N-acetylglucosaminyltransferase enzymes with altered substrate specificity and their use in fermentative oligosaccharide production

Abstract

Provided are variants of a β-1,3-N-acetylglucosaminyltransferase polypeptide which possess a higher substrate specificity for lactose than for lacto-N-neotetraose as compared to Neisseria meningitidis LgtA, microbial cells possessing such a variant β-1,3-N-acetylglucosaminyltransferase polypeptide, and their use in producing oligosaccharides of interest which contain an N-acetylglucosamine moiety.

Description

[0001] N-acetylglucosaminyltransferase enzymes with altered substrate specificity and their use in fermentative oligosaccharide production

[0002] The present invention relates to the field of fermentative production of oligosaccharides containing an N-acetylglucosamine moiety, and concerns variants of an N-acetylglucosaminyltransferase as well as the production oligosaccharides containing an N-acetylglucosamine moiety utilizing said N-acetylglucosaminyl- transferase variants.

[0003] Background

[0004] Human milk contains large amounts of sugars. The predominant sugar in human milk is the disaccharide lactose which constitutes the energy source for a breast fed infant. Human milk further contains more than 150 structurally distinct oligosaccharides, the so-called human milk oligosaccharides (HMOs). HMOs are not digested and metabolized by the infant. However, HMOs are known to provide several health benefits to the infant. For example, numerous HMOs exhibit prebiotic effects and thereby promote the infant’s development of a healthy gut microbiome. Certain HMOs interfere with the adhesion of viral or bacterial pathogens to epithelial cells in the gut and thereby prevent enteric diseases. HMOs were shown to modulate the innate immune system, thus participating in the maturation of an infant’s immune system. Furthermore, certain HMOs are contributing to the development of the infant’s nervous system and cognitive capabilities.

[0005] Various processes were developed for the industrial scale production of selected HMOs such that these HMOs become available as ingredients to infant formula at reasonable costs. The fermentative HMO production using metabolically engineered microorganism, such as recombinant yeast or E. coli, for intracellular biosynthesis of a particular HMO, and subsequent recovery of the HMO from the culture medium or microbial cell proved to be the unsurpassed way of industrial scale HMO production from a technical as well as from an economic perspective.

[0006] HMOs contain a lactose moiety at their reducing end which is supplemented with at least one of a fucose (Fuc) moiety, an N-acetylneuraminic acid (NeuNAc) moiety, an N-acetylglucosamine (GIcNAc) moiety and / or a galactose (Gal) moiety. Exemplary HMOs containing an N-acetylglucosamine moiety are for example lacto-N-triose II (LNT-II; LNT-2; GlcNAc(pi ,3)Gal(pi ,4)Glc), lacto-N-tetraose (LNT; Gal(pi ,3)GlcNAc (pi ,3)Gal(pi ,4)Glc), lacto-N-neotetraose (LNnT; Gal(pi ,4)GlcNAc(pi ,3)Gal(pi ,4) Glc), and the fucosylated and / or sialylated derivatives thereof, e.g. lacto-N-fuco- pentaose I (LNFP I), lacto-N-fucopentose II (LNFP II), lacto-N-fucopentaose III (LNFP III), lacto-N-difucohexaose I (LDFH I), lacto-N-difucohexaose II (LDHF II), si alyl lacto-N-tetraose a (LST a), sialyllacto-N-tetraose b (LST b), sialyllacto-N-tetra- ose c (LST c) disialyllacto-N-tetraose (DSLNT).

[0007] Lacto-N-tetraose has prebiotic effects, immune modulatory effects, anti-inflammatory effects, intestinal cell responses regulatory effects, antibacterial and antiviral activity. Lacto-N-neotetraose is known to exhibit anti-inflammatory properties, to induce type 2 immune responses, to induce angiogenesis, and to possess antibacterial effects.

[0008] Biosynthesis of LNnT by metabolically engineered bacteria has been described e.g. in US 2009 / 0082307 A1. In principle, exogenous lactose is internalized by the bacterial cell. As the bacterial cell is LacZ", it does not hydrolyze the internalized lactose. Expression of a recombinant IgtA gene allows the production of the LgtA enzyme, a p-1 ,3-N-acetylglucosaminyltransferase, which intracellularly transfers a GIcNAc moiety from UDP-GIcNAc onto a lactose molecule to generate lacto-N-triose II. The trisaccharide LNT-II may then be utilized as an educt by e.g. a galactosyltransferase such as the p-1 ,4-galactosyltransferase LgtB from Neisseria menin- gitides, which transfers a galactose moiety from UDP-Gal to the N-acetylgluco- samine moiety of LNT-II to form lacto-N-neo-tetraose (P-D-Gal-[1-4]-p-D-GlcNAc- [1-3]-p-D-Gal-[1-4]-p-D-Glc).

[0009] In other embodiments, a p-1 ,3-galactosyltransferase such as E. coli WbgO, which transfers a galactose moiety from UDP-Gal to the N-acetylglucosamine moiety of LNT-II to form lacto-N-tetraose (P-D-Gal-[1-3]-p-D-GlcNAc-[1-3]-p-D-Gal-[1-4]-p-D- Glc) may be used to intracellularly synthesize LNT.

[0010] The p-1 ,3-N-acetylglucosaminyltransferase LgtA of Neisseria meningitidis ( / V.m.LgtA) was expressed at high levels in E. coli and characterized as catalyst in the synthesis of GlcNAc(pi-3)Gal linkages (Blixt, E. et al. (1999) Glycobiology 9: 1061-1071). N. meningitides LgtA appeared to be unusual in that it displays a broad acceptor specificity in vitro towards both a- and p-galactosides, whether structurally related to N- or O-protein-, or lipid-linked oligosaccharides. Although lactose was found to be a highly preferred acceptor molecule, the recombinant enzyme also acts efficiently on monomeric and dimeric N-acetyllactosaminoglycans. N. meningitides LgtA showed a high donor promiscuity towards UDP-GalNAc, but not towards other UDP-sugars, and can catalyze the introduction of GalNAc in p-1,3-linkage to an a- or p-Gal moiety in an acceptor molecule.

[0011] Despite the preference of / V.m.LgtA for lactose as acceptor molecule, the enzyme may also initiate to generate undesired by-products, including conversion of a desired HMO such as LNnT into longer-chain oligosaccharides, e.g. para-lacto-N- neohexaose (pLNnH). Para-lacto-N-neohexaose (pLNnH) is a neutral hexasaccharide exhibiting the structure: Gal(pi,4)GlcNAc-(pi ,3)Gal(pi ,4)GlcNAc(pi ,3) Gal(pi ,4)Glc, yet an undesired by-product in the production of e.g. LNnT.

[0012] Therefore, there was a need for improved p-1 ,3-N-acetylglucosaminyltransferase enzymes that possess an even higher substrate specificity for lactose as compared to LNT or LNnT to reduce the level of undesired by-products such as e.g. pLNnH.

[0013] Certain variants of the p-1 ,3-N-acetylglucosaminyltransferase LgtA possessing higher substrate specificity for binding to its intended substrates relative to longer- chain oligosaccharides are disclosed in WO 2022 / 133 093 A1.

[0014] However, the need for improved p-1,3-N-acetylglucosamine transferase enzymes that result in an improved biosynthesis of LNT-II, LNT, LNnT as compared to pLNnH remains.

[0015] Summary

[0016] The present disclosure provides variant p-1,3-N-acetylglucosaminyltransferase polypeptides, nucleic acid molecules comprising at least one nucleotide sequence which encodes one of the variant p-1 ,3-N-acetylglucosaminyltransferase polypeptides, microbial cells containing at least one of the variant p-1 ,3-N-acetylgluco- saminyltransferase polypeptides and / or one of the nucleotide sequences encoding one of the variant p-1 ,3-N-acetylglucosaminyltransferase polypeptides, the use of the variant p-1,3-N-acetylglucosaminyltransferase polypeptides and / or the microbial cells for the production of an oligosaccharide of interest which comprises at least one N-acetylglucosamine moiety, methods for producing an oligosaccharide of interest which contains at least one N-acetylglucosamine moiety, wherein the method utilizes one of the variant p-1,3-N-acetylglucosaminyltransferase polypeptides or a microbial host cell containing at least one of the variant p-1 ,3-N-acetyl- glucosaminyltransferase polypeptides. Further disclosed is a method of altering the substrate specificity of a p-1 ,3-N-acetylglucosaminyltransferase polypeptide.

[0017] In a first aspect, provided are variants of a p-1,3-N-acetylglucosaminyltransferase polypeptide, wherein the amino acid sequences of the variant p-1 ,3-N-acetyl- glucosaminyltransferase polypeptides comprise at least one amino acid substitution relative to the amino acid sequence of the p-1 ,3-N-acetylglucosaminyltransferase polypeptide as set forth in SEQ ID NO: 1, wherein the at least one amino acid substitution comprises an amino acid substitution of at least one of the amino acids at amino acid positions 158, 249 and 256 of the amino acid sequence set forth in SEQ ID NO: 1 with an amino acid that possesses a hydrophobic side chain.

[0018] In a second aspect, provided are nucleic acid molecules comprising a nucleotide sequence that encodes one of the variant p-1,3-N-acetylglucosaminyltransferase polypeptides which comprise at least one amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 1 , wherein the at least one amino acid substitution comprises an amino acid substitution of at least one of the amino acids at amino acid positions 158, 249 and 256 of the amino acid sequence as set forth in SEQ ID NO: 1 with an amino acid that possesses a hydrophobic side chain.

[0019] In a third aspect, provided are genetically-engineered microbial cells comprising a variant p-1,3-N-acetylglucosaminyltransferase polypeptide which comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, wherein the at least one amino acid substitution comprises an amino acid substitution of at least one of the amino acids at amino acid positions 158, 249 and 256 of the amino acid sequence as set forth in SEQ ID NO: 1 with an amino acid that possesses a hydrophobic side chain. In a fourth aspect, provided is the use of a genetically-engineered microbial cells comprising a variant p-1 ,3-N-acetylglucosaminyltransferase polypeptide which comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, wherein the at least one amino acid substitution comprises an amino acid substitution of at least one of the amino acids at amino acid positions 158, 249 and 256 of the amino acid sequence as set forth in SEQ ID NO: 1 with an amino acid that possesses a hydrophobic side chain, for the production of an oligosaccharide of interest comprising at least one N-acetylgluco- samine moiety.

[0020] In a fifth aspect, provided is a method for producing an oligosaccharide of interest, wherein said oligosaccharide of interest comprises at least one N-acetylglucosamine moiety, the method comprises culturing a population of genetically engineered microbial cells comprising a variant p-1,3-N-acetylglucosaminyltransferase polypeptide which comprises at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO: 1, wherein the at least one amino acid substitution comprises an amino acid substitution of at least one of the amino acids at amino acid positions 158, 249 and 256 of the amino acid sequence as set forth in SEQ ID NO: 1 with an amino acid that possesses a hydrophobic side chain in a culture medium and under conditions that are permissive for the microbial cells to synthesize the oligosaccharide of interest comprising at least one N- acetylglucosamine moiety.

[0021] In a further aspect, provided are fermentation compositions comprising a population of genetically engineered microbial cells comprising a variant p-1,3-N-acetylgluco- saminyltransferase polypeptide which comprises at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO: 1 , wherein the at least one amino acid substitution comprises an amino acid substitution of at least one of the amino acids at amino acid positions 158, 249 and 256 of the amino acid sequence as set forth in SEQ ID NO: 1 with an amino acid that possesses a hydrophobic side chain, and an oligosaccharide of interest comprising at least one N-acetylgluco- samine moiety, wherein the oligosaccharide of interest has been synthesized by the genetically engineered microbial cells. In yet another aspect, provided is a method for altering the substrate specificity of a P-1 ,3-N-acetylglucosaminyltransferase polypeptide possessing an amino acid sequence as set forth in SEQ ID NO: 1.

[0022] Brief description of the drawings

[0023] FIG. 1 displays the nucleotide sequence of the Neisseria meningitidis (3-1 ,3-N- acetylglucosaminyltransferase LgtA encoding gene IgtA (lowercase), and the deduced amino acid sequence of the Neisseria meningitidis p-1 ,3-N-acetyl- glucosaminyl-transferase LgtA (upper case).

[0024] FIG. 2 demonstrates the LNnT productivity of different E. coli strains which only differ in the amino acid sequence of their exogenous p-1 ,3-N-acetyl- glucosaminyl-transferase.

[0025] FIG. 3 illustrates the relative biosynthesis of LNT-II, LNnT and hexaoses by E. coli strains which differ in the amino acid sequence of their p-1 ,3-N- acetylglucosaminyl-transferase.

[0026] FIG. 4 shows the Michaelis Menten kinetics of / V.m.LgtA and the variant p-1 ,3-N- acetylglucosaminyltransferase polypeptide / V.m.LgtA(H158W, H249F, Q256W) for lactose.

[0027] FIG. 5 shows the Michaelis Menten kinetics of / V.m.LgtA and the p-1 ,3-N-acetyl- glucosaminyltransferase polypeptide variant / V.m.LgtA(H158W, H249F, Q256W) for LNnT.

[0028] Detailed description

[0029] According to the first aspect, provided are variants of a p-1 ,3-N-acetylglucosaminyl- transferase polypeptide, wherein the variant p-1 ,3-N-acetylglucosaminyltransferase polypeptide comprises at least one amino acid substitutions relative to the amino acid sequence of the p-1 ,3-N-acetylglucosaminyltransferase polypeptide as set forth in SEQ ID NO: 1. The at least one amino acid substitution comprises a substitution of at least one of the amino acids at amino acid positions 158, 249 and 256 of the amino acid sequence set forth in SEQ ID NO: 1 with an amino acid that possess a hydrophobic side chain.

[0030] A p-1 ,3-N-acetylglucosaminyltransferase catalyzes the introduction of GIcNAc from uridine diphosphate N-acetylglucosamine (UDP-GIcNAc) in a pi ,3-linkage to accepting Gal residues within an oligosaccharide. The amino acid sequence set forth in SEQ ID NO: 1 is the amino acid sequence of the Neisseria meningitidis p- 1,3-N-acetylglucosaminyltransferase LgtA (UniProtKB Entry No. Q9JXQ6) (https: / / www.uniprot.org / uniprotkb / ) based on NCBI Reference Sequence Database RefSeq protein record WP_002257440.1 as of 15-NOV-2022. The nucleotide sequence of the Neisseria meningitidis p-1,3-N-acetylglucosaminyltransferase LgtA amino acid sequence of the Neisseria meningitidis p-1 ,3-N-acetylglucosaminyl- transferase gene IgtA and the deduced amino acid sequence of Neisseria meningitidis p-1,3-N-acetylglucosaminyltransferase LgtA is shown in FIG. 1.

[0031] The variant p-1 ,3-N-acetylglucosaminyltransferase polypeptide has a sequence identity of at least 80% to the amino acid sequence as set forth in SEQ ID NO: 1. Notwithstanding the sequence identity of at least 80%, the variant p-1 ,3-N-acety- Iglucosaminyltransferase polypeptide comprises an amino acid that possesses a hydrophobic side chain at one or more of amino acid positions 158, 249 and 256 with respect to the amino acid sequence set forth in SEQ ID NO: 1.

[0032] The variant p-1 ,3-N-acetylglucosaminyltransferase polypeptide that possesses an amino acid having a hydrophobic side chain at one of the amino acid positions 158. 249 and 256 of an amino acid sequence as set forth in SEQ ID NO: 1 exhibits an increased substrate specificity for lactose over lacto-N-neotetraose or lacto-N- neohexaose as compared to the substrate specificity of a p-1 ,3-N- acetylglucosaminyltransferase polypeptide having the amino acid sequence as set forth in SEQ ID NO: 1.

[0033] Substrate specificity is determined by steady-state kinetic parameters derived from the Michaelis-Menten equation and measured experimentally, the kcat and Kmvalues, are widely used to characterize enzymatic reactions. Specifically, kcat is a term that defines the maximal rate, Kmis the substrate concentration at which reaction reaches half of its maximal rate, and kcat / Kmis considered a measure of the catalytic efficiency or substrate specificity. A substrate with a higher fcat / m value is considered a better or preferred substrate.

[0034] In certain embodiments, the variant p-1 ,3-N-acetylglucosaminyltransferase polypeptide comprises at least two amino acid substitution relative to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the amino acid substitution comprise substitution of at least two of the amino acids at amino acid positions 158, 249 and 256 of the amino acid sequence as set forth in SEQ ID NO: 1 with amino acids that possess hydrophobic side chains.

[0035] In additional and / or alternative embodiments, the variant p-1 ,3-N-acetylgluco- saminyltransferase polypeptide comprises amino acid substitutions of the amino acids at amino acid positions 249 and 256 of the amino acid sequence of SEQ ID NO: 1. In additional embodiments, the variant p-1 ,3-N-acetylglucosaminyl- transferase polypeptide comprises a further amino acid substitution at amino acid position 158 of the amino acid sequence set forth in SEQ ID NO. 1.

[0036] In some embodiments, the variant p-1 ,3-N-acetylglucosaminyltransferase polypeptide comprises an amino acid substitution of the amino acid residue at position 249 of SEQ ID NO: 1 , wherein the amino acid residue at position 249 of SEQ ID NO: 1 is substituted with a hydrophobic amino acid residue. The amino acid residue at amino acid position 249 of SEQ ID NO: 1 is a histidine (H) residue. Thus, in some embodiments, the variant p-1 ,3-N-acetylglucosaminyltransferase polypeptide comprises a hydrophobic amino acid residue instead of the histidine residue at the amino acid position of or corresponding to amino acid position 249 of SEQ ID NO: 1.

[0037] In additional and / or alternative embodiments, the variant p-1 ,3-N-acetylgluco- saminyltransferase polypeptide comprises an amino acid substitution of the amino acid residue at position 256 of SEQ ID NO: 1 , wherein the amino acid residue at position 256 of SEQ ID NO: 1 is substituted with a hydrophobic amino acid residue. The amino acid residue at amino acid position 256 of SEQ ID NO: 1 is a glutamine residue. Thus, in some embodiments, the variant p-1 ,3-N-acetylglucosaminyl- transferase polypeptide comprises a hydrophobic amino acid instead of the glutamine residue at the amino acid position of or corresponding to amino acid position 256 of SEQ ID NO: 1.

[0038] In additional and / or alternative embodiments, the variant p-1,3-N-acetylgluco- saminyltransferase polypeptide comprises an amino acid substitution of the amino acid residue at position 158 of SEQ ID NO: 1, wherein the amino acid residue at position 158 of SEQ ID NO: 1 is substituted with a hydrophobic amino acid residue. The amino acid residue at amino acid position 158 of SEQ ID NO: 1 is a histidine residue. Thus, in some embodiments, the variant p-1,3-N-acetylglucosaminyl- transferase polypeptide comprises a hydrophobic amino acid residue instead of the histidine residue at the amino acid position of or corresponding to amino acid position 158 of SEQ ID NO: 1.

[0039] Each of the hydrophobic amino acid residues substituting one or more of the amino acid residues at the amino acid positions 249, 256 and 158 of SEQ ID NO: 1 is selected from the group of amino acid residues consisting of glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M) and tryptophane (W). In some embodiments, the hydrophobic amino acid is selected from phenylalanine (F) and tryptophane (W).

[0040] In additional and / or alternative embodiments, the variant p-1,3-N-acetylgluco- saminyltransferase polypeptide comprises a substitution of the amino acid residue at amino acid position 249 of SEQ ID NO: 1 with a phenylalanine (F) and / or a substitution of the amino acid residue at amino acid position 256 of SEQ ID NO: 1 with tryptophane residue.

[0041] In additional and / or alternative embodiments, the variant p-1,3-N-acetylgluco- saminyltransferase polypeptide further comprises a substitution of the amino acid residue at amino acid position 158 of SEQ ID NO: 1 with a tryptophane residue.

[0042] In certain embodiments, the variant p-1,3-N-acetylglucosaminyltransferase polypeptide has an amino acid sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the variant p-1,3-N-acetylglucosaminyltransferase polypeptide has an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 1 only by way of (i) the two acid residue substitutions at amino acid positions 249 and 256 of the amino acid sequence set forth in SEQ ID NO: 1 or the three amino acid residue substitutions at amino acid positions 158, 249 and 256 of the amino acid sequence set forth in SEQ ID NO: 1 with hydrophobic amino acid residues, and, optionally, (ii) by one or more additional, conservative amino acid residue substitutions or amino acid residue deletions. In yet other embodiments, the variant p-1,3-N-acetylglucosaminyltransferase polypeptide has an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 1 only by way of the two or three amino acid residue substitutions, i.e. the amino acid residue substitutions H249F, Q256W or H158W, H249F, Q256W.

[0043] In other embodiments, the variant p-1,3-N-acetylglucosaminyltransferase polypeptide possessing the at least two amino acid substitutions of at least two of the amino acid residues selected from the amino acid residues at amino acid positions 158, 249 and 256 of SEQ ID NO: 1 has an amino acid sequence that is at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 % at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % identical to the amino acid sequence set forth in SEQ ID NO: 1.

[0044] The term “sequence identity of [a certain] %” in the context of two or more nucleotide sequences or amino acid sequences refers to a relationship between the sequences of two polypeptides or polynucleotides, as determined by sequence comparison (alignment). As used herein, “sequence identity” is determined across the entire length of a sequence. “Sequence identity” means that the two or more sequences have nucleotides or amino acid residues in common in the given percentage when compared and aligned. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model, algorithms, or computer program.

[0045] Percent sequence identity of nucleotide sequences or amino acid sequences can be readily calculated by any of the methods known to one of ordinary skill in the art. In preferred embodiments, the “percent identity” of two sequences (e.g., polynucleotide or amino acid sequences) is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST® and XBLAST® programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. Where gaps exist between two sequences, Gapped BLAST ® can be utilized, for example, as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST® programs, the default parameters of the respective programs (e.g., XBLAST® and NBLAST®) can be used, or the parameters can be adjusted appropriately as would be understood by one of ordinary skill in the art.

[0046] Another local alignment technique which may be used, for example, is based on the Smith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981) J. Mol.

[0047] Biol. 147:195-197). A general global alignment technique which may be used, for example, is the Needleman-Wunsch algorithm (Needleman, S. B. & Wunsch, C. D. (1970) J. Mol. Biol. 48:443-453), which is based on dynamic programming.

[0048] Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0049] For multiple sequence alignments, computer programs including Clustal Omega (Sievers et al., Mol Syst Biol. 2011 Oct. 11 ; 7:539) may be used. In some embodiments, a sequence, including a polynucleotide or amino acid sequence, is found to have a specified percent identity to a reference sequence, such as a sequence disclosed in this application and / or recited in the claims when sequence identity is determined using Clustal Omega (Sievers et al., Mol Syst Biol. 2011 Oct. 11; 7:539).

[0050] Examples of sequence alignment algorithms are CLUSTAL Omega (http: / / www.ebi. ac.uk / Tools / msa / clustalo / ), EMBOSS Needle (http: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ), MAFFT (http: / / mafft.cbrc.jp / alignment / server / ) or MUSCLE (http: / / www.ebi.ac.uk / Tools / msa / muscle / ).

[0051] For purposes of the present invention, the “sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277,), preferably version 5.0.0 or later (available at https: / / www.ebi.ac.uk / Tools / psa / emboss needle / ). The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment).

[0052] For purposes of the present invention, the “sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1 970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), 10 preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides x 100) / (Length of Alignment — Total Number of Gaps in Alignment).

[0053] In some embodiments, the variant p-1,3-N-acetylglucosaminyltransferase polypeptide comprises functional fragments of any one of the variant polypeptides described herein before. Said functional fragments comprise variants of the variant P-1 ,3-N-acetylglucosaminyltransferase polypeptides that are truncated by one or more amino acid residues at the N-terminal and / or C-terminal end as compared to any one of the various variant p-1 ,3-N-acetylglucosaminyltransferase polypeptides.

[0054] The variant N-acetylglucosaminyltransferase polypeptides described herein before are advantageous as compared to the p-1,3-N-acetylglucosaminyltransferase polypeptide of SEQ ID NO: 1 in that the variant p-1,3-N-acetylglucosaminyl- transferase polypeptides exhibit increased substrate specificity for lactose over a longer chain oligosaccharide. The longer chain oligosaccharide is either LNnT or p- LNnH. The increased substrate specificity of the variant p-1,3-N-acetylgluco- saminyltransferase polypeptides for lactose over LNnT or pLNnH provides a better biosynthesis of LNT II, LNT, LNnT and fucosylated and / or sialylated derivatives of LNT or LNnT, because the variant p-1,3-N-acetylglucosaminyltransferase polypeptides preferably synthesize LNT II from lactose than a pentaose from LNT or LNnT. Thus, the amount of undesired by-product in the enzymatic synthesis of the tetrasaccharides LNT, LNnT or of their fucosylated and / or sialylated derivatives is reduced as compared to the use of / V.m.LgtA for synthesizing LNT or LNnT, especially when metabolically engineered microbial cells bearing a variant (3-1 ,3-N- acetylglucosaminyltransferase polypeptide instead of / V.m.LgtA are used for the in vivo biosynthesis of LNT or LNnT. Thus, producing LNT, LNnT or one of their fucosylated and / or sialylated derivatives in higher purity by using said variant (3-1,3- N-acetylglucosaminyltransferase polypeptide, for example by using microbial cells possessing such variant p-1,3-N-acetylglucosaminyltransferase polypeptides as disclosed herein before, is facilitated.

[0055] According to the second aspect, provided are nucleic acid molecules comprising a nucleotide sequence that encodes a variant p-1 ,3-N-acetylglucosaminyltransferase polypeptide as disclosed herein before, i.e. a variant of / V.m.LgtA which variant comprises substitutions of one, two or all three of the amino acid residues at amino acid positions 158, 249 and 256 of the amino acid sequence as set forth in SEQ ID NO: 1 with an amino acid or with amino acids that possess a hydrophobic side chain.

[0056] The nucleic acid molecule is either a linear nucleic acid molecule or a circular nucleic acid molecule. Exemplary linear nucleic acid molecules are ribonucleic acids, chromosomes, and gene duplicates. Examples of circular nucleic acid molecules are plasmids, cosmids, bacterial artificial chromosomes, and yeast artificial chromosomes; bacterial chromosomes.

[0057] In some embodiments, the nucleotide sequence encoding the variant p-1 ,3-N- acetylglucosaminyltransferase polypeptide is operably linked to at least one expression control sequence. The term "operably linked" as used herein, shall mean a functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of transcription factor binding sites) and a second nucleotide sequence such as a nucleotide sequence encoding a (3-1 ,3-N- acetylglucosaminyltransferase polypeptide, wherein the expression control sequence effects transcription and / or translation of the second nucleotide sequence. Accordingly, the term "promoter" designates nucleotide sequences which usually "precede" a protein-coding nucleotide sequence in a polynucleotide and e.g. provide a site for initiation of the transcription into mRNA. "Regulator" sequences, also usually located "upstream" of (i.e. , preceding) a protein-coding nucleotide sequence in a given polynucleotide, bind proteins that determine the frequency (or rate) of transcriptional initiation. Collectively referred to as "promoter / regulator" or "control" nucleotide sequence, these sequences which precede a selected protein-coding nucleotide sequence (or series of protein-coding nucleotide sequences) in a nucleic acid molecule cooperate to determine whether the transcription (and eventual expression) of a protein-coding nucleotide sequence will occur. Nucleotide sequences which "follow" a protein-coding nucleotide sequence in a polynucleotide and provide a signal for termination of the transcription into mRNA are referred to as transcription "terminator" sequences. It is understood that the nucleotide sequence encoding the variant p-1 ,3-N-acetylglucosaminyltransferase polypeptide and being operably linked to expression control sequences will be transcribed - and eventually expressed - under permissive conditions.

[0058] In some embodiments, the promoter is an inducible promoter, i.e. a promoter enabling to switch on expression of the second nucleotide sequence by the presence of an inductor such as a small molecule. In alternative embodiments, the promoter is a negatively regulated promoter, i.e. a promoter whose activity is negatively regulated in response to a small molecule.

[0059] Being operably linked to expression control sequences enables expression of the nucleotide sequence encoding the variant p-1 ,3-N-acetylglucosaminyltransferase polypeptide in vitro or in vivo, and thereby enables synthesis of the variant p-1 ,3-N- acetylglucosaminyltransferase polypeptide for its use in the synthesis of an oligosaccharide of interest that comprises an N-acetylglucosamine moiety.

[0060] According to the third aspect, provided are genetically engineered microbial cells comprising at least one variant N-acetylglucosaminyltransferase polypeptide as described herein before, i.e. variant of a p-1,3-N-acetylglucosaminyltransferase polypeptide possessing an amino acid sequence that has a sequence identity of at least 80% to the amino acid sequence as set forth in SEQ ID NO: 1, wherein the variant comprises at least one amino acid substitution relative to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the at least one amino acid substitution comprises an amino acid substitution of at least one of the amino acids at amino acid positions 158, 249 and 256 of the amino acid sequence as set forth in SEQ ID NO: 1 with an amino acid that possesses a hydrophobic side chain and / or at least one nucleic acid molecule comprising a nucleotide sequence which encodes such variant p-1,3-N-acetylglucosaminyltransferase polypeptide.

[0061] A “genetically engineered microbial cell" is understood as a microbial cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide. Thus, the nucleotide sequences as used in the invention, may, e.g., be comprised in a vector which is to be stably transformed / transfected or otherwise introduced into host microbial cells. A great variety of expression systems can be used to produce polypeptides. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and to synthesize a polypeptide in a microbial cell may be used for expression in this regard. The appropriate polynucleotide may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., “Molecular Cloning, A laboratory Manual,” 2ndEdition, Cold Spring Harbor Laboratory Press, 1989. Preferably, polynucleotides containing the appropriate nucleotide sequence(s) are stably introduced into the genome of the microbial cell. Genomic integration can be achieved by recombination or transposition. The genetically engineered microbial cell is either a prokaryotic cell or a eukaryotic cell. Suitable microbial cells include bacterial cells, archaebacterial cells, yeast cells, and fungal cells.

[0062] In some embodiments, the genetically engineered microbial cell is a prokaryotic cell, more specifically a bacterial cell. The bacterial cell can be selected from the group consisting of the genera of Bacillus, Lactobacillus, Lactococcus, Enterococcus, Bifidobacterium, Sporolactobacillus spp., Micromonospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas. Suitable bacterial species are Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, Bacillus circulans, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium bifidum, Citrobacter freundii, Clostridium cellulolyticum, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium acetobutylicum, Corynebacterium glutamicum, Enterococcus faecium, Enterococcus thermophiles, Escherichia coli, Erwinia herbicola (Pantoea agglomerans), Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, Lactococcus lactis, Pantoea citrea, Pecto bacterium carotovorum, Proprionibacterium freudenreichii, Pseudomonas fluorescens, Pseudomonas aeruginosa, Streptococcus thermophiles and Xanthomonas campestris.

[0063] Bacterial cells can be genetically engineered by well established molecular biological methods, have little demands on the nutritional composition of the culture medium, multiply rapidly, and fermentation conditions for in vivo production can be controlled.

[0064] In other embodiments, the genetically engineered microbial cell is a eukaryotic cell such as a yeast cell. The yeast cell may be selected from the group consisting of Saccharomyces sp., in particular Saccharomyces cerevisiae, Saccharomycopsis sp., Pichia sp. (aka Hanensula sp.), in particular Pichia pastoris, Kluyveromyces sp., Yarrowia sp., Rhodotorula sp., and Schizosaccharomyces sp. Y1

[0065] Although not multiplying as rapidly as bacterial cells, and being more demanding with respect to their culture medium, eukaryotic microorganisms can be genetically engineered and fermentation conditions for in vivo production can be controlled.

[0066] In some embodiments, the genetically engineered microbial cell contains a plasmid which comprises the nucleotide sequence encoding at least one of the variant (3-1 ,3- N-acetylglucosaminyltransferase polypeptides. In additional or alternative embody- ments, the nucleotide sequence encoding the variant p-1 ,3-N-acetylglucosaminyl- transferase polypeptide is present integrated into the genomic DNA of the microbial cell.

[0067] In certain embodiments, the genetically engineered microbial cell is a microbial cell for the production of an oligosaccharide of interest, wherein the oligosaccharide of interest comprises at least one N-acetylglucosamine moiety. Thus, such microbial cell is able to synthesize an N-acetylglucosamine-containing (GIcNAc-containing) oligosaccharide intracellularly.

[0068] The terms “is capable of’ and “is able to” are used synonymously and are to be understood such that the microbial cell synthesizes the GIcNAc-containing oligosaccharide of interest intracellularly when cultured in a medium and under conditions that are permissive for the microbial cell to synthesize the GIcNAc- containing oligosaccharide of interest.

[0069] In certain embodiments, the GIcNAc-containing oligosaccharide of interest is a human milk oligosaccharide (HMO). Exemplary HMOs that certain of the genetically engineered microbial cells may produce are lacto- N-triose II (LNT II), lacto-N- tetraose (LNT), lacto- N-neotetraose (LNnT), para-lacto-N-hexaose (pLNH), para- Lacto-N-neohexaose (pLNnH). Additional exemplary HMOs that certain of the genetically engineered microbial cells may produce are the fucosylated and / or sialylated derivatives of LNT or LNnT, such as e.g. lacto-N-fucopentaose I (LNFP I), lacto- N-neofucopentaose I (LNnFP I), lacto-N-fucopentaose II (LNFP II), lacto-N- fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), lacto-N- neofucopentaose V (LNnFP V), lacto-N-difucohexaose I (LNDFH I), lacto-N- difucohexaose II (LNDFH II), difucosyllacto-N-hexaose I (DF-LNH I), difucosyllacto- N-hexaose II (DF-LNH II), lacto- / V-sialylpentaose a (LST a), lacto-ZV-sialylpentaose b (LST b), lacto- / V-sialylpentaose c (LST c), fucosyl-lacto-ZV-sialylpentaose a (F-LST a), fucosyl-lacto-ZV-sialylpentaose b (F-LST b), fucosyl-lacto-ZV-sialylpentaose c (F- LST c), disialyl-lacto-ZV-tetraose (DS-LNT).

[0070] In additional and / or alternative embodiments, the genetically engineered microbial cell for producing a GIcNAc-containing oligosaccharide of interest further comprises one or more exogenous and / or heterologic nucleotide sequences that each, independently, encode one or more enzymes that is / are necessary for the metabolic pathway to synthesize the GIcNAc-containing oligosaccharide of interest.

[0071] For intracellular biosynthesis of a GIcNAc-containing oligosaccharide of interest, the genetically engineered microbial cell possesses a metabolic pathway for intracellular biosynthesis of UDP-GIcNAc. In some embodiments, the metabolic pathway for intracellular biosynthesis of UDP-GIcNAc comprises the enzymatic activities for the de novo biosynthesis of UDP-GIcNAc from fructose-6-phsophate. This de novo biosynthesis pathway for UDP-GIcNAc comprises the enzymatic activities of a glucosamine-fructose-6-phosphate transaminase, a phosphoglucosamine mutase, and an N-acetylglucosamine-1-phosphate uridyltransferase. Providing a microbial cell that possesses a de novo biosynthesis pathway for UDP-GIcNAc is advantageous, because it is not necessary to culture the microbial cell in the presence of exogenous GIcNAc.

[0072] A glucosamine-fructose-6-phosphate transaminase catalyzes the conversion of D- fructose-6-phosphate and L-glutamine to D-glucosamine-6-phosphate and L-gluta- mate. An exemplary glucosamine-fructose-6-phosphate transaminase that can be used in the microbial cells disclosed herein is E. coli GlmS (UniProtKB Entry No. P17169). In certain embodiments, the genetically engineered microbial cell overexpresses and / or expresses an exogenous glucosamine-fructose-6-phosphate transaminase gene, such as the E. coli glmS gene or glucosamine-fructose-6- phosphate transaminase gene from a different species.

[0073] A phosphoglucosamine mutase catalyzes the conversion of glucosamine-6- phosphate to glucosamine-1-phosphate. An exemplary phosphoglucosamine mutase that can be used in the microbial cells disclosed herein is E. coli GlmM (UniProtKB Entry No. P31120). All references to UniProtKB Entry Nos. refer to UniProtKB (www.uniprot.org) Release 2023_03 released on June 28, 2023. In certain embodiments, the genetically engineered microbial cell overexpresses and / or expresses an exogenous phosphoglucosamine mutase gene, such as the E. coli glmM gene or a phosphoglucosamine mutase gene from a different species.

[0074] An N-acetylglucosamine-1 -phosphate uridyltransferase catalyzes the transfer of an acetyl group from acetyl coenzyme A to glucosamine-1-phosphate to produce N- acetylglucosmaine-1-phoshate, and then converts N-acetylglucosamine-1 -phoshate to UDP-GIcNAc utilizing UTP. An exemplary N-acetylglucosamine-1 -phosphate uridyltransferase that can be used in the microbial cells disclosed herein is E. coli GlmU (UniProtKB Entry No. P0ACC7).

[0075] In certain embodiments, the genetically engineered microbial cell overexpresses and / or expresses an exogenous N-acetylglucosamine-1 -phosphate uridyltransferase gene, such as the E. coli glmU gene or an N-acetylglucosamine-1-phosphate uridyltransferase gene from a different species.

[0076] The E. coli N-acetylglucosamine-1 -phosphate uridyltransferase is a bifunctional protein, wherein the C-terminal domain catalyzes the first enzymatic reaction, and the N-terminal domain catalyzes the latter enzymatic rection.

[0077] In an alternative, the two enzymatic steps for converting GlcNAc-1-P to UDP - GIcNAc can be performed by two distinct polypeptides, a glucosamine-1 -phosphate acetyltransferase for transferring an acetyl group to GlcNAc-1-P, and an UDP-N- acetylglucosamine diphosphorylase to catalyze the formation of UDP-GIcNAc from N-acetylglucosamine-1 -phosphate and UTP. In some embodiments, the microbial cell has been genetically engineered to possess an UDP-N-acetylglucosamine diphosphorylase or to increase the UDP-N-acetylglucosamine diphosphorylase activity within the microbial cell.

[0078] In some embodiments, especially wherein the oligosaccharide of interest is LNT, LNnT or a fucosylated and / or sialylated derivative of LNT or LNnT, the genetically engineered microbial cell comprises a LNT-2 accepting galactosyltransferase for galactosylating LNT-2. In some embodiments, the LNT-2 accepting galactosyltransferase is a (3-1,3- galactosyltransferase. An exemplary LNT-2 accepting p-1,3-galactosyltransferase that can be used in the microbial cells disclosed herein is E. coli WbgO (UniProtKB Entry No. B1B4W3).

[0079] In some embodiments, the microbial cell has been genetically engineered to possess a LNT-2 accepting p-1 ,3-galactosyltransferase. Genetically engineering the microbial cell to possess a LNT-2 accepting p-1,3-galactosyltransferase allows to provide a genetically engineered microbial cell and a method for producing LNT.

[0080] In some embodiments, the LNT-2 accepting galactosyltransferase is a p-1, 4- galactosyltransferase. An exemplary LNT-2 accepting p-1,4-galactosyltransferase that can be used in the microbial cells disclosed herein is a Neisseria meningitidis LgtB (UniProtKB Entry No. Q51116 and others).

[0081] In some embodiments, the microbial cell has been genetically engineered to possess a LNT-2 accepting p-1 ,4-galactosyltransferase. Genetically engineering the microbial cell to possess a LNT-2 accepting p-1,4-galactosyltransferase allows to provide a genetically engineered microbial cell and a method for producing LNnT.

[0082] In additional and / or alternative embodiments, the genetically engineered microbial cell possess a metabolic pathway for the biosynthesis of UDP-galactose. In some embodiments, the genetically engineered microbial cell has been genetically engineered to possess a metabolic pathway for the biosynthesis of UDP-galactose or for enhancing the metabolic pathway for the biosynthesis of UDP-galactose.

[0083] An exemplary metabolic pathway for intracellular biosynthesis of UDP-galactose starts from glucose-6-phosphate. This metabolic pathway comprises the enzymatic activities of a phosphoglucomutase, an UTP — glucose-1 -phosphate uridylyltransferase, and an UDP-glucose 4-epimerase. One or more of these enzymatic activities can be provided by a heterologous enzyme.

[0084] In some embodiments, the genetically engineered microbial cell possesses at least one exogenous nucleotide sequence that encodes one or more of the enzymes that are involved in the intracellular biosynthesis of UDP-galactose. The enzyme phosphoglucomutase converts glucose- 1-phsophate to glucose-6- phosphate. The phosphoglucomutase is encoded by a phosphoglucomutase gene.

[0085] A suitable example of a phosphoglucomutase gene is the pmg gene of E. coli (strain K12) encoding the E. coli phosphoglucomutase PgM (UniProtKB Entry No. P36938).

[0086] The enzyme UTP — glucose-1 -phosphate uridylyltransferase converts glucose-1- phosphate to UDP-glucose. The UTP — glucose-1 -phosphate uridylyltransferase is encoded by a UTP — glucose-1 -phosphate uridylyltransferase gene. A suitable example of a UTP — glucose-1 -phosphate uridylyltransferase gene is the galU gene of E. coli (strain K12) encoding the E. coli UTP — glucose-1 -phosphate uridylyltransferase GalU (UniProtKB Entry No. P0AEP3).

[0087] The enzyme UDP-glucose 4-epimerase converts UDP-glucose to UDP-galactose.

[0088] The UDP-glucose 4-epimerase is encoded by a UDP-glucose 4-epimerase gene. An example of a suitable UDP-glucose 4-epimerase gene is the galE gene of E. coli (strain K12) encoding the E. coli UDP-glucose 4-epimerase GalE (UniProtKB entry No. P09147).

[0089] Expression of one or more exogenous and / or endogenous genes encoding one or the enzymes necessary for intracellular UDP-galactose biosynthesis implements or enhances intracellular GDP-galactose biosynthesis in the genetically engineered microbial cell, and provides the donor substrate for the galactosyltransferases involved in the biosynthesis of LNT or LNnT.

[0090] In embodiments, wherein the oligosaccharide of interest is a fucosylated derivative of LNT or LNnT, such as e.g. LNFP I, LNnFP I, LNFP II, LNFP III, LNFP V, LNnFP V, LNDFH I, LNDFH II, DF-LNH I, DF-LNH II, F-LST a, F-LST b, and F-LST c, the microbial cell further comprises at least one fucosyltransferase for transferring at least one L-fucose moiety from GDP-L-fucose as donor substrate to LNT, LNnT or their fucosylated and / or sialylated derivatives as acceptor molecule. These genetically engineered microbial cells further comprise a metabolic pathway for intracellular biosynthesis of GDP-L-fucose.

[0091] The term “fucosyltransferase” as used herein, refers to polypeptides which are capable of catalyzing the transfer of a fucose residue from a donor substrate to an acceptor molecule. The donor substrate for the transfer of a fucose residue to an acceptor molecule is typically guanosine-diphosphate L-fucose (GDP-L-fucose). The acceptor molecule is at least one of LNT, LNnT and their fucosylated and / or sialylated derivatives.

[0092] The term “fucosyltransferase” as used herein is also understood to encompass functional variants of said fucosyltransferase, functional fragments of said fucosyltransferases and functional fragments of said functional variants. The term “functional” indicates that said variants and fragments are also capable of catalysing the transfer of a fucose residue from the donor substrate to the acceptor molecule, i.e. they possess fucosyltransferase activity.

[0093] The term “functional fragment” as used herein refers to a truncated polypeptide as compared to the naturally occurring fucosyltransferase, and which possesses the same fucosyltransferase activity as the naturally occurring polypeptide said fragment originates from.

[0094] The term “functional variant” as used herein refers to a polypeptide that possesses the same fucosyltransferase activity as the naturally occurring polypeptide said derivative originates from, but which has an altered amino acid sequence as compared to the naturally occurring polypeptide.

[0095] The fucosyltransferase is capable of transferring a fucose residue from a donor substrate to an acceptor molecule. The term “capable of” with respect to the fucosyltransferase refers to the fucosyltransferase activity of the heterologous fucosyltransferase and the provision that suitable reaction conditions are required for the heterologous fucosyltransferase to possess its enzymatic activity. In the absence of suitable reaction conditions, the fucosyltransferase does not exhibit its enzymatic activity, but retains its enzymatic activity and possesses its enzymatic activity when suitable reaction conditions are restored. Suitable reaction conditions include the presence of a suitable donor substrate, the presence of suitable acceptor molecules, the presence of essential cofactors such as - for example - monovalent or divalent ions, a pH value in an appropriate range, a suitable temperature and the like. It is not necessary that the optimum values for each and every factor effecting the enzymatic reaction of the fucosyltransferase is met, but the reaction conditions have to be such that the fucosyltransferase performs its enzymatic activity. Accordingly, the term “capable of’ excludes any conditions upon which the enzymatic activity of the fucosyltransferase has been irreversibly impaired, and also excluded exposure of the fucosyltransferase to any such condition. Instead, “capable of” means that the fucosyltransferase is enzymatically active, i.e. possesses its fucosyltransferase activity, if suitable reactions conditions (where all requirements being necessary for the fucosyltransferase to perform its enzymatic activity) are provided.

[0096] Fucosyltransferases form a-1,2-, a-1,3-, a-1,4-, or a-1,6-glycosidic linkages between fucose and a saccharide moiety of the acceptor molecule. Accordingly, the term "alpha-1 , 2-fucosyltransferase” refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate to an acceptor molecule forming an alpha- 1,2-linkage of the fucose residue and the saccharide residue of the acceptor molecule. The term "alpha-1 , 3-fucosyltranferase " refers to a glycosyltransferase that catalyses the transfer of fucose from a donor substrate to an acceptor molecule in an alpha-1, 3-linkage of the fucose residue and a saccharide residue of the acceptor molecule. The term "alpha- 1,4-fucosyltranferase " refers to a glycosyltransferase that catalyses the transfer of fucose from a donor substrate to an acceptor molecule in an alpha-1 , 4-linkage of the fucose residue and a saccharide residue of the acceptor molecule; and the term "alpha- 1,6- fucosy Itranferase " refers to a glycosyltransferase that catalyses the transfer of fucose from a donor substrate to an acceptor molecule in an alpha-1 , 6-linkage of the fucose residue and a saccharide residue of the acceptor molecule.

[0097] The term “donor substrate” with respect to transferring a fucose residue from the donor substrate to an acceptor molecule refers to a molecule comprising a fucose residue, said molecule being utilized by the heterologous fucosyltransferase a source of fucose which is to be transferred to a specific acceptor molecule. Typically, the donor substrate is GDP-L-fucose.

[0098] The term “acceptor molecule” as used herein refers to a molecule which receives the fucose residue from the donor substrate by the enzymatic activity of the fucosyltransferase. As used herein, the term “acceptor molecule” more specifically refers to at least one of LNT, LNnT and their fucosylated and / or sialylated derivatives.

[0099] In some embodiments, the fucosyltransferase is capable of transferring a fucose residue to a lactotetraose as an acceptor molecule. The term “lactotetraose” as used herein refers to a tetrasaccharide, i.e. an oligosaccharide consisting of 4 monosaccharide residues, wherein the tetrasaccharide comprises a lactose motif (Gal(pi,4)Glc) at its reducing end.

[0100] In an embodiment, the lactotetraose is selected from the group consisting of Lacto- ZV-tetraose (LNT; Gal(pi ,3)GlcNAc(pi,3)Gal(pi ,4)Glc) and Lacto- / V-neotetraose (LNnT ; Gal(pi ,3)GlcNAc(pi ,4)Gal(pi ,4)Glc). The enzymatic activity of the fucosyltransferase leads to a fucosylated oligosaccharide, more specifically to a fucosylated lactotetraose, i.e. a lactofucopentaose. Said lactofucopentaose is a pentasaccharide preferably selected from the group consisting of lacto-ZV-fucopentaose I (LNFP-I), lacto-ZV-neofucopentaose I (LNnFP-l), lacto-ZV-fucopentaose II (LNFP II), lacto- / V-neofucopentaose III (LNnFP-ll I), lacto-ZV-fucopentaose V (LNFP-V) and lacto-ZV-neofucopentaose V (LNnFP-V).

[0101] Polypeptides which were identified of possessing fucosyltransferase activity for transferring a fucose residue from a donor substrate to a lactotetraose are for example those disclosed in WO 2019 / 008133 A1 (incorporated herein by reference) such as the polypeptides of Helicobacter hepaticus ATCC 51449 (GenBank accession: AAP76669), Brachyspira pilosicoli WesB (WESB_1374) (GenBank accession: CCG56842), Yersinia sp. A125 KOH2 (WbcH-like) (GenBank accession: CAI39173), Gramella forsetii KT0803 (GenBank accession: WP_011708479), Francisella philomiragia ssp. philomiragia ATCC 25015 (FTPG_00102) (GenBank accession: EET21243), Pseudogulbenkiania ferrooxidans 2002 (FuraDRAFT_0420) (GenBank accession: EEG10438), Sideroxydans lithotrophicus ES-11 (Slit_2889) (GenBank accession: ADE13114), Providencia alcalifaciens (WdcS) (GenBank accession: AFH02807), Pseudoalteromonas haloplanktis ANT / 505 (PH505_ae00940) (GenBank accession: EGI74693), Roseovarius nubinhibens ISM (ISM_09170) (GenBank accession: EAP78457), Thalassospira profundimaris WP0211 (TH2_05058) (GenBank accession: EKF09232), Desulfovibrio alaskensis G20 (Dde_2877) (GenBank accession: ABB39672), Thermosynechococcus elongates BP-1 (t110994) (GenBank accession: BAC08546), Bacteroides fragilis strain ATCC 25285 (BF9343_3370) (GenBank accession: CAH09151), and Escherichia coli 0126 (WbgL) (GenBank accession: ABE98421).

[0102] In additional and / or alternative embodiments, the genetically engineered microbial cell producing a fucosylated derivative of LNT or LNnT comprises a metabolic pathway for intracellular biosynthesis of GDP-L-fucose. The metabolic pathway for intracellular GDP-L-fucose biosynthesis may be a salvage pathway or a de novo pathway.

[0103] The salvage pathway for intracellular biosynthesis of GDP-L-fucose comprises the enzymatic activities of fucose kinase for phosphorylation of L-fucose to provide fucose-1-phosphate as well as of a fucose-1-phosphate guanylyltransferase to convert fucose- 1 -phosphate to GDP-L-fucose. Hence, the genetically engineered microbial cell possessing the salvage pathway for intracellular biosynthesis of GDP- L-fucose comprises a fucose kinase and a fucose-1 -phosphate guanylyltransferase. In certain embodiments, the enzymatic activities of the fucose kinase and the fucose-1-phosphate guanylyltransferase are provided by a bifunctional enzyme that exhibits both enzymatic activities.

[0104] Examples of a suitable bifunctional fucose kinase I fucose-1 -phosphate guanylyltransferase is encoded by the fkp gene of Bacteroides fragilis (UniProtKB Entry No. Q58T34). Alternatively, genes encoding a fucose kinase, a fucose-1- phosphate guanylyltransferase and / or a bifunctional fucose kinase / a fucose-1 - phosphate guanylyltransferase can be obtained from the genera Lentisphaera, Ruminococcus, Solibacter, Arabidopsis, Oryza, Physcomitrella, Vitis, Danio, Bos, Equus, Macaca, Pan, Homo, Rattus, Mus and Xenopus.

[0105] Possessing the salvage pathway for intracellular biosynthesis of GDP-L-fucose enables the genetically engineered microbial cell to utilize intracellular free L-fucose for GDP-L-fucose biosynthesis.

[0106] In some embodiments, the genetically engineered microbial cell possessing the salvage pathway for intracellular biosynthesis of GDP-L-fucose further comprises a fucose permease for internalization of exogenous L-fucose by the microbial cell. A suitable fucose permease is the L-fucose-proton symporter FucP of E. coli (strain K12) (UniProt Entry No. P11551 and / or GenBank accession CP000948).

[0107] In some embodiments, the genetically engineered microbial cell possesses a de novo pathway for the intracellular biosynthesis of GDP-L-fucose. The de novo pathway for intracellular biosynthesis of GDP-L-fucose starts with an isomerisation of fructose-6-phosphate to mannose-6-phosphate by a mannose-6-phosphatae isomerase. Mannose-6-phosphate is then converted to mannose-1-phosphate by a phosphomannomutase. Mannose- 1 -phosphate is reacted with GTP by a mannose- 1-phosphate guanylyltransferase to yield GDP-alpha-D-mannose which is further converted by a GDP-mannose-4,6-dehydratase to GDP-4-keto-6-deoxymannose. GDP-4-keto-6-deoxymannose it then converted to GDP-L-fucose by two steps involving an epimerase activity and a reductase activity, which are both provided by a GDP-L-fucose synthase.

[0108] Hence, a microbial cell that possesses the de novo pathway for intracellular biosynthesis of GDP-L-fucose comprises a mannose-6-phosphate isomerase phosphomannomutase, a mannose-1 -phosphate guanylyltransferase, a GDP- mannose-4,6-dehydratase and a GDP-L-fucose synthase.

[0109] A suitable mannose-6-phosphate isomerase is e.g. the mannose-6-phosphate isomerase of E. coli (strain K12) (UniProtKB Entry No. P00946), encoded by the E. coli (K12) manA gene.

[0110] A suitable phosphomannomutase is e.g. the phosphomannomutase of E. coli (strain K12) (UniProtKB Entry No. P24175), encoded by the E. coli (K12) manB gene.

[0111] A suitable mannose-1 -phosphate guanylyltransferase is e.g. the mannose-1- phosphate guanylyltransferase of E. coli (strain K12) (UniProtKB Entry No. P24174), encoded by the E. coli (K12) manC gene.

[0112] A suitable GDP-mannose-4,6-dehydratase is e.g. the GDP-mannose-4,6- dehydratase of E. coli (strain K12) (UniProt KB Entry No. P0AC88), encoded by the E. coli (K12) gmd gene. A suitable GDP-L-fucose synthase is e.g. the E. coli (strain K12) GDP-L-fucose synthase (UniProtKB Entry No. P32055), encoded by the E. coli (K12) i / caG gene.

[0113] Thus, in some embodiments the genetically-engineered microbial cell has been genetically engineered to contain and express one or more genes encoding at least one of the enzymes involved in the salvage pathway and / or the de novo pathway for intracellular biosynthesis of GDP-L-fucose.

[0114] In some embodiments, one or more of the genes encoding at least one of the enzymes involved in the salvage pathway and / or the de novo pathway for intracellular biosynthesis of GDP-L-fucose is a heterologous gene.

[0115] In some embodiments, the GIcNAc-containing oligosaccharide of interest further comprises at least one NeuNAc moiety.

[0116] In embodiments, wherein the oligosaccharide of interest is a sialylated derivative of LNT or LNnT, such as e.g. LST-a, LST-b, LST-s, F-LST a, F-LST b, and F-LST c, the microbial cell comprises at least one sialyltransferase for transferring a NeuNAc moiety from CMP-NeuNAc as donor substrate to an acceptor saccharide, and a metabolic pathway for intracellular biosynthesis of CMP-NeuNAc

[0117] The term “sialyltransferase” as used herein, refers to polypeptides which are capable of catalyzing the transfer of a N-acetylneuraminic acid (NeuNAc) moiety from a donor substrate to an acceptor molecule. The donor substrate for the transfer of a NeuNAc moiety to an acceptor molecule is typically cytidine monophosphate N- acetylneuraminic acid (CMP-NeuNAc). The acceptor molecule is at least one of LNT, LNnT and their fucosylated and / or sialylated derivatives.

[0118] The term “sialyltransferase” as used herein is also understood to encompass functional variants of said sialyltransferase, functional fragments of said sialyltransferases and functional fragments of said functional variants. The term “functional” indicates that said variants and fragments are also capable of catalysing the transfer of a NeuNAc residue from the donor substrate to the acceptor molecule, i.e. they possess sialyltransferase activity. The term “sialyltransferase” as used herein refers to polypeptides being capable of possessing sialyltransferase activity. “Sialyltransferase activity” refers to the transfer of a sialic acid residue, preferably of an / V-acetylneuraminic acid (Neu5Ac) residue, from a donor substrate to an acceptor molecule. The term “sialyltransferase” comprises functional fragments of the sialyltransferases described herein, functional variants of the sialyltransferases described herein, and functional fragments of the functional variants. “Functional” in this regard means that the fragments and / or variants are capable of possessing sialyltransferase activity. Functional fragments of a sialyltransferase encompass truncated versions of a sialyltransferase as encoded by it naturally occurring gene, which truncated version is capable of possessing sialyltransferase activity. Examples of truncated versions are sialyltransferases which do not comprise a so-called leader sequence which typically directs the polypeptide to a specific subcellular localization. Typically, such leader sequences are removed from the polypeptide during its subcellular transport, and are also absent in the naturally occurring mature sialyltransferase.

[0119] The sialyltransferase is capable of transferring a sialic acid residue from a donor substrate to an acceptor molecule. The term “capable of” with respect to the sialyltransferase refers to the sialyltransferase activity of the sialyltransferase and the provision that suitable reaction conditions are required for the sialyltransferase to possess its enzymatic activity. In the absence of suitable reaction conditions, the sialyltransferase does not possess its enzymatic activity, but retains its enzymatic activity and possesses its enzymatic activity when suitable reaction conditions are restored. Suitable reaction conditions include the presence of a suitable donor substrate, the presence of suitable acceptor molecules, the presence of essential cofactors such as - for example - monovalent or divalent ions, a pH value in an appropriate range, a suitable temperature and the like. It is not necessary that the optimum values for each and every factor effecting the enzymatic reaction of the heterologous sialyltransferase is met, but the reaction conditions have to be such that the heterologous sialyltransferase performs its enzymatic activity. Accordingly, the term “capable of’ excludes any conditions upon which the enzymatic activity of the heterologous sialyltransferase has been irreversibly impaired and also excluded exposure of the heterologous sialyltransferase to any such condition. Instead, “capable of’ means that the sialyltransferase is enzymatically active, i.e. possesses its sialyltransferase activity, if permissive reactions conditions (where all requirements being necessary for the sialyltransferase to perform its enzymatic activity) are provided to the sialyltransferase.

[0120] Sialyltransferases can be distinguished on the type of sugar linkage they form. As used herein, the terms “a-2,3-sialyltransferase” and “a-2,3-sialyltransferase activity” refer to polypeptides and their enzymatic activity which add a sialic acid residue with an a-2,3 linkage to galactose, / V-acetylgalactosamine or a galactose or / V-acetyl- galactosamine residue of the acceptor molecule. Likewise, the terms “a-2,6-sialyl- transferase” and “a-2,6-sialyltransferase activity” refer to polypeptides and their enzymatic activity which add a sialic acid residue with an a-2,6 linkage to galactose, / V-acetylgalactosamine or a galactose or / V-acetylgalactosamine residue of the acceptor molecule. Likewise, the terms “a-2,8-sialyltransferase” and “a-2,8- sialyltransferase activity” refer to polypeptides and their enzymatic activity which add a sialic acid residue with an a-2,8 linkage to galactose, / V-acetylgalactosamine or a galactose or / V-acetylgalactosamine residue of the acceptor molecule.

[0121] In an additional and / or alternative embodiment, the genetically engineered microbial cell contains at least one sialyltransferase, preferably at least one heterologous sialyltransferase, wherein said sialyltransferase is capable of possessing an a-2,3- sialyltransferase activity and / or an a-2,6-sialyltransferase activity and / or an a-2,8-sialyltransferase activity for transferring the NeuNAc moiety from CMP- NeuNAc as donor substrate to the acceptor saccharide.

[0122] Exemplary sialyltransferases that can be employed in the production of sialylated derivatives of LNT or LNnT are e.g. disclosed in WO 2019 / 020707 A1 (enclosed herein by reference) such as the polypeptides of Neisseria meningitidis (UniProtKB Entry No. U60660), Campylobacter jejuni (UniProtKB Entry Nos. Q5W603, Q9L9Q5 and Q9RGF1), Helicobacter acinonychis (UniProtKB Entry No. Q17WF9), Photobacterium sp. JT-ISH-224 (UniProtKB Entry No. BAF92026), Pasteurella dagmatis (UniProt Entry NO. K9UUI6), Vibrio sp. (UniProtKB Entry No. A8R0Y0), Pasteurella multocida (UniProt Entry Nos. Q9CP67 and Q9CNC4), Photobacterium damselae (UniProtKB Entry Nos. 066375 and D0YWU3), Streptococcus agalactiae (UniProtKB Entry No. Q9AQI8); Haemophilus somnus (UniProtKB Entry No. B0LIWB1), Haemophilus ducreyi (UniProtKB Entry Nos. Q9X4A1 and Q7VPL1), Photobacterium phosphoreum (UniProtKB Entry No. A5LHX0), Photobacterium leiognathi (UniProtKB Entry Nos. D0VYB7 and A8R0T5), Campylobacter coli (UniProtKB Entry No. T2LJB7), Vibrio harveyi (UniProtKB Entry No. A0A8B3DCA5), Streptococcus entericus (UniParc Entry No. UPI000363B79E), Avibacterium paragallinarum (UniProtKB Entry No. A0A377IUJ3), Haemophilus parahaemolyticus (UniProtKB Entry No. I3DHL4), Alistipes sp. (UniProtKB Entry No. R6WNL1 and UniParc Entry NO.UPI00050A31C8), Campylobacter jejuni (UniProtKB Entry No. Q9L9Q5), Alistipes shahii (UniProtKB Entry No. D4IM12), Actinobacillus suis (UniProtKB Entry No. K0G612), Actinobacillus capsulatus (UniParc Entry No. U PI 0003711362), Bibersteinia trehalosi (UniProtKB Entry No. W0R3X2), and Haemophilus somnus (UniProtKB Entry No. B0UUE7).

[0123] In additional and / or alternative embodiments, the genetically engineered microbial cell producing a sialylated derivative of LNT or LNnT comprises a metabolic pathway for intracellular biosynthesis of CMP-NeuNAc as donor substrate for the NeuNAc moiety. The metabolic pathway for intracellular biosynthesis of CMP- NeuNAc may be a de novo pathway.

[0124] The de novo biosynthesis pathway for intracellular biosynthesis of CMP-NeuNAc comprises the enzymatic activities of a glutamine:fructose-6-phosphate aminotransferase, an / V-acetylneuraminic acid synthase, and a CMP-NeuNAc synthase.

[0125] The glutamine:fructose-6-phosphate aminotransferase catalyses the conversion of fructose-6-phosphate (Fru-6P) to glucosamine-6-phosphate (GlcN-6P) using glutamine. This enzymatic reaction is typically considered to be the first step in the CMP-NeuNAc biosynthesis pathway. Alternative names of the glutamine:fructose-6- phosphate aminotransferase are D-fructose-6-phosphate aminotransferase, GFAT, glucosamine-6-phosphate synthase, hexosephosphate aminotransferase, and L- glutamine-D-fructose-6-phosphate aminotransferase.

[0126] In an additional and / or alternative embodiment, the genetically engineered microbial cell possesses a glutamine:fructose-6-phosphate aminotransferase. The glutamine:fructose-6-phosphate aminotransferase may be a heterologous glutamine:fructose-6-phosphate aminotransferase. An example of a suitable glutamine:fructose-6-phosphate aminotransferase is the glutamine:fructose-6- phosphate aminotransferase which is derived from E. coli (E. coli GlmS (UniProtKB Entry No. P17169), or a functional variant of the E. coli GlmS. The functional variant is a version of the E. coli GlmS which shows significantly reduced sensitivity to glucosamine-6-phosphate inhibition as the wild-type enzyme referenced herein before does.

[0127] Thus, in an additional and / or alternative embodiment, the genetically engineered microbial cell contains a nucleic acid molecule comprising and expressing a nucleotide sequence encoding a glutamine:fructose-6-phosphate aminotransferase, preferably the glutamine:fructose-6-phosphate aminotransferase GlmS of E. coli (K12) or a functional variant or functional fragment thereof.

[0128] In an additional and / or alternative embodiment, the genetically engineered microbial cell comprises sialic acid synthase activity. The sialic acid synthase catalyzes the condensation of ManNAc and phosphoenolpyruvate (PEP) to / V-acetylneuraminic acid (NeuNAc).

[0129] In an additional and / or alternative embodiment, the genetically engineered microbial cell comprises a sialic acid synthase or a functional variant thereof, preferably a heterologous sialic acid synthase. Examples of sialic acid synthases are known from a variety of bacterial species such as Campylobacter jejuni, Streptococcus agalactiae, Butyrivibrio proteoclasticus, Methanobrevibacter ruminatium, Aceto bacterium woodii, Desulfobacula toluolica, Escherichia coli, Prevotella nigescens, Halorhabdus tiamatea, Desulfotignum phosphitoxidans, or Candidatus Scalindua sp., Idomarina loihiensis, Fusobacterium nucleatum or Neisseria meningitidis. Preferably, the sialic acid synthase is the / V-acetylneuraminic acid synthase NeuB of C. jejuni (UniProtKB Entry No. Q4L130) as encoded by the C. jejuni neuB gene.

[0130] Thus, in an additional and / or alternative embodiment, the genetically engineered microbial cell contains a nucleic acid molecule comprising and expressing a nucleotide sequence encoding a sialic acid synthase, preferably the N- acetylneuraminic acid synthase NeuB of C. jejuni or a functional variant or functional fragment thereof.

[0131] The genetically engineered microbial cell possesses cytidine 5’-monophospho- (CMP)- / V-acetylneuraminic acid synthetase activity for transferring cytidine 5’- monophosphate onto / V-acetylneuraminic acid to generate a CMP-activated N- acetylneuraminic acid (CMP-NeuNAc). This enzymatic reaction is the last one in the metabolic pathway for intracellular biosynthesis of CMP-NeuNAc. Several 5’- monophospho-(CMP)-sialic acid synthetases are known in the art and have been described, e.g. 5’-monophospho- (CMP)-sialic acid synthetases from E. coli, Neisseria meningitidis, Campylobacter jejuni, Streptococcus sp., etc.

[0132] In an additional and / or alternative embodiment, the genetically engineered microbial cell contains a cytidine 5’-monophospho- (CMP)- / V-acetylneuraminic acid synthetase, preferably a heterologous cytidine 5’-monophospho- (CMP)- / V-acetyl- neuraminic acid synthetase, more preferably the / V-acetylneuraminate cytidyltransferase NeuA from E. coli. E. coli NeuA (UnitProtKB - P13266) is encoded by the E. coli neu / X gene.

[0133] Thus, in an additional and / or alternative embodiment, the genetically engineered microbial cell contains a nucleic acid molecule comprising and expressing a nucleotide sequence encoding a cytidine 5’-monophospho- (CMP)- / V- acetylneuraminic acid synthetase, preferably the cytidine 5’-monophospho- (CMP)- / V-acetylneuraminic acid synthetase NeuA of E. coli or a functional variant or functional fragment thereof.

[0134] In some embodiments, the biosynthesis pathway for intracellular biosynthesis of CMP-NeuNAc comprises the enzymatic activities of a phosphoglucosamine mutase to convert glucosamine-6-phosphate to glucosamine-1-phosphate, a bifunctional polypeptide that catalyzes the transfer of an acetyl group from acetyl coenzyme A to glucosamine-1-phosphate to produce N-acetylglucosamine-1-phosphate (GlcNAc-1- P), which is converted into UDP-GIcNAc by the transfer of uridine 5-monophosphate (from uridine 5-tri phosphate), a reaction catalyzed by the N-terminal domain of the same polypeptide, and of a UDP-N-acetylglucosamine 2-epimerase which converts UDP-GIcNAc to N-acetylmannosamine (ManNAc). An example of a suitable phosphoglucosamine mutase is E. coli (strain K12) GlmM (UniProtKB Entry No. P31120) being encoded by the E. coli (K12) glmM gene.

[0135] Thus, in an additional and / or alternative embodiment, the genetically engineered microbial cell contains a nucleic acid molecule comprising and expressing a nucleotide sequence encoding a phosphoglucosamine mutase, preferably the phosphoglucosamine mutase GlmM of E. coli (K12) or a functional variant or functional fragment thereof.

[0136] An example of a suitable bifunctional polypeptide that catalyzes the transfer of an acetyl group from acetyl coenzyme A to glucosamine-1-phosphate to produce GlcNAc-1-P, and further converts GlcNAc-1-P into UDP-GIcNAc is E. coli Gimli (UniProtKB Entry No. P0ACC7) as encoded by the E. coli (K12) glmU gene.

[0137] Thus, in an additional and / or alternative embodiment, the genetically engineered microbial cell contains a nucleic acid molecule comprising and expressing a nucleotide sequence encoding a bifunctional polypeptide that catalyzes the transfer of an acetyl group from acetyl coenzyme A to glucosamine-1-phosphate to produce GlcNAc-1-P, and further converts GlcNAc-1-P into UDP-GIcNAc, preferably the bifunctional polypeptide GlmU of E. coli (K12) or a functional variant or functional fragment thereof.

[0138] An example of a suitable UDP-N-acetylglucosamine 2-epimerase is Campyllobacter jejuni NeuC (UniProtKB Entry No. Q9F9F3) as encoded by the C. jejuni neuC gene.

[0139] Thus, in an additional and / or alternative embodiment, the genetically engineered microbial cell contains a nucleic acid molecule comprising and expressing a nucleotide sequence encoding a UDP-N-acetylglucosamine 2-epimerase, preferably the UDP-N-acetylglucosamine 2-epimerase NeuC of C. jejuni or a functional variant or functional fragment thereof.

[0140] In some embodiments, the biosynthesis pathway for the intracellular biosynthesis of CMP-NeuNAc comprises a glucosamine-6-phosphate / V-acetyltransferase.

[0141] Thus, in an additional and / or alternative embodiment, the genetically engineered microbial cell contains a nucleic acid molecule comprising and expressing a nucleotide sequence encoding a phosphoglucosamine mutase, preferably the phosphoglucosamine mutase GlmM of E. coli (K12) or a functional variant or functional fragment thereof.

[0142] The genetically engineered microbial cell comprises a biosynthesis pathway for the intracellular biosynthesis of CMP-NeuNAc which does not utilize UDP-GIcNAc. The genetically engineered microbial cell comprises a biosynthesis pathway for the intracellular biosynthesis of a CMP-NeuNAc which comprises a glucosamine-6- phosphate / V-acetyltransferase activity. A CMP-NeuNAc biosynthesis pathway using a glucosamine-6-phosphate / V-acetyltransferase activity for intracellular biosynthesis of CMP-NeuNAc does not utilize UDP-GIcNAc.

[0143] The CMP-NeuNAc biosynthesis pathway which utilizes the glucosamine-6- phosphate / V-acetyltransferase further comprises a) the enzymatic activities of a glucosamine-6-phosphate / V-acetyltransferase, an N- acetylglucosamine-6-phosphate phosphatase and an / V-acetylglucosamine 2-epi- merase; and / or b) the enzymatic activities of a glucosamine-6-phosphate / V-acetyltransferase, an N- acetylglucosamine-6-phosphate epimerase and an / V-acetylmannosamine-6- phosphate phosphatase. Therefore, it is not necessary that the genetically engineered microbial cell comprises the enzymatic activities of a phosphoglucosamine mutase, an / V-acetylglucosamine-1 -phosphate uridyltransferase and an UDP / V-acetylglucosamine 2-epimerase with concomitant release of UDP for intracellular sialic acid biosynthesis. Thus, in an additional and / or alternative embodiment, the genetically engineered microbial cell being capable of synthesizing sialic acid does not comprise one or more enzymatic activities selected from the group consisting of the enzymatic activities of a phosphoglucosamine mutase, an / V-acetylglucosamine-1 -phosphate uridyltransferase and an UDP / V-acetylglucosamine 2-epimerase with concomitant release of UDP.

[0144] In an additional and / or alternative embodiment, the genetically engineered microbial cell possesses glucosamine-6-phosphate / V-acetyltransferase activity. Said glucosamine-6-phosphate / V-acetyltransferase activity converts GlcN-6P to / V-ace- tylglucosamine-6-phosphate (GlcNAc-6P). An example of a glucosamine-6-phos- phate / V-acetyltransferase is the Saccharomyces cerevisiae Gna1 (UniProtKB Entry No. P43577).

[0145] In an additional and / or alternative embodiment, the genetically engineered microbial cell contains a glucosamine-6-phosphate / V-acetyltransferase, preferably a heterologous glucosamine-6-phosphate / V-acetyltransferase, more preferably S. cerevisiae Gna1 (UniProtKB Entry No. P43577) or a functional variant thereof.

[0146] Thus, in an additional and / or alternative embodiment, the genetically engineered microbial cell contains a nucleic acid molecule comprising and expressing a nucleotide sequence encoding a glucosamine-6-phosphate / V-acetyltransferase, preferably the glucosamine-6-phosphate / V-acetyltransferase Gna! of S. cerevisiae or a functional variant or functional fragment thereof.

[0147] In an additional and / or alternative embodiment, the genetically engineered microbial cell possesses a / V-acetylglucosamine-6-phosphate phosphatase activity. Said N- acetylglucosamine-6-phosphate phosphatase activity converts GlcNAc-6P to N- acetylglucosamine (GIcNAc). Examples of an / V-acetylglucosamine-6-phosphate phosphatase are sugar phosphatases of the HAD-like superfamily which catalyze the conversion of GlcNAc6P to GIcNAc. The HAD-like superfamily of enzymes is named after the bacterial enzyme haloacid dehydrogenase and includes phosphatases. A suitable phosphatase of the HAD-like superfamily catalyzing the conversion of GlcNAc6P to GIcNAc may be selected from the group consisting of fructose-1-phosphate phosphatase (YqaB, UniProtKB Entry No. P77475) and alpha- D-glucose 1-phosphate phosphatase (YihX, UniProtKB Entry No. P0A8Y3). The E. coli YqaB and E. coliY\hX enzymes are considered to also act on GlcNAc6P (Lee, S.-W. and Oh, M.-K. (2015) Metabolic Engineering 28: 143-150).

[0148] In an additional and / or alternative embodiment, the sugar phosphatase of the HAD- like superfamily catalyzing the conversion of GlcNAc-6P to GIcNAc is a heterologous enzyme in the genetically engineered microbial cell. In an additional and / or alternative embodiment, the sugar phosphatase of the HAD-like superfamily catalyzing the conversion of GlcNAc6P to GIcNAc is selected from the group consisting of E. coli YqaB, E. coliY\hX, and functional variants thereof. In an additional and / or alternative embodiment, the genetically engineered microbial cell contains a nucleic acid molecule which comprises and expresses a nucleotide sequence encoding a sugar phosphatase of the HAD-like superfamily catalyzing the conversion of GlcNAc6P to GIcNAc. In an additional and / or alternative embodiment, the nucleotide sequence encoding the sugar phosphatase of the HAD-like super- family catalyzing the conversion of GlcNAc6P to GIcNAc is a heterologous nucleotide sequence. In an additional and / or alternative embodiment, the nucleotide sequence encoding the sugar phosphatase of the HAD-like superfamily catalyzing the conversion of GlcNAc6P to GIcNAc encodes the E. coli fructose- 1 -phosphate phosphatase or the E. coli alpha-D-glucose 1-phosphate phosphatase. In an additional and / or alternative embodiment, the non-naturally-occurring microorganism has been genetically engineered to contain a nucleic acid molecule comprising and expressing a nucleotide sequence encoding a sugar phosphatase of the HAD-like superfamily catalyzing the conversion of GlcNAc6P to GIcNAc or a functional fragment of said HAD phosphatase and / or to comprise a sugar phosphatase of the HAD-like superfamily.

[0149] In an additional and / or alternative embodiment, the genetically engineered microbial cell possesses / V-acetylglucosamine 2-epimerase activity. / V-acetylglucosamine 2- epimerase (EC 5.1.3.8) is an enzyme that catalyzes the conversion of / V-acetyl- glucosamine (GIcNAc) to / V-acetylmannosamine (ManNAc). The enzyme is a racemase acting on carbohydrates and their derivatives. The systematic name of this enzyme class is / V-acyl-D-glucosamine 2-epimerase. This enzyme participates in amino-sugar metabolism and nucleotide-sugar metabolism, preferably a heterologous / V-acetylglucosamine 2-epimerase.

[0150] In an additional and / or alternative embodiment, the genetically engineered microbial cell comprises an / V-acetylglucosamine 2-epimerase, preferably a heterologous N- acetylglucosamine 2-epimerase. Examples of / V-acetylglucosamine 2-epimerases were described from Anabena variabilis, Acaryochloris sp., Nostoc sp., Nostoc punctiforme, Bacteroides ovatus or Synechocystis sp. An example of a suitable N- acetylglucosamine 2-epimerase is the / V-acetylglucosamine 2-epimerase of B. ovatus ATCC 8483 (UniProtKB Entry No. A7LVG6) as encoded by gene BACOVA_01816. Another example is the / V-acetylglucosamine 2-epimerase of Synechocystis sp. (strain PCC 6803) (UniProtKB Entry No. P74124) which is also known as renin-binding protein and is encoded by the slr1975 gene.

[0151] In an additional and / or alternative embodiment, the genetically engineered microbial cell contains a nucleic acid molecule comprising a nucleotide sequence which encodes an / V-acetylglucosamine 2-epimerase, preferably the / V-acetylglucosamine 2-epimerase of B. ovatus ATCC 8483 or Synechocystis sp. (strain PCC 6803) or a functional variant thereof.

[0152] In an additional and / or alternative embodiment, the genetically engineered microbial cell possesses / V-acetylglucosamine-6-phosphate epimerase activity and N- acetylmannosamine-6-phosphate phosphatase activity. / V-acetylglucosamine-6- phosphatase epimerase converts / V-acetylglucosamine-6-phosphate (GlcNAc-6P) to / V-acetylmannosamine-6-phosphate (ManNAc-6P), whereas / V-acetylmannosamine- 6-phosphate phosphatase dephosphorylates ManNAc-6P to give N- acetylmannosamine (ManNAc). Possessing / V-acetylglucosamine-6-phosphate epimerase activity and / V-acetylmannosamine-6-phosphate phosphatase activity provides an additional or alternative way for providing ManNAc for Neu5Ac production.

[0153] In an additional and / or alternative embodiment, the genetically engineered microbial cell contains an / V-acetylglucosamine-6-phosphate epimerase. An example of a suitable / V-acetylglucosamine-6-phosphate epimerase is E. coli NanE (UniProtKB Entry No. P0A761) as encoded by the E. coli nanE gene.

[0154] Thus, in an additional and / or alternative embodiment, the genetically engineered microbial cell contains a nucleic acid molecule comprising and expressing a nucleotide sequence encoding an / V-acetylglucosamine-6-phosphate epimerase, preferably a nucleotide sequence encoding E. coli NanE.

[0155] In an additional and / or alternative embodiment, the genetically engineered microbial cell contains an / V-acetylmannosamine-6-phosphate phosphatase.

[0156] Thus, in additional and / or alternative embodiment, the genetically engineered microbial cell contains a nucleic acid molecule comprising and expressing a nucleotide sequence encoding an / V-acetylmannosamine-6-phosphate phosphatase. The genetically engineered microbial cell possesses sialyltransferase activity, preferably a heterologous sialyltransferase activity, and more preferably a sialyltransferase activity selected from the group consisting of a-2,3-sialyltransferase activity, a-2,6-sialyltransferase activity and / or a-2,8-sialyltransferase activity. The sialyltransferase activity is capable of transferring the / V-acetylneuraminic acid moiety from the CMP-NeuNAc to an acceptor molecule, wherein said acceptor molecule is a saccharide molecule, to provide a sialylated saccharide.

[0157] In some embodiments, the oligosaccharide of interest is a derivative of LNT or LNnT which is fucosylated and sialylated. It is understood that the genetically engineered microbial cells for producing a fucosylated and sialylated derivative of LNT or LNnT possesses at least one fucosyltransferase, a metabolic pathway for biosynthesis of GDP-L-fucose, at least one sialyltransferase and a metabolic pathway for CMP- NeuNAc biosynthesis as outlined herein before

[0158] In additional and / or alternative embodiments, the genetically engineered microbial cell comprises a lactose permease or a lactose importer for the internalization of exogenous lactose. In certain embodiments, lactose permease or lactose importer is a non-endogenous or heterologous permease or importer. In some embodiments the genetically engineered microbial cell contains a polynucleotide comprising a nucleotide sequence that encodes the non-endogenous or heterologous lactose permease or lactose importer.

[0159] Possessing a lactose permease enables the genetically engineered microbial cell to internalize exogenous lactose and utilize it e.g. as an educt in the intracellular biosynthesis of the oligosaccharide of interest. A suitable lactose permease is E. coli (K12) lactose permease LacY (UniProtKB Entry No. P02920) as encoded by the E. coli K12 lacY gene.

[0160] In an additional and / or alternative embodiment, the genetically engineered microbial cell contains a nucleic acid molecule which comprises and expresses a nucleotide sequence encoding a lactose permease, such as e.g. the E. coli K12 lactose permease LacY or functional fragments or functional variants thereof. In additional and / or alternative embodiments, the genetically engineered microbial cell synthesizes lactose intracellularly.

[0161] In an additional and / or alternative embodiment, the genetically engineered microbial cell has reduced, deleted or functionally impaired enzymatic activity that hydrolyzes lactose, such as e.g. p-galactosidase activity. For example, in E. coli (strain K12) the P-galactosidase is LacZ (UniProtKB Entry No. P00722) as encoded by the lacZ gene.

[0162] Having deleted or at least reduced the intracellular p-galactosidase activity improves yield of the desired oligosaccharide as the intracellular pool of lactose not diminished by intracellular p-galactosidase activity. Hence, more lactose - regardless of whether internalized lactose or lactose being produced intracellularly by the genetically engineered microbial cell that produces the oligosaccharide of interest - is available for production of the oligosaccharide of interest.

[0163] In additional and / or alternative embodiments, the genetically engineered microbial cell has reduced, deleted or functionally impaired enzymatic activities which acetylates lactose. Deletion or at least reduction of galactoside O-acetyltransferase activity improves yield of the desired oligosaccharide as the intracellular pool of lactose is not diminished. For example, in E. coli (strain K12) the galactoside O- acetyltransferase is LacA (UniProt Entry No. P07464) as encoded by the lacZ gene.

[0164] According to the fourth aspect, provided is the use of a variant p-1 ,3-N- acetylglucosaminyltransferase polypeptide as described herein before for synthesis of a GIcNAc-containing oligosaccharide of interest, preferably of a GIcNAc- containing human milk oligosaccharide, and of a genetically engineered microbial cell that is able to intracellularly synthesize a GIcNAc moiety-containing oligosaccharide of interest as described herein before for the production of a GIcNAc-containing oligosaccharide of interest, preferably of a GIcNAc-containing human milk oligosaccharide

[0165] According to the fifth aspect, provided is a method for producing a GIcNAc- containing oligosaccharide of interest. The method comprises culturing a population of genetically engineered microbial cells as described herein before in a culture medium and under conditions that are permissive for the genetically engineered microbial cell to produce the GIcNAc-containing oligosaccharide of interest.

[0166] The expression “culture medium that is permissive for the genetically engineered microbial cell to produce the GIcNAc containing oligosaccharide of interest” as used herein refers to a culture medium that is adapted to cell growth, the culture medium comprising nutrients and growth factors essential to growth of the genetically engineered microbial cells and is free of toxic agents that can cause cell death, such as cell growth inhibitors, or wherein toxic agents are present in an amount that is not lethal to said cells. In addition, the culture medium contains factors essential to the intracellular synthesis of the desired oligosaccharide such as, e.g. educts and / or precursors of the oligosaccharide of interest.

[0167] The expression “conditions that are permissive for the genetically engineered microbial cell to produce the GIcNAc containing oligosaccharide of interest are understood to be conditions relating to physical and / or chemical parameters including but not limited to temperature, pH, pressure, osmotic pressure and product / precursor / acceptor concentration.

[0168] In some embodiments, the GIcNAc-containing oligosaccharide of interest is a human milk oligosaccharide. In additional and / or alternative embodiments, the HMO is selected from the group consisting of LNnT, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DF-LNH I, DF- LNH II, DF-LNnH, DF-para-LNH, DF-para-LNnH, TF-LNH, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS-LNnH I, and FDS-LNH II.

[0169] In some embodiments, the method comprises culturing the population of genetically engineered microbial cells in the presence of exogenous lactose. Lactose is a disaccharide. Lactose is added to the culture medium if the genetically engineered microbial cell can internalize exogenous lactose and / or is not able to intracellularly synthesize lactose. The internalized lactose can serve as educt for the intracellular biosynthesis of the oligosaccharide of interest. Lactose serves as preferred substrate for the variant p-1 ,3-N-acetylglucosaminyltransferase polypeptide. In additional and / or alternative embodiments, the method comprises culturing the genetically engineered microbial cell in the presence of at least one of glucose, fructose, sucrose and glycerol. Any one of these compounds serves as a carbon source and energy source for the microbial cells. In some embodiments, the genetically engineered microbial cell is cultivated in the presence of a mixture of glucose and fructose. The mixture may be an equimolar mixture of glucose and fructose, and may be obtained by hydrolysis of sucrose.

[0170] The method, optionally, further comprises recovering the GIcNAc-containing oligosaccharide of interest from the culture medium and / or the genetically engineered microbial cells.

[0171] The recovery of the GIcNAc-containing oligosaccharide of interest comprises at least one of the following steps: clarification, centrifugation, microfiltration, ultrafiltration, nanofiltration, diafiltrations, two-phase partitioning, reverse osmosis, microfiltration, activated charcoal or carbon treatment, treatment with non-ionic surfactants, enzymatic digestion, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange treatment, hydrophobic interaction chromatography and / or gel filtration, ligand exchange chromatography, electrodialysis, drying such as e.g. rotary evaporation, free-fall evaporation, freeze-drying, spray-drying, drum-drying, roller-drying, vacuum-drying.

[0172] The present invention will be described with respect to embodiments and with reference to drawings, but the invention is not limited thereto but only by the claims. Furthermore, the terms first, second and the like in the description and in the claims are used for distinguishing between similar elements and not necessarily for describing a sequence, either temporally, spatially, in ranking or in any other manner. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments described herein are capable of operation in other sequences than described or illustrated herein.

[0173] It is to be noticed that the term “comprising”, used in the description and the claims, should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. It is thus to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude 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 only of components A and B. It means that with respect to the present invention, the only relevant components of the device are A and B.

[0174] Reference throughout this specification 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” 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, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

[0175] Similarly, it should be appreciated that in the description of exemplary embodiments, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, 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 this invention.

[0176] Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features or different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination. Furthermore, an element described herein of an apparatus embodiment is an example of a means for carrying out the function performed by the element for the purpose of carrying out the invention.

[0177] In the description and drawings provided herein, numerous specific details are set forth. However, it is understood that embodiments 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.

[0178] The invention will now be described by a detailed description of several embodiments of the invention. It is clear that other embodiments of the invention can be configured according to the knowledge of persons skilled in the art without departing from the true spirit or technical teaching of the invention, the invention being limited only by the terms of the appended claims.

[0179] Example 1 : Genetic engineering of LNnT-producing E. coli bearing variant P-1,3-N-acetylglucosaminyltransferase polypeptides

[0180] A bacterial production strain producing LNT-II was generated by insertion of the 1,3- N-acetylglucosaminyl-transferase gene IgtA from Neisseria meningitidis into the araBA locus of an E. coli DH1 strain in which the genes wcaJ, lacZ, lacA, nagB, waaB, and mdoH were deleted. In addition, the E. coli lacY gene was expressed heterologously in this strain. The bacterium’s metabolic pathway to generate UDP- galactose was enhanced by chromosomal integration of E. coli genes pgm, galE and galU. Furthermore, the strain possesses a p-1,4-galactosyltransferase gene lex- 1 from Aggregatibacter segnis which was placed under control of a constitutive promoter and a rrnB terminator. The resulting strain is designated “E. coli LNnT”. An operon containing the gene encoding the saccharide transporter LmrC of Streptomyces lincolnensis and the gene encoding the LambB porin of E. coli was constructed and inserted via lambda-Red recombination at the asl locus yielding E. coli strain “E. coli LNnT TP”.

[0181] Various amino acid substitutions were introduced into the coding sequence of IgtA within the strain “E. coli LNnT TP” via single-stranded DNA recombineering. To this end, E. coli cells of the strain were made lambda-Red- and electro-competent, and DNA oligonucleotides possessing a nucleotide sequence of 80 to 90 nucleotides being complementary to the coding region of IgtA wherein each oligonucleotide carried the desired mutation(s) of the LgtA-encoding nucleotide sequence were electroporated. The nucleotide sequences of these primers are provided in Table 1.

[0182] Table 1: MAGE primers used to generate variants of N.m. IgtA.

[0183] After recovery, bacterial cells were plated to pick single colonies for analysis of the nucleotide sequence of their IgtA gene by PGR. Amplification products obtained by PGR using primer pairs, wherein one primer was 3'-complementary to either the wild-type or the mutated IgtA nucleotide and the other primer matching a nucleotide sequence 500 bp downstream of the mutated site were subjected to Sanger sequence analysis. Clonal lines of “E. coli LNnT TP” bearing the desired IgtA variants were subjected to a microtiterplate screening assay for oligosaccharide production. After 24 hours in the main culture containing 10 mM lactose, the supernatants were harvested and a 1 / 20 dilution of each supernatant used for LC-MS quantification of LNT2, LNnT and pLNnH.

[0184] The effect of the LgtA(H158_H249F_Q256W) variant on LNnT production strain was analyzed in small scale fermentations (3-L-fermenters) running for 100 hours. Oligosaccharide analysis of the culture supernatants in these fermentations revealed a reduction of pLNnH relative to LNnT by a factor of four for “E. coli LNnT TP” LgtA(H158_H249F_Q256W) as compared to “E. coli LNnT TP”

[0185] Example 2: Substrate specificity of LgtA variants (in vitro)

[0186] Substrate specificity of LgtA and its variants towards lactose and LNnT were assessed in vitro by determining Michaelis Menten kinetics using purified proteins. For better solubility, the coding sequences of lgtA_WT or lgtA_(H158W_H249F_ Q256W) were fused to the coding sequence of the lex-1 gene from Aaggregati- bacter aphrophilus [WP_005701792.1] further providing a C-terminal (His)6tag to the fusion proteins. The coding sequence of the lgtA-lex1 fusion proteins were placed under control of the tetracycline promotor within the plasmid pINT (see e.g. EP 3 929 300 A1) resulting in plasmids plNT-lgtA-lex1 and plNT-lgtA_(H158W_ H249F_Q256W)-lex1. The plasmids were transformed in SoluBL21 cells (Gelantis, San Diego, CA). Plasmid pl NT encodes also the tetracycline repressor. Hence, a low basal expression of the fusion proteins was induced by the addition of anhydrotetracycline to 0.2 pg / ml. The main cultures of 200 ml Studier medium ZYP- 5052 with 100 pg / ml ampicillin were grown at 37 °C until an ODeoo of 0.8 was reached. Temperature of the cultures was shifted to 16 °C, anhydrotetracyline was then added and cultures were shaken for another 16 hours. Cells were harvested by centrifugation and a soluble lysate under native conditions was prepared in lysis buffer [50 mM sodium phosphate, 300 mM NaCI, 5 mM imidazole, 10 mM p-mer- captoethanol. Briefly, cells were resuspended in lysis buffer, incubated with 5 mg lysozyme for 20 min, sonicated in a cell-disruptor and centrifuged for 20 min at 20.000 x g. The clarified lysate was applied to a Ni-NTA column, washed with lysis buffer containing 40 mM imidazole and eluted with lysis buffer containing 200 mM imidazole.

[0187] Enzymatic reactions were performed in a reaction solution [20 mM Tris-HCI, pH 7.4, 200 mM NaCI, 4 mM MnCI2, 4 mM UDP-GIcNAc and 10 ng / pl purified fusion protein with different concentrations of the acceptor substrates lactose or LNnT. At time points of 0, 15, 30, 60, 90, 135 min, aliquots were quenched in 0.1 % SDS, 10 mM EDTA stop solution. A 1 :100 dilution was used for LC-MS quantification of lactose and LNT-II as well as LNnT and GIcNAc-LNnT. Time courses were performed for acceptor substate concentrations of 5, 10, 20, 30, 60, 90 and 120 mM (lactose or LNnT) supplemented in the reaction solution. Formed LNT-II or GIcNAc-LNnT was plotted against incubation time; thereby initial velocities were obtained through linear regression. These initial velocities were then plotted against the acceptor substrate concentration as shown in FIG. 4 and FIG. 5.

[0188] The calculated Kmof LgtA for lactose of around 40 mM is in the range of published results of 28 mM (Blixt et al. (1999) supra). The Kmfor lactose was about the same for / V.m.LgtA (•) and / V.m.LgtA(H158W_H249F_Q256W) (♦) as shown in FIG. 4. Furthermore, the / V.m.LgtA and the variant enzymes seem to be comparably active towards lactose as substrate. With LNnT as substrate (FIG. 5), some activity could be shown for / V.m.LgtA (•). However, the / V.m.LgtA variant (H158W_H249F_Q256W) (♦) appeared to be 10-30-fold less active towards LNnT.

[0189] Example 3: Substrate specificity of LgtA variants (in vivo)

[0190] The different LNnT production strains described in Example 1 were cultured for assessing LNnT production.

[0191] Culture medium

[0192] The culture medium used to grow the cells for the production of the desired oligosaccharide contained: 3 g / L KH2PO4, 12 g / L K2HPO4, 5 g / L (NH4)SO4, 0.3 g / L citric acid, 0.1 g / L NaCI, 2 g / L MgSO4x7 H2O and 0.015 g / L CaChx6 H2O, supplemented with 10 mg / L thiamine and 1 ml / L trace element solution (54.4 g / L ammonium ferric citrate, 9.8 g / L MnChx4 H2O, 1.6 g / L C0CL2x6 H2O, 1 g / L CuChx2 H2O, 1.9 g / L MnChx4 H2O, 1.1 g / L Na2MoO4x2 H2O, 1.5 g / L Na2SeO3, 1.5 g / L N iSC>4x6 H2O). The pH of the medium was adjusted to 7.0. For synthesis of the desired oligosaccharide lactose was added to the medium to a concentration of 10 mM.

[0193] LC / MS analytics

[0194] Product identity was determined by multiple reaction monitoring (MRM) using an LC triple-quadrupole MS detection system (Shimadzu LCMS-8050). Precursor ions were selected and analyzed in quadrupole 1, followed by collision-induced fragmentation (CID) with argon and selection of fragment ions in quadrupole 3. Selected transitions and collision energies for intermediates and end-product metabolites are listed in Table 2. LNT2, LNnT, and pLNnH in particle free culture supernatant or cytoplasmic fractions were separated by high performance liquid chromatography (HPLC) using a Waters XBridge Amide HPLC column (3.5 pm, 2.1 A 50 mm). Neutral sugars were eluted with isocratic flow of H2O with 0.1% (v / v) ammonium hydroxide. Before LC / MS analysis, the samples of neutral oligosaccharides were prepared by filtration (0.22 pm pore size) and cleared by solid-phase extraction on a Strata ABW ion exchange matrix (Phenomenex). The HPLC system encompasses a Shimadzu Nexera X2 SIL-30ACMP autosampler run at 8 °C, a Shimadzu LC-20AD pump, and a Shimadzu CTO-20AC column oven running at 35 °C for elution of neutral sugars. For the analysis of neutral sugars 1 pl was injected into the instrument. Flow rate was set 0,3 mL / min (neutral), corresponding to a run time of 5 min. Whereas neutral sugars were analyzed in negative ion mode. The mass spectrometer was operated at unit resolution.

[0195] Collision energy, Q1 and Q3 pre-bias were optimized for each analyte. Quantification methods were established using commercially available standards (Carbosynth and Elicityl).

[0196] Some results of these experiments are displayed in FIG. 2 and FIG 3 which illustrate that the relative titer of LNnT obtained from culturing E. coli strains containing / V.m.LgtA(H249F, Q256W) or / V.m.LgtA(H158W, H249F, Q256W) provide threefold or fourfold amounts of LNnT after 90 hours of fermentation as compared to otherwise genetically identical E. coli strains containing the wild-type / V.m.LgtA (Fig. 2).

[0197] Table 2 List of diagnostic MRM transitions and parameters for the identification of LNnT, pLNnH (CE = Collision energy) of the Shimadzu LCMS-8050 Triple Quadrupole (QQQ) Mass Analyzer.

[0198] While the relative amounts of LNT-2 and LNnT are nearly the same when culturing E. coli strains containing the wild-type / V.m.LgtA (C), the variant (3-1 ,3-N- acetylglucosaminyltransferase / V.m.LgtA(H249F, Q256W) (A) or the (3-1 ,3-N- acetylglucosaminyltransferase / V.m.LgtA (H158W, H249F, Q256W) (B), a significant reduction of pLNH in the cultures is achieved.

Claims

Claims1. A variant of a p-1 ,3-N-acetylglucosaminyltransferase polypeptide possessing an amino acid sequence that has a sequence identity of at least 80% to the amino acid sequence as set forth in SEQ ID NO: 1, wherein the variant comprises at least one amino acid substitution relative to the amino acid sequence as set forth in SEQ ID NO: 1, wherein the at least one amino acid substitution comprises an amino acid substitution of at least one of the amino acids at amino acid positions 158, 249 and 256 of the amino acid sequence set forth in SEQ ID NO: 1 with a hydrophobic amino acid.

2. The variant p-1 ,3-N-acetylglucosaminyltransferase polypeptide of claim 1 , wherein the variant comprises two or more amino acid substitutions relative to the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the two or more amino acid substitutions comprise amino acid substitutions of at least two of the amino acids at amino acid positions 158, 249 and 256 of the amino acid sequence set forth in SEQ ID NO: 1 with a hydrophobic amino acids.

3. The variant p-1 ,3-N-acetylglucosaminyltransferase polypeptide of claim 1 or 2, wherein the hydrophobic amino acid is selected from glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M) and tryptophane (W).

4. The variant p-1 ,3-N-acetylglucosaminyltransferase polypeptide of any one of claims 1 to 3, wherein the polypeptide exhibits increased substrate specificity for lactose over lacto-N-neotetraose or lacto-N-neohexaose as compared to the substrate specificity of a p-1,3-N-acetylglucosaminyltransferase polypeptide having the amino acid sequence as set forth in SEQ ID NO: 1.

5. A nucleic acid molecule comprising a nucleotide sequence which encodes a variant p-1,3-N-acetylglucosaminyltransferase polypeptide according to any one of claims 1 to 3.

6. The nucleic acid molecule of claim 5, wherein the nucleotide sequence encoding the variant p-1 ,3-N-acetylglucosaminyltransferase polypeptide is operably linked to at least one expression control sequence.

7. A genetically engineered microbial cell for intracellular biosynthesis of an oligosaccharide of interest containing an N-acetylglucosamine moiety, the microbial cell comprising a variant p-1 ,3-N-acetylglucosaminyltransferase polypeptide according to any one of claims 1 to 4 and / or a nucleic acid molecule according to any one of claims 5 and 6.

8. The microbial cell of claim 7, wherein the microbial cell is able to intracellularly synthesize a human milk oligosaccharide, preferably a human milk oligosaccharide selected from the group consisting of lacto-N-triose II, lacto-N- tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-neofucopenta- ose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difuco- hexaose II, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, difucosyllacto-N-hexaose I, difucosyllacto-N-hexa- ose II, lacto-ZV-sialylpentaose a, lacto- / V-sialylpentaose b, lacto-ZV-sialylpenta- ose c, fucosyl-lacto-ZV-sialylpentaose a, fucosyl-lacto-ZV-sialylpentaose b, fucosyl-lacto-ZV-sialylpentaose c, disialyl-lacto-ZV-tetraose.

9. Use of a variant p-1 ,3-N-acetylglucosaminyltransferase polypeptide of any one of claims 1 to 4 or of a genetically engineered microbial cell of claim 7 or 8 for the synthesis of an oligosaccharide of interest containing an N- acetylglucosamine moiety.

10. A method of producing an oligosaccharide of interest containing an N- acetylglucosamine moiety, the method comprising the steps of culturing a population of genetically engineered microbial cells according to claim 7 or 8 in a culture medium and under conditions that are permissive for the microbial cells to intracellularly synthesize the oligosaccharide of interest, and optionally recovering the oligosaccharide of interest.

11. The method of claim 10, wherein the oligosaccharide of interest is a human milk oligosaccharide, preferably a human milk oligosaccharide selected from the group consisting of lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, lacto-N-hexaose, lacto-N- neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, difucosyllacto- N-hexaose I, difucosyllacto-N-hexaose II, lacto-ZV-sialylpentaose a, lacto- / V- sialylpentaose b, lacto- / V-sialylpentaose c, fucosyl-lacto-ZV-sialylpentaose a, fucosyl-lacto-ZV-sialylpentaose b, fucosyl-lacto-ZV-sialylpentaose c, disialyl- lacto-ZV-tetraose.

12. A method for altering the substrate specificity of a p-1 ,3-N-acetylglucosaminyl- transferase polypeptide possessing an amino acid sequence as set forth in SEQ ID NO: 1, wherein the method comprises substituting one, two or all of the amino acid residues at amino acid positions 158, 249 and 256 with a hydrophobic amino acid, preferably by providing a nucleic acid molecule comprising a nucleotide sequence which encodes the p-1 ,3-N- acetylglucosaminyltransferase polypeptide possessing an amino acid sequence as set forth in SEQ ID NO: 1, altering the nucleotide sequence of one, two or all the codons encoding the amino acid residues at positions 158, 249 and 256 of the amino acid sequence set forth in SEQ ID NO: 1 to the nucleotide sequence of codon(s) encoding a hydrophobic amino acid; and expressing the nucleotide sequence encoding the resulting variant p-1 ,3-N- acetylglucosaminyltransferase polypeptide to provide a p-1 ,3-N- acetylglucosaminyltransferase polypeptide that exhibits an increased substrate specificity for lactose over lacto-N-neotetraose or lacto- N- neohexaose as compared to the substrate specificity of the p-1 ,3-N- acetylglucosaminyltransferase polypeptide having the amino acid sequence as set forth in SEQ ID NO: 1.

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