaJ - The Escherichia coli strain is metabolically engineered to efficiently produce 2'-fucocarbonyl lactose through total fermentation using a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) as the primary carbon and energy source. Consequently, expression of the glucokinase gene glk, the glucose dehydrogenase gene gcd, and / or the glucose PTS permease gene ptsG is reduced and / or eliminated. Furthermore, a glucose permease gene is expressed or overexpressed in the E. coli strain.

[0102] In addition, at least one of the E. coli genes manA, manC, manB, gmd, wcaG, pgm, galU and galE is expressed / overexpressed, as well as heterologous β-1,4-galactosyltransferase (which is capable of transferring galactose from UDP-galactose to glucose to produce lactose), and α-1,2-fucosyltransferase (which is capable of transferring fucose from GDP-fucose to lactose to produce 2'-fucosyllactose).

[0103] In a preferred embodiment, the production strain is further engineered by reducing and / or decreasing the expression of the phosphofructokinase gene 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 production strain thus engineered to be cultured on a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) as the primary carbon and energy source, while avoiding hindering the metabolism of the strain, but increasing the precursor supply (glucose and fructose-6-phosphate and glucose-6-phosphate) for the production of 2'-fucosyllactose by full fermentation.

[0104] Reference Figure 1, schematically showing an exemplary microbial cell of the present invention. The microbial cell is capable of producing 2'-FL when cultured on a mixed feedstock consisting of glucose (Glu) and fructose (Fru), but the mixed feedstock does not contain lactose (Lac). The microbial cell expresses polynucleotides encoding a glucose transporter (Glf) and a fructose transporter, which are used to import glucose and fructose into the cell, respectively. Since the expression of the glucose kinase Glk has been eliminated by deletion or functional inactivation of the glk gene, any glucose imported by the cell can be used as a substrate for the β1,4-galactosyltransferase GalTpm1141 to produce lactose (Lac) within the cell through the UDP-galactose biosynthesis pathway.

[0105] Imported fructose is phosphorylated by the cell's fructose-6 kinase to generate an intracellular pool of Fru-6-P. 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, producing Lac. Another portion of the fru-6-P pool is used in the GDP-L-Fuc biosynthetic pathway to produce GDP-L-fucose. This GDP-L-fucose serves as a fucose donor for the 2'-fucosyltransferase WbgL. A third portion of the intracellular Fru-6-P pool is used for energy and biomass production by converting fru-6-P to Fru-1,6-diP via the cellular phosphofructokinases PfkA and / or PfkB.

[0106] Figure 2 Schematically shows another exemplary microbial cell of the present invention, which 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). Figure 1 In the exemplary microbial cell shown in , the microbial cell also 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 used by the microbial cell to produce UDP-Gal or Fru-6-P. In a variant of the microbial cell (not shown), the pgi gene of the cell encoding glucose-6-phosphate isomerase (Pgi) is deleted. Along with the deletion of the glk gene, the microorganism has been genetically engineered so that the free glucose monomers obtained by Glf can be used as substrates for β1,4-galactosyltransferase, and any Glu-6-P obtained by PtsG can be used for UDP-Gal biosynthesis.

[0107] Figure 3Schematically shows another exemplary microbial cell of the present invention, which 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). Figure 2 The exemplary microbial cell shown in , 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-bisphosphatase. The microbial cell has fructose-1,6-bisphosphatase (GlpX) activity. Thus, the genetically engineered microbial cell is metabolically engineered so that fructose and / or fructose-1-P can be used by the cell for UDP-Gal biosynthesis.

[0108] Furthermore, deletion or functional inactivation of the phosphofructokinase gene renders the cells devoid of phosphofructokinase (PfkA / PfkB) activity. Deletion or functional inactivation of the phosphofructokinase gene impairs the conversion of Fru-6P to Fru-1,6-P, thereby preventing the use of Fru-6-P to generate high-energy triphosphates and increasing 2'-FL production.

[0109] Example

[0110] Example 1 - Preparation of mixed monosaccharide raw materials

[0111] By dissolving 500g sucrose in water, prepare 50% (w / v) sucrose solution.The final volume of solution is 1 liter.Under 30 ℃ to 35 ℃ temperature, by using 96% (v / v) sulfuric acid to adjust pH.Then, by solution vertical autoclave (Systec VX-65, Linden, Germany) at 121 ℃ sterilization 45 minutes.Sampling before and after heat sterilization, and keeping freezing before carrying out high performance liquid chromatography (HPLC) analysis.Employing is connected to RID-10A refractive index detector (Shimadzu, Germany) and Waters XBridge Amide post 3.5 μ m (250x4.6mm) (Eschborn, Germany) of Shimadzu HPLC system to carry out HPLC. Isocratic elution was performed at 35°C 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 a flow rate of 1.4 mL min -1The samples were cleaned up by solid phase extraction on an ion exchange matrix (Strata ABW, Phenomenex). 10 μl of sample (1:5 dilution) was applied to the column. Finally, the relative amounts of detected sugars were determined. As shown in Table 1, before heat treatment, the conversion of sucrose to the monosaccharides glucose and fructose increased with decreasing solution pH. When acidified with sulfuric acid, complete sucrose cleavage was observed at pH ≤ 3.50.

[0112]

[0113] Table 1: Relative amounts of sugars detected in a 50% (w / v) sucrose solution before and after pH adjustment by heat sterilization. pH adjustment was performed with 96% (v / v) sulfuric acid. The percentage amounts of sugars detected by HPLC (area under the curve; AUC) are depicted.

[0114] Example 2 - Feedstock-dependent growth of various gene-deleted strains

[0115] The wild type E. coli BL21(DE3) strain and the mutant E. coli pfkA were compared. - (△pfkA), Escherichia coli pfkB - (△pfkB), Escherichia coli pfkA - pfB - Growth behavior of (ΔpfkAΔpfkA). 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 shake flasks containing 20 mL of mineral salts medium containing 7 g L - 1 NH4H2PO4, 7g·L -1 K2HPO4, 2g·L -1 KOH, 0.3 g·L -1 Citric acid, 2g·L -1 MgSO4×7·H2O and 0.015g·L -1 CaCl2×6·H2O, supplement 1mL·L -1 Trace element solution (54.4 g·L -1 Ammonium ferric citrate, 9.8 g·L -1 MnCl2×4·H2O、1.6g·L -1 CoCl2×6·H2O、1g·L -1 CuCl2×2·H2O, 1.9g·L -1 H3BO3,9g·L -1ZnSO4×7·H2O、1.1g·L -1 Na2MoO4×2·H2O、1.5g·L -1 Na2SeO3, 1.5g·L -1 NiSO4×6·H2O) and containing 2% (m / v) glucose (A) or 1% (w / v) glucose / 1% (w / v) fructose (B) as carbon source. The culture was inoculated to OD 0.1 and the OD 600 Measure and monitor growth and development over 26 hours. Figure 2 As shown, when glucose was provided as the sole carbon and energy source, E. coli pfkA - pfB - Little growth was shown, and when mixed monosaccharide feedstocks were available, growth was indistinguishable from that of the wild-type strain and the single deletion mutants.

[0116] Example 3 - Full fermentation of 2'-fucosyllactose by engineered E. coli strains grown on mixed monosaccharide feedstocks

[0117] The genotype pfkA was further genetically engineered by overexpressing enzymes for de novo GDP-fucose synthesis (ManB, ManC, Gmd, WcaG), the 2'-fucosyltransferase gene wbgL from Escherichia coli:O126, the sugar efflux transporter gene yberc0001_9420 from Yersinia bercovieri ATCC 43970, the glucose facilitating factor 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. - , lacZ - 、fuclK - 、wcaJ - 、glk - 、gcd - 、pstG -Escherichia coli BL21 (DE3) strain was used. 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. EZ-Tn5™ transposase (Epicentre, USA) was used to integrate linear DNA fragments, or the hyperactive C9-mutant of the mariner transposase Himar1 (Proc. Natl. Acad. Sci. 1999, USA 96:11428-11433) was used for transposition. These genes were codon-optimized for expression in E. coli and synthetically prepared by GenScript.

[0118] The resulting E. coli strain was cultured in a 3 L fermentor (New Brunswick, Edison, USA) at 30°C, starting with 1000 mL of mineral salts medium containing 7 g L -1 NH4H2PO4, 7g·L -1 K2HPO4, 2g·L - 1 KOH, 0.3 g·L -1 Citric acid, 2g·L -1 MgSO4×7·H2O and 0.015g·L -1 CaCl2×6·H2O, supplement 1mL·L -1 Trace element solution (54.4 g·L -1 Ammonium ferric citrate, 9.8 g·L -1 MnCl2×4·H2O、1.6g·L -1 CoCl2×6·H2O、1g·L -1 CuCl2×2·H2O, 1.9g·L -1 H3BO3,9g·L -1 ZnSO4×7·H2O、1.1g·L -1 Na2MoO4×2·H2O、1.5g·L -1 Na2SeO3, 1.5g·L -1 NiSO4 × 6·H2O) and containing 2% (m / v) hydrolyzed sucrose as a carbon source. The culture was started by adding a 2.5% (v / v) inoculum from a preculture grown in the same medium. The end of the batch phase was characterized by a rise in dissolved oxygen levels. Immediately after leaving the batch phase, a carbon feed consisting of fully hydrolyzed sucrose was applied, supplemented with 2 g·L -1 MgSO4×7·H2O, 0.015g·L -1 CaCl2×6·H2O and 1mL·L-1 Trace element solution. Feed rate is 12.0-15.0 mL·L -1 ·h -1 , refer to the starting volume. Inflation is maintained at 3 L min -1 .

[0119] The dissolved oxygen was maintained at 20-30% saturation by controlling the stirring rate. The pH was maintained at 6.7 by adding 25% ammonia solution. The culture was continued for 86 hours, and a large amount of 2'-FL was produced in the culture supernatant.

Claims

1. A genetically engineered microbial cell for producing lactose or an oligosaccharide containing a galactose-β1,4-glucose moiety at its reducing end from a mixed feedstock of glucose and fructose, wherein the microbial cell has: at least one glucose transporter for transporting glucose from the culture medium into the cytoplasm of the microbial cells, thereby making the free glucose available for the intracellular biosynthesis of lactose; -UDP-galactose biosynthetic pathway; and - at least one β-1,4-galactosyltransferase capable of galactosylating free glucose to produce lactose within the cell, The glucokinase gene of the microbial cell has been deleted or functionally inactivated, The microbial cells are bacterial cells selected from the following bacterial species: Escherichia coli, The microbial cell expresses or overexpresses a gene selected from Zymomonas mobilis, wherein the gene selected from glf encodes a glucose transporter for transferring glucose from the culture medium to the cytoplasm of the microbial cell, thereby making the free glucose available for the biosynthesis of lactose in the cell. The β-1,4-galactosyltransferase is encoded by a gene selected from the group consisting of Pasteurella multocida galTpm1141, GenBank: AEC04686.

2. The genetically engineered microbial cell according to claim 1, wherein the microbial cell has phosphoglucomutase, UTP-glucose-1-phosphate-uridyl transferase, and UDP-glucose 4-epimerase.

3. The genetically engineered microbial cell according to claim 1 or 2, wherein the microbial cell has at least one additional glycosyltransferase.

4. The genetically engineered microbial cell of claim 3, wherein the at least one additional glycosyltransferase is selected from the group consisting of fucosyltransferases, sialyltransferases, glucosaminyltransferases, and galactosyltransferases.

5. The genetically engineered microbial cell of claim 1, wherein the microbial cell comprises a glucose transporter phosphotransferase system. The genetically engineered microbial cell of claim 1 , wherein the microbial cell has a fructose transporter.

7. The genetically engineered microbial cell of claim 1, wherein the microbial cell has a fructose-specific phosphotransferase system, and wherein the cell further comprises 1-phosphofructokinase.

8. The genetically engineered microbial cell of claim 6, wherein the microbial cell comprises fructokinase-6 activity and 6-phosphofructokinase-1 activity.

9. The genetically engineered microbial cell of claim 7, wherein the microbial cell comprises fructose-1,6-bisphosphatase.

10. The genetically engineered microbial cell according to claim 7 or 9, wherein the microbial cell comprises a deletion or functional inactivation of its glucose-6-phosphate isomerase.

11. The genetically engineered microbial cell of claim 3, wherein the additional glycosyltransferase is a fucosyltransferase, and wherein the microbial cell has mannose-6-phosphate isomerase, phosphomannose mutase, mannose-1-phosphate-guanylyltransferase, GDP-mannose-4,6-dehydratase, GDP-L-fucose synthase.

12. The genetically engineered microbial cell of claim 1, wherein the microbial cell comprises an exporter or permease that exports the oligosaccharide of interest from the cell.

13. The genetically engineered microbial cell according to claim 1, wherein The microbial cell comprises a sugar efflux transporter.

14. Use of the genetically engineered microbial cell according to any one of claims 1 to 13 for the production of lactose or an oligosaccharide comprising a galactose-β1,4-glucose moiety at its reducing end.

15. A method for producing lactose or an oligosaccharide of interest containing a galactose-β1,4-glucose moiety at its reducing end, the method comprising the following steps: a) providing a genetically engineered microbial cell according to any one of claims 1 to 13; b) cultivating the microbial cells in a culture medium under conditions that allow production of the lactose or oligosaccharide of interest, wherein the culture medium contains as a main carbon source 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; and c) recovering lactose or target oligosaccharides from the culture medium and / or the microbial cells.

16. The method according to claim 15, wherein the target oligosaccharide is a human milk oligosaccharide selected from the following: 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, 3'-sialyllactose, 6'-sialyllactose, 3-fucosyl-3'-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-difucosylhexose I, lacto-N-difucosylhexose II, lacto-N-sialylpentose LSTa, LSTb, LSTc.

17. The method according to claim 15 or 16, wherein the mixture of glucose and at least one additional monosaccharide is a mixed feed of glucose and fructose.

18. The method according to claim 17, wherein The mixed raw material of glucose and fructose is obtained by hydrolyzing sucrose.

19. The method of claim 15 or 16, wherein the microbial cells are cultured in the absence of an exogenous supply of lactose.

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