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VARIANT SUCROSE PERMEASE POLYPEPTIDES

20240035004 · 2024-02-01

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the present invention is in the technical field of fermentation of metabolically engineered cells. The present invention describes new sucrose permease polypeptides, and their applications. The present invention also describes a metabolically engineered cell for the production of a glycosylated product using the novel sucrose permease polypeptides. Furthermore, the present invention provides a method for the production of a glycosylated product by a cell using the novel sucrose permease polypeptides as well as the purification of said glycosylated product from the cultivation.

    Claims

    1.-45. (canceled)

    46. A sucrose permease having sucrose permease activity and comprising a polypeptide that has at least 80% overall sequence identity to SEQ ID NO: 1 and that i) differs from SEQ ID NO: 1 by having a different amino acid for proline at position 169 (P169a), for valine at position 316 (V316c) or for phenylalanine at position 371 (F371e) wherein a, c, and e can be any amino acid residue excluding histidine for residue a, or ii) shares the serine at position 246 of SEQ ID NO: 1 and differs from SEQ ID NO: 1 by having a different amino acid for tryptophan at position 230 (W230b) or for cysteine at position 327 (C327d), wherein b and d can be any amino acid residue, or iii) differs from SEQ ID NO: 1 by having at least two amino acid differences selected from the group consisting of proline at position 169 (P169f), tryptophan at position 230 (W230b), valine at position 316 (V316c), cysteine at position 327 (C327d) and phenylalanine at position 371 (F371e), wherein f, b, c, d, and e can be any amino acid residue, or iv) differs from SEQ ID NO: 1 by having three amino acid differences selected from the group consisting of proline at position 169 (P169f), tryptophan at position 230 (W230b), valine at position 316 (V316c), cysteine at position 327 (C327d) and phenylalanine at position 371 (F371e), or v) differs from SEQ ID NO: 1 by having four amino acid differences selected from the group consisting of proline at position 169 (P169f), tryptophan at position 230 (W230b), valine at position 316 (V316c), cysteine at position 327 (C327d) and phenylalanine at position 371 (F371e), wherein f, b, c, d, and e can be any amino acid residue, or vi) differs from SEQ ID NO: 1 by having five amino acid differences selected from the group consisting of proline at position 169 (P169f), tryptophan at position 230 (W230b), valine at position 316 (V316c), cysteine at position 327 (C327d) and phenylalanine at position 371 (F371e), wherein f, b, c, d, and e can be any amino acid residue.

    47. The sucrose permease of claim 46, wherein the sucrose permease: i) differs from SEQ ID NO: 1 by having a different amino acid for proline at position 169 (P169a), for valine at position 316 (V316c) or for phenylalanine at position 371 (F371e) wherein a and e can be any amino acid residue excluding histidine for residue a, and wherein c can be any amino acid residue excluding methionine, or ii) shares the serine at position 246 of SEQ ID NO: 1 and differs from SEQ ID NO: 1 by having a different amino acid for tryptophan at position 230 (W230b) or for cysteine at position 327 (C327d) wherein b and d can be any amino acid residue, or iii) differs from SEQ ID NO: 1 by having at least two amino acid differences selected from the group consisting of proline at position 169 (P169f), tryptophan at position 230 (W230b), valine at position 316 (V316c), cysteine at position 327 (C327d) and phenylalanine at position 371 (F371e), wherein f, b, c, d, and e can be any amino acid residue, or iv) differs from SEQ ID NO: 1 by having three amino acid differences selected from the group consisting of proline at position 169 (P169f), tryptophan at position 230 (W230b), valine at position 316 (V316c), cysteine at position 327 (C327d) and phenylalanine at position 371 (F371e), or v) differs from SEQ ID NO: 1 by having four amino acid differences selected from the group consisting of proline at position 169 (P169f), tryptophan at position 230 (W230b), valine at position 316 (V316c), cysteine at position 327 (C327d) and phenylalanine at position 371 (F371e), wherein f, b, c, d, and e can be any amino acid residue, or vi) differs from SEQ ID NO: 1 by having five amino acid differences selected from the group consisting of proline at position 169 (P169f), tryptophan at position 230 (W230b), valine at position 316 (V316c), cysteine at position 327 (C327d) and phenylalanine at position 371 (F371e), wherein f, b, c, d, and e can be any amino acid residue.

    48. The sucrose permease of claim 46, wherein the sucrose permease: i) differs from SEQ ID NO: 1 by having a different amino acid for proline at position 169 (P169a), for valine at position 316 (V316c), or for phenylalanine at position 371 (F371e), wherein a and e can be any amino acid residue excluding histidine for residue a, and wherein c is selected from the group consisting of alanine, cysteine and phenylalanine, or ii) shares the serine at position 246 of SEQ ID NO: 1 and differs from SEQ ID NO: 1 by having a different amino acid for tryptophan at position 230 (W230b) or for cysteine at position 327 (C327d) wherein b and d can be any amino acid residue, or iii) differs from SEQ ID NO: 1 by having at least two amino acid differences selected from the group consisting of proline at position 169 (P169f), tryptophan at position 230 (W230b), valine at position 316 (V316c), cysteine at position 327 (C327d) and phenylalanine at position 371 (F371e), wherein f, b, c, d, and e can be any amino acid residue, or iv) differs from SEQ ID NO: 1 by having at least three amino acid differences selected from the group consisting of proline at position 169 (P169f), tryptophan at position 230 (W230b), valine at position 316 (V316c), cysteine at position 327 (C327d) and phenylalanine at position 371 (F371e), wherein f, b, c, d, and e can be any amino acid residue, or v) differs from SEQ ID NO: 1 by having four amino acid differences selected from the group consisting of proline at position 169 (P169f), tryptophan at position 230 (W230b), valine at position 316 (V316c), cysteine at position 327 (C327d) and phenylalanine at position 371 (F371e), wherein f, b, c, d, and e can be any amino acid residue, or vi) differs from SEQ ID NO: 1 by having five amino acid differences selected from the group consisting of proline at position 169 (P169f), tryptophan at position 230 (W230b), valine at position 316 (V316c), cysteine at position 327 (C327d) and phenylalanine at position 371 (F371e), wherein f, b, c, d, and e can be any amino acid residue.

    49. The sucrose permease of claim 46, wherein the sucrose permease has an improved affinity for sucrose compared to the sucrose permease of SEQ ID NO: 1.

    50. The sucrose permease of claim 46, wherein the sucrose permease i) comprises the polypeptide of any one of SEQ ID NOs: 26, 5, 16, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31 or 32, or ii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 26, 5, 16, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31 or 32, having at least 80% overall sequence identity to the full length of any one of the polypeptides with SEQ ID NOs: 26, 5, 16, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31 or 32 and having sucrose permease activity.

    51. The sucrose permease of claim 46, wherein the sucrose permease i) comprises the polypeptide of SEQ ID NO: 32, or ii) is a functional homologue, variant or derivative of SEQ ID NO: 32, having at least 80% overall sequence identity to the full length of polypeptides with SEQ ID NO: 32 and having sucrose permease activity.

    52. The sucrose permease of claim 46, wherein the sucrose permease i) comprises the polypeptide of SEQ ID NOs: 27, 28, 29, 30 or 31, or ii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 27, 28, 29, 30 or 31, having at least 80% overall sequence identity to the full length of any one of the polypeptides with SEQ ID NOs: 27, 28, 29, 30 or 31 and having sucrose permease activity.

    53. The sucrose permease of claim 46, wherein the sucrose permease i) comprises the polypeptide of SEQ ID NOs: 26, 17, 18, 19, 20, 21, 22, 23, 24 or or ii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 26, 17, 18, 19, 20, 21, 22, 23, 24 or 25, having at least 80% overall sequence identity to the full length of any one of the polypeptides with SEQ ID NOs: 26, 17, 18, 19, 20, 21, 22, 23, 24 or 25 and having sucrose permease activity.

    54. The sucrose permease of claim 50, wherein the sucrose permease with SEQ ID NO: 26: differs from SEQ ID NO: 1 by having a different amino acid for valine at position 316, for cysteine at position 327 and for phenylalanine at position 371, or is a functional homologue, variant or derivative of any one of SEQ ID NOs: 33, 110, 111, 112, 113 or 114, having at least 80% overall sequence identity to the full length of any one of the polypeptides with SEQ ID NOs: 33, 110, 111, 112, 113 or 114 and having sucrose permease activity.

    55. The sucrose permease of claim 46, wherein the sucrose permease i) comprises the polypeptide of SEQ ID NOs: 16, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or ii) is a functional homologue, variant, or derivative of any one of SEQ ID NOs: 16, 7, 8, 9, 10, 11, 12, 13, 14 or 15, having at least 80% overall sequence identity to the full length of any one of the polypeptides with SEQ ID NOs: 16, 7, 8, 9, 10, 11, 12, 13, 14 or 15 and having sucrose permease activity.

    56. The sucrose permease of claim 50, wherein the sucrose permease with SEQ ID NO: 16: differs from SEQ ID NO: 1 by having a different amino acid for cysteine at position 327 and for phenylalanine at position 371, or is a functional homologue, variant or derivative of any one of SEQ ID NOs: 40, 117 or 118, having at least 80% overall sequence identity to the full length of any one of the polypeptides with SEQ ID NOs: 40, 117 or 118 and having sucrose permease activity.

    57. The sucrose permease of claim 46, wherein the sucrose permease i) comprises the polypeptide of SEQ ID NOs: 5, 2, 3, 4 or 6, or ii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 5, 2, 3, 4 or 6, having at least 80% overall sequence identity to the full length of any one of the polypeptides with SEQ ID NOs: 5, 2, 3, 4 or 6 and having sucrose permease activity.

    58. The sucrose permease of claim 50, wherein the sucrose permease with SEQ ID NO: 5: differs from SEQ ID NO: 1 by having a different amino acid for cysteine at position 327, or is a functional homologue, variant or derivative of SEQ ID NO: 36 or 100, having at least 80% overall sequence identity to the full length of the polypeptides with SEQ ID NO: 36 or 100 and having sucrose permease activity.

    59. The sucrose permease of claim 46, wherein the sucrose permease further differs from SEQ ID NO: 1 by having at least one further amino acid difference selected from the group consisting of: leucine at position 61 is substituted with proline, tryptophan, histidine, phenylalanine, or tyrosine, phenylalanine at position 159 is substituted with leucine, glycine at position 162 is substituted with cysteine, proline at position 169 is substituted with histidine, arginine at position 300 is substituted with alanine or leucine, glutamine at position 353 is substituted with histidine, truncation of amino acid residues 404 to 415, and truncation of amino acid residues 409 to 415.

    60. The sucrose permease of claim 59, wherein the sucrose permease is: represented by SEQ ID NO: 118 or 119, or is a functional homologue, variant or derivative of SEQ ID NO: 118 or 119, having at least 80% overall sequence identity to the full length of the polypeptides with SEQ ID NO: 118 or 119 and having sucrose permease activity.

    61. The sucrose permease of claim 46, wherein the sucrose permease comprises of at least 350 amino acids.

    62. The sucrose permease of claim 46, wherein the sucrose permease comprises of less than 450 amino acids.

    63. A metabolically engineered cell for producing a glycosylated product, the cell comprising a pathway for production of the glycosylated product, wherein the cell is capable of expressing at least one sucrose permease that has sucrose permease activity and comprises a polypeptide that differs from SEQ ID NO: 1 by (i) having at least one amino acid difference selected from the group consisting of: proline at position 169 (P169f), tryptophan at position 230 (W230b), valine at position 316 (V316c), cysteine at position 327 (C327d), and phenylalanine at position 371 (F371e) wherein f, b, c, d, and e can be any amino acid residue and/or (ii) a truncation of amino acid residues 403 to 415, 404 to 415, 405 to 415, 406 to 415, 407 to 415, 408 to 415, 409 to 415, 410 to 415, 411 to 415, 412 to 415, 413 to 415 or 414 to 415.

    64. The cell of claim 63, wherein the sucrose permease has an improved affinity for sucrose compared to the sucrose permease of SEQ ID NO: 1.

    65. The cell of claim 63, wherein the sucrose permease i) comprises the polypeptide of any one of SEQ ID NOs: 26, 5, 16, 34, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 35, 36, 37, 38, 39, 40, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or 121, or ii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 26, 5, 16, 34, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 35, 36, 37, 38, 39, 40, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 or 121, having at least 80% overall sequence identity to the full length of any one of the polypeptides with SEQ ID NOs: 26, 5, 16, 34, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 35, 36, 37, 38, 39, 40, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 or 121 and having sucrose permease activity.

    66. The cell of claim 63, wherein the sucrose permease further differs from SEQ ID NO: 1 by having at least one further amino acid difference selected from the group consisting of: leucine at position 61 is substituted with proline, tryptophan, histidine, phenylalanine or tyrosine, phenylalanine at position 159 is substituted with leucine, glycine at position 162 is substituted with cysteine, proline at position 169 is substituted with histidine, arginine at position 300 is substituted with alanine or leucine, glutamine at position 353 is substituted with histidine, truncation of amino acid residues 404 to 415, and truncation of amino acid residues 409 to 415.

    67. The cell of claim 63, wherein the cell is a microorganism, a bacterium, an Escherichia coli strain, an E. coli K-12 strain, or E. coli MG1655.

    68. The cell of claim 63, wherein the glycosylated product is selected from the group consisting of di- or oligosaccharides, nucleosides, glycosides and glycolipids, mammalian milk di- or oligosaccharides, and human milk di- or oligosaccharides.

    69. A method of producing a glycosylated product by the cell of claim 63, the method comprising: i) cultivating the cell under conditions permissive to produce the glycosylated product, ii) optionally, separating the glycosylated product from the cultivation, and iii) optionally, purifying the glycosylated product from the cell.

    Description

    [0390] The invention will be described in more detail in the examples and the attached figures, in which

    [0391] FIG. 1 shows the normalized maximal growth rate (Max in %) of an E. coli 3-FL production strain comprising the wild type CscB sucrose permease with SEQ ID NO 01 from E. coli W when grown on minimal medium comprising 4, 8 or 30 g/L sucrose.

    [0392] FIG. 2 shows the normalized maximal growth rate (Max in %) of E. coli 2-FL production strains expressing the a1,2-fucosyltransferase with SEQ ID NO 65 from H. pylori and either wild-type CscB from E. coli W with SEQ ID NO 01 or a variant thereof comprising a V316A, C327G and/or F371V mutation as specified in SEQ ID NOs 33, 35, 36, 37, 38, 39, 40, when grown on minimal medium comprising 4, 8 or 30 g/L sucrose.

    [0393] FIG. 3 shows the normalized maximal growth rate (Max in %) of E. coli 3-FL production strains expressing the a1,3-fucosyltransferase with SEQ ID NO 69 from B. psittacipulmonis and either wild-type CscB from E. coli W with SEQ ID NO 01 or a variant thereof comprising a V316A, C327G and/or F371V mutation as specified in SEQ ID NOs 33, 35, 36, 37, 38, 39, 40, when grown on minimal medium comprising 4, 8 or 30 g/L sucrose.

    [0394] FIG. 4 shows the normalized maximal growth rate (Max in %) of E. coli 3-FL production strains expressing the a1,3-fucosyltransferase with SEQ ID NO 70 from A. oryzae and either wild-type CscB from E. coli W with SEQ ID NO 01 or a variant thereof comprising a V316A, C327G and/or F371V mutation as specified in SEQ ID NOs 33, 35, 36, 37, 38, 39, 40, when grown on minimal medium comprising 4, 8 or 30 g/L sucrose.

    [0395] FIG. 5 shows the normalized maximal growth rate (Max in %) of E. coli 3-FL production strains expressing the a1,3-fucosyltransferase with SEQ ID NO 69 from B. psittacipulmonis and either wild-type CscB from E. coli W with SEQ ID NO 01 (WT) or a variant thereof differing by an amino acid change at position C327 into A (SEQ ID NO 100) or G (SEQ ID NO 36), at position F371 into E (SEQ ID NO 102), N (SEQ ID NO 121), M (SEQ ID NO 120), A (SEQ ID NO 105), C (SEQ ID NO 107), Q (SEQ ID NO 103), G (SEQ ID NO 119), L (SEQ ID NO 104), T (SEQ ID NO 106), S (SEQ ID NO 108), I (SEQ ID NO 109) or V (SEQ ID NO 37), or at position V316 into F (SEQ ID NO 99), A (SEQ ID NO 35) or C (SEQ ID NO 98), when grown on minimal medium comprising 4, 8 or 30 g/L sucrose.

    [0396] FIG. 6 shows the normalized maximal growth rate (Max in %) of E. coli 3-FL production strains expressing the a1,3-fucosyltransferase with SEQ ID NO 69 from B. psittacipulmonis and either wild-type CscB from E. coli W with SEQ ID NO 01 (WT) or a variant thereof with either SEQ ID NO 110 (differing from SEQ ID NO 01 by a V3161, C327A, F371T mutation), SEQ ID NO 111 (differing from SEQ ID NO 01 by a V316M, C327G and F371G mutation), SEQ ID NO 112 (differing from SEQ ID NO 01 by a V316L, C327G and F371S mutation), SEQ ID NO 113 (differing from SEQ ID NO 01 by a V316F, C327G and F371V mutation) or SEQ ID NO 114 (differing from SEQ ID NO 01 by a V3161, C327G and F371C mutation), when grown on minimal medium comprising 5 or 30 g/L sucrose.

    [0397] FIG. 7 shows the normalized maximal growth rate (Max in %) of E. coli 3-FL production strains expressing the a1,3-fucosyltransferase with SEQ ID NO 69 from B. psittacipulmonis and either wild-type CscB from E. coli W with SEQ ID NO 01 (WT) or a variant thereof with either SEQ ID NO 115 (differing from SEQ ID NO 01 by a C-terminal truncation of 12 amino acid residues), SEQ ID NO 116 (differing from SEQ ID NO 01 by a C-terminal truncation of 7 amino acid residues), SEQ ID NO 40 (differing from SEQ ID NO 01 by a C327G and a F371V mutation), SEQ ID NO 117 (differing from SEQ ID NO 01 by a C327G and a F371V mutation and by a C-terminal truncation of 12 amino acid residues) or SEQ ID NO 118 (differing from SEQ ID NO 01 by a C327G and a F371V mutation by a C-terminal truncation of 7 amino acid residues), when grown on minimal medium comprising 5 or 30 g/L sucrose.

    [0398] FIG. 8 shows the normalized maximal growth rate (Max in %) of E. coli 3-FL production strains expressing the a1,3-fucosyltransferase with SEQ ID NO 69 from B. psittacipulmonis and either wild-type CscB from E. coli W with SEQ ID NO 01 (WT) or a variant thereof with either SEQ ID NO 36 or 40 from the genome or from an expression plasmid, when grown on minimal medium comprising 5 or 30 g/L sucrose.

    [0399] The following examples will serve as further illustration and clarification of the present invention and are not intended to be limiting.

    EXAMPLES

    Example 1. Materials and Methods Escherichia coli

    Media

    [0400] The Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). The minimal medium used in the cultivation experiments in 96-well plates or in shake flasks contained 2.00 g/L NH4Cl, 5.00 g/L (NH4)2SO4, 2.993 g/L KH2PO4, 7.315 g/L K2HPO4, 8.372 g/L MOPS, 0.5 g/L NaCl, 0.5 g/L MgSO4.Math.7H2O, 30 g/L sucrose or 30 g/L glycerol, 1 ml/L vitamin solution, 100 IA molybdate solution, and 1 mL/L selenium solution. As specified in the respective examples, 0.30 g/L sialic acid and/or 20 g/L lactose were additionally added to the medium as precursor(s). The minimal medium was set to a pH of 7 with 1M KOH. Vitamin solution consisted of 3.6 g/L FeCl2.Math.4H2O, 5 g/L CaCl2.Math.2H2O, 1.3 g/L MnCl2.Math.2H2O, 0.38 g/L CuCl2.Math.2H2O, 0.5 g/L CoCl2.Math.6H2O, 0.94 g/L ZnCl2, 0.0311 g/L H3BO4, 0.4 g/L Na2EDTA.Math.2H2O and 1.01 g/L thiamine.Math.HCl. The molybdate solution contained 0.967 g/L NaMoO4.Math.2H2O. The selenium solution contained 42 g/L Seo2. The minimal medium for fermentations contained 6.75 g/L NH4C1, 1.25 g/L (NH4)2SO4, 2.93 g/L KH2PO4 and 7.31 g/L KH2PO4, 0.5 g/L NaCl, 0.5 g/L MgSO4.Math.7H2O, 30 g/L sucrose or 30 g/L glycerol, 1 mL/L vitamin solution, 100 L/L molybdate solution, and 1 mL/L selenium solution with the same composition as described above. As specified in the respective examples, 0.30 g/L sialic acid and/or 20 g/L lactose were additionally added to the medium as precursor(s).

    [0401] Complex medium was sterilized by autoclaving (121 C., 21 min) and minimal medium by filtration (0.22 m Sartorius). When necessary, the medium was made selective by adding an antibiotic: e.g. chloramphenicol (20 mg/L), carbenicillin (100 mg/L), spectinomycin (40 mg/L) and/or kanamycin (50 mg/L).

    Plasmids

    [0402] pKD46 (Red helper plasmid, Ampicillin resistance), pKD3 (contains an FRT-flanked chloramphenicol resistance (cat) gene), pKD4 (contains an FRT-flanked kanamycin resistance (kan) gene), and pCP20 (expresses FLP recombinase activity) plasmids were obtained from Prof. R. Cunin (Vrije Universiteit Brussel, Belgium in 2007). Plasmids were maintained in the host E. coli DH5alpha (F.sup., phi80dlacZM15, (lacZYA-argF) U169, deoR, recA1, endA1, hsdR17(rk.sup., mk.sup.+), phoA, supE44, lambda.sup., thi-1, gyrA96, relA1) bought from Invitrogen.

    Strains and Mutations

    [0403] Escherichia coli K12 MG1655 [.sup., F.sup., rph-1] was obtained from the Coli Genetic Stock Center (US), CGSC Strain #: 7740, in March 2007. Gene disruptions, gene introductions and gene replacements were performed using the technique published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645). This technique is based on antibiotic selection after homologous recombination performed by lambda Red recombinase. Subsequent catalysis of a flippase recombinase ensures removal of the antibiotic selection cassette in the final production strain. Transformants carrying a Red helper plasmid pKD46 were grown in mL LB media with ampicillin, (100 mg/L) and L-arabinose (10 mM) at 30 C. to an OD.sub.600nm of 0.6. The cells were made electrocompetent by washing them with 50 mL of ice-cold water, a first time, and with 1 mL ice cold water, a second time. Then, the cells were resuspended in 50 L of ice-cold water. Electroporation was done with 50 L of cells and 10-100 ng of linear double-stranded-DNA product by using a Gene Pulser (BioRad) (600, 25 FD, and 250 volts). After electroporation, cells were added to 1 mL LB media incubated 1 h at 37 C., and finally spread onto LB-agar containing 25 mg/L of chloramphenicol or 50 mg/L of kanamycin to select antibiotic resistant transformants. The selected mutants were verified by PCR with primers upstream and downstream of the modified region and were grown in LB-agar at 42 C. for the loss of the helper plasmid. The mutants were tested for ampicillin sensitivity. The linear ds-DNA amplicons were obtained by PCR using pKD3, pKD4 and their derivates as template. The primers used had a part of the sequence complementary to the template and another part complementary to the side on the chromosomal DNA where the recombination must take place. For the genomic knock-out, the region of homology was designed 50-nt upstream and 50-nt downstream of the start and stop codon of the gene of interest. For the genomic knock-in, the transcriptional starting point (+1) had to be respected. PCR products were PCR-purified, digested with Dpnl, re-purified from an agarose gel, and suspended in elution buffer (5 mM Tris, pH 8.0). Selected mutants were transformed with pCP20 plasmid, which is an ampicillin and chloramphenicol resistant plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis. The ampicillin-resistant transformants were selected at 30 C., after which a few were colony purified in LB at 42 C. and then tested for loss of all antibiotic resistance and of the FLP helper plasmid. The gene knock outs and knock ins are checked with control primers.

    [0404] For GDP-fucose production upon sucrose, the mutant strain was derived from E. coli K12 MG1655 comprising knock-outs of the E. coli wcaJ and thyA genes and genomic knock-ins of constitutive transcriptional units containing a sucrose permease chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121, a fructose kinase (Frk) originating from Zymomonas mobilis with SEQ ID NO 63 and a sucrose phosphorylase (SP) originating from Bifidobacterium adolescentis with SEQ ID NO 64. For production of fucosylated oligosaccharides, the mutant GDP-fucose production strain was additionally modified with expression plasmids comprising constitutive transcriptional units for an alpha-1,2-fucosyltransferase chosen from the list comprising SEQ ID NO 65 (H. pylori), SEQ ID NO 66 (Helicobacter sp.), SEQ ID NO 67 (Porphyromonas catoniae ATCC 51270), and SEQ ID NO 68 (Akkermansia muciniphila) and/or an alpha-1,3-fucosyltransferase chosen from the list comprising SEQ ID NO 69 (Basilea psittacipulmonis JF4266) and SEQ ID NO 70 (Azospirillum oryzae A2P) and with a constitutive transcriptional unit for the E. coli thyA with SEQ ID NO 71 as selective marker. The constitutive transcriptional units of the fucosyltransferase genes could also be present in the mutant E. coli strain via genomic knock-ins. GDP-fucose production can further be optimized in the mutant E. coli strain by genomic knock-outs of the E. coli genes comprising glgC, agp, pfkA, pfkB, pgi, arcA, iclR, pgi and Ion as described in WO2016075243 and WO2012007481. GDP-fucose production can additionally be optimized comprising genomic knock-ins of constitutive transcriptional units for the E. coli manA with SEQ ID NO 72, manB with SEQ ID NO 73, manC with SEQ ID NO 74, gmd with SEQ ID NO 75 and fcl with SEQ ID NO 76. GDP-fucose production can also be obtained by genomic knock-outs of the E. coli fucK and fucI genes and genomic knock-ins of constitutive transcriptional units containing the fucose permease (fucP) from E. coli with SEQ ID NO 77 and the bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase (fkp) from Bacteroides fragilis with SEQ NO ID 78. If the mutant strains producing GDP-fucose were intended to make fucosylated lactose structures, the strains were additionally modified with genomic knock-outs of the E. coli LacZ, LacY and LacA genes and with a genomic knock-in of a constitutive transcriptional unit for the E. coli LacY with SEQ ID NO 79.

    [0405] For sialic acid production upon sucrose, the mutant strain was derived from E. coli K12 MG1655 comprising knock-outs of the E. coli nagA and nagB genes and genomic knock-ins of constitutive transcriptional units containing a sucrose permease chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121, a fructose kinase (Frk) originating from Z. mobilis with SEQ ID NO 63, a sucrose phosphorylase (SP) originating from B. adolescentis with SEQ ID NO 64, a glucosamine 6-phosphate N-acetyltransferase (GNA1) from Saccharomyces cerevisiae with SEQ ID NO 80, an N-acetylglucosamine 2-epimerase (AGE) from Bacteroides ovatus with SEQ ID NO 81 and an N-acetylneuraminate (Neu5Ac) synthase (NeuB) from Neisseria meningitidis with SEQ ID NO 82. Sialic acid production can further be optimized in the mutant E. coli strain with genomic knock-outs of the E. coli genes comprising nagC, nagD, nagE, nanA, nanE, nanK, manX, manY and manZ as described in WO18122225 and with genomic knock-ins of constitutive transcriptional units comprising a mutated variant of the L-glutamine-D-fructose-6-phosphate aminotransferase (glmS*54) from E. coli with SEQ ID NO 83 (differing from the wild-type E. coli glmS by an A39T, an R250C and an G472S mutation) and the phosphatase yqaB from E. coli with SEQ ID NO 84. Sialic acid production upon sucrose can also be obtained by knock-outs of the E. coli nagA and nagB genes and genomic knock-ins of constitutive transcriptional units containing a sucrose permease chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121, a fructose kinase (Frk) originating from Z. mobilis with SEQ ID NO 63, a sucrose phosphorylase (SP) originating from B. adolescentis with SEQ ID NO 64, the phosphoglucosamine mutase (glmM) from E. coli with SEQ ID NO 96, the N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase (gImU) from E. coli with SEQ ID NO 97, the UDP-N-acetylglucosamine 2-epimerase (NeuC) from Campylobacter jejuni with SEQ ID NO 85 and the N-acetylneuraminate synthase (NeuB) from N. meningitidis with SEQ ID NO 82. Also in this mutant strain, sialic acid production can further be optimized with genomic knock-ins of constitutive transcriptional units comprising the mutant glmS*54 from E. coli with SEQ ID NO 83 and the phosphatase yqaB from E. coli with SEQ ID NO 84. For sialylated oligosaccharide production, the sialic acid production strains further need to express an N-acylneuraminate cytidylyltransferase (NeuA) from Pasteurella multocida with SEQ ID NO 86, and a beta-galactoside alpha-2,3-sialyltransferase comprising SEQ ID NO 87 (PmultST3) from P. multocida and/or SEQ ID NO 88 (NmeniST3) from N. meningitidis, and/or a beta-galactoside alpha-2,6-sialyltransferase comprising SEQ ID NO 89 (PdST6) from Photobacterium damselae and/or SEQ ID NO 90 (P-JT-ISH-224-5T6) from Photobacterium sp. JT-ISH-224. Constitutive transcriptional units of PmNeuA and the sialyltransferases can be delivered to the mutant strain either via genomic knock-in or via expression plasmids. If the mutant strains producing sialic acid and CMP-sialic acid were intended to make sialylated lactose structures, the strains were additionally modified with genomic knock-outs of the E. coli LacZ, LacY and LacA genes and with a genomic knock-in of a constitutive transcriptional unit for the E. coli LacY with SEQ ID NO 79.

    [0406] To produce LN3 (GlcNAc-b1,3-Gal-b1,4-Glc) and oligosaccharides originating thereof comprising lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) upon sucrose, the mutant strain was derived from E. coli K12 MG1655 and modified with a knock-out of the E. coli LacZ and nagB genes and with genomic knock-ins of constitutive transcriptional units containing a sucrose permease chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121, a fructose kinase (Frk) originating from Z. mobilis with SEQ ID NO 63, a sucrose phosphorylase (SP) originating from B. adolescentis with SEQ ID NO 64 and the galactoside beta-1,3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis with SEQ ID NO 91. For LNT or LNnT production, the mutant strain is further modified with constitutive transcriptional units for the N-acetylglucosamine beta-1,3-galactosyltransferase (WbgO) from E. coli 055:H7 with SEQ ID NO 92 or the N-acetylglucosamine beta-1,4-galactosyltransferase (LgtB) from N. meningitidis with SEQ ID NO 93, respectively, that can be delivered to the strain either via genomic knock-in or from an expression plasmid. Optionally, multiple copies of the LgtA, wbgO and/or LgtB genes could be added to the mutant E. coli strains. Also, LNT and/or LNnT production can be enhanced by improved UDP-GlcNAc production by modification of the strains with one or more genomic knock-ins of a constitutive transcriptional unit for glmS*54 from E. coli with SEQ ID NO 83. In addition, the strains can optionally be modified for enhanced UDP-galactose production with genomic knock-outs of the E. coli ushA and galT genes. The mutant E. coli strains can also optionally be adapted with a genomic knock-in of a constitutive transcriptional unit for the UDP-glucose-4-epimerase (galE) from E. coli with SEQ ID NO 94, the phosphoglucosamine mutase (glmM) from E. coli with SEQ ID NO 96 and the N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase (glmU) from E. coli with SEQ ID NO 97.

    [0407] Preferably but not necessarily, the glycosyltransferases were N-terminally fused to an MBP-tag to enhance their solubility (Fox et al., Protein Sci. 2001, 10(3), 622-630).

    [0408] All constitutive promoters, UTRs and terminator sequences originated from the libraries described by Mutalik et al. (Nat. Methods 2013, No. 10, 354-360) and Cambray et al. (Nucleic Acids Res. 2013, 41(9), 5139-5148): the genes were expressed using promoters MutalikP5 (PROM0005_MutalikP5) and apFAB82 (PROM0050_apFAB82) as described by Mutalik et al. (Nat. Methods 2013, No. 10, 354-360), UTRs used comprised GalE_BCD12 (UTR0010_GalE_BCD12) and GalE_LeuAB (UTR0014_GalE_LeuAB) as described by Mutalik et al. (Nat. Methods 2013, No. 10, 354-360), and terminator sequence used was ilvGEDA (TER0007_ilvGEDA) as described by Cambray et al. (Nucleic Acids Res. 2013, 41(9), 5139-5148). All genes were ordered synthetically at Twist Bioscience (twistbioscience.com) or IDT (eu.idtdna.com) and the codon usage was adapted using the tools of the supplier.

    [0409] All strains were stored in cryovials at 80 C. (overnight LB culture mixed in a 1:1 ratio with 70% glycerol).

    Cultivation Conditions

    [0410] A preculture of 96-well microtiter plate experiments was started from a cryovial, in 150 L LB and was incubated overnight at 37 C. on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96well square microtiter plate, with 400 L minimal medium by diluting 400. These final 96-well culture plates were then incubated at 37 C. on an orbital shaker at 800 rpm for 72h, or shorter, or longer. To measure sugar concentrations at the end of the cultivation experiment whole broth samples were taken from each well by boiling the culture broth for 15 min at 60 C. before spinning down the cells (=average of intra- and extracellular sugar concentrations).

    [0411] A preculture for the bioreactor was started from an entire 1 mL cryovial of a certain strain, inoculated in 250 mL or 500 mL minimal medium in a 1 L or 2.5 L shake flask and incubated for 24 h at 37 C. on an orbital shaker at 200 rpm. A 5 L bioreactor was then inoculated (250 mL inoculum in 2 L batch medium); the process was controlled by MFCS control software (Sartorius Stedim Biotech, Melsungen, Germany). Culturing condition were set to 37 C., and maximal stirring; pressure gas flow rates were dependent on the strain and bioreactor. The pH was controlled at 6.8 using 0.5 M H2SO4 and 20% NH.sub.4OH. The exhaust gas was cooled. 10% solution of silicone antifoaming agent was added when foaming raised during the fermentation.

    Optical Density

    [0412] Cell density of the cultures was frequently monitored by measuring optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium or with a Spark 10M microplate reader, Tecan, Switzerland).

    Analytical Analysis

    [0413] Standards such as but not limited to sucrose, lactose, TEL, 3-FL, DiFL, 3SL, 6SL, lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neo-tetraose (LNnT), LNEP-1, LNEP-11, LNEP-111, LNFP-V, LNEP-VI, LSTa, LSTc and LSTd were purchased from Carbosynth (UK), Elicityl (France) and IsoSep (Sweden). Other compounds were analysed with in-house made standards.

    [0414] Neutral oligosaccharides were analysed on a Waters Acquity H-class UPLC with Evaporative Light Scattering Detector (ELSD) or a Refractive Index (RI) detection. A volume of 0.7 L sample was injected on a Waters Acquity UPLC BEH Amide column (2.1100 mm; 130 ; 1.7 m) column with an Acquity UPLC BEH Amide VanGuard column, 130 , 2.15 mm. The column temperature was 50 C. The mobile phase consisted of a water and % acetonitrile solution to which 0.2% triethylamine was added. The method was isocratic with a flow of 0.130 mL/min. The ELS detector had a drift tube temperature of 50 C. and the N2 gas pressure was 50 psi, the gain 200 and the data rate 10 pps. The temperature of the RI detector was set at 35 C.

    [0415] Sialylated oligosaccharides were analysed on a Waters Acquity H-class UPLC with Refractive Index (RI) detection. A volume of 0.5 L sample was injected on a Waters Acquity UPLC BEH Amide column (2.1100 mm; 130 ; 1.7 m). The column temperature was 50 C. The mobile phase consisted of a mixture of % acetonitrile, 26% ammonium acetate buffer (150 mM) and 4% methanol to which 0.05% pyrrolidine was added. The method was isocratic with a flow of 0.150 mL/min. The temperature of the RI detector was set at 35 C.

    [0416] Both neutral and sialylated sugars were analysed on a Waters Acquity H-class UPLC with Refractive Index (RI) detection. A volume of 0.5 L sample was injected on a Waters Acquity UPLC BEH Amide column (2.1100 mm; 130 ; 1.7 m). The column temperature was 50 C. The mobile phase consisted of a mixture of 72% acetonitrile and 28% ammonium acetate buffer (100 mM) to which 0.1% triethylamine was added. The method was isocratic with a flow of 0.260 mL/min. The temperature of the RI detector was set at 35 C.

    [0417] For analysis on a mass spectrometer, a Waters Xevo TQ-MS with Electron Spray Ionisation (ESI) was used with a desolvation temperature of 450 C., a nitrogen desolvation gas flow of 650 L/h and a cone voltage of 20 V. The MS was operated in selected ion monitoring (SIM) in negative mode for all oligosaccharides. Separation was performed on a Waters Acquity UPLC with a Thermo Hypercarb column (2.1100 mm; 3 m) on 35 C. A gradient was used wherein eluent A was ultrapure water with 0.1% formic acid and wherein eluent B was acetonitrile with 0.1% formic acid. The oligosaccharides were separated in 55 min using the following gradient: an initial increase from 2 to 12% of eluent B over 21 min, a second increase from 12 to 40% of eluent B over 11 min and a third increase from 40 to 100% of eluent B over 5 min. As a washing step 100% of eluent B was used for 5 min. For column equilibration, the initial condition of 2% of eluent B was restored in 1 min and maintained for 12 min.

    [0418] Both neutral and sialylated sugars at low concentrations (below 50 mg/L) were analysed on a Dionex HPAEC system with pulsed amperometric detection (PAD). A volume of 5 L of sample was injected on a Dionex CarboPac PA200 column 4250 mm with a Dionex CarboPac PA200 guard column 450 mm. The column temperature was set to 30 C. A gradient was used wherein eluent A was deionized water, wherein eluent B was 200 mM Sodium hydroxide and wherein eluent C was 500 mM Sodium acetate. The oligosaccharides were separated in 60 min while maintaining a constant ratio of 25% of eluent B using the following gradient: an initial isocratic step maintained for 10 min of 75% of eluent A, an initial increase from 0 to 4% of eluent C over 8 min, a second isocratic step maintained for 6 min of 71% of eluent A and 4% of eluent C, a second increase from 4 to 12% of eluent C over 2.6 min, a third isocratic step maintained for 3.4 min of 63% of eluent A and 12% of eluent C and a third increase from 12 to 48% of eluent C over 5 min. As a washing step 48% of eluent C was used for 3 min. For column equilibration, the initial condition of 75% of eluent A and 0% of eluent C was restored in 1 min and maintained for 11 min. The applied flow was 0.5 mL/min.

    Example 2. Evaluation of Growth of an E. coli 3-Fucosyllactose Production Strain Expressing the Wild Type CscB Sucrose Permease from E. coli W when Cultivated in Medium with Decreasing Sucrose Concentrations

    [0419] An E. coli strain modified for GDP-fucose production as described in Example 1 was adapted for growth on sucrose by genomic knock-ins of constitutive transcriptional units for the wild-type sucrose permease CscB with SEQ ID NO 01 from E. coli W, the fructose kinase (Frk) from Zymomonas mobilis with SEQ ID NO 63 and the sucrose phosphorylase (SP) from Bifidobacterium adolescentis with SEQ ID NO 64. In a next step, the strain was further transformed with an expression plasmid having a constitutive transcriptional unit to express the a1,3-fucosyltransferase with SEQ ID NO 69 from Basilea psittacipulmonis. The novel strain was evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium comprised 30, 8 or 4 g/L sucrose. The strain was grown in three biological replicates in a 96-well plate. Based on the absorbance measurements at 600 nm, the maximal growth rate (Max) was calculated and normalized to the average Max observed when grown in medium comprising g/L of sucrose. FIG. 1 demonstrates that the normalized maximal growth rate (Max in %) of the strain decreases with decreasing sucrose concentration in the medium. Such a strain thus grows slower if lower amounts of sucrose are available in the medium.

    Example 3. Evaluation of Growth of E. coli Strains Expressing Various Sucrose Permeases when Cultivated in Medium with Decreasing Sucrose Concentrations

    [0420] An E. coli strain modified for GDP-fucose production as described in Example 1 was adapted for growth on sucrose by genomic knock-ins of constitutive transcriptional units for the fructose kinase (Frk) from Z. mobilis with SEQ ID NO 63, the sucrose phosphorylase (SP) from B. adolescentis with SEQ ID NO 64 and either the wild-type E. coli W cscB sucrose permease with SEQ ID NO 01 or a variant thereof containing a V316A, C327G and/or F371V mutation as specified in SEQ ID NOs 33, 35, 36, 37, 38, 39, 40. In a next step, the strains were further transformed with an expression plasmid having a constitutive transcriptional unit to express either an a1,2-fucosyltransferase with SEQ ID NO 65 from H. pylori or an a1,3-fucosyltransferase selected from B. psittacipulmonis (SEQ ID NO 69) or from A. oryzae (SEQ ID NO 70). The novel strains were evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium comprised 30, 8 or 4 g/L sucrose. The strains were grown in three biological replicates in a 96-well plate. Based on the absorbance measurements at 600 nm, the maximal growth rate (Max) was calculated and normalized to the average Max observed for the reference strain having the wild-type CscB gene from E. coli W with SEQ ID NO 01 when grown in medium comprising 30 g/L of sucrose. FIGS. 2, 3 and 4 show that in conditions where the sucrose concentration in the medium was low (in this experiment 4 or 8 g/L), the normalized maximal growth rate (Max in %) of the strains expressing a sucrose permease with SEQ ID NO 33, 35, 36, 37, 38, 39 or 40 was higher than the normalized maximal growth rate of the respective reference strain expressing the wild type CscB from E. coli W with SEQ ID NO 01. This observation was independent of the fucosyltransferase expressed. Additionally, in conditions where the sucrose concentration in the medium was high (30 g/L), the normalized maximal growth rate of the strains expressing the sucrose permease with SEQ ID NO 40, differing by a C327G and a F371V mutation compared to the wild-type CscB from E. coli W with SEQ ID NO 01, was higher than the normalized maximal growth rate of the respective reference strain expressing said wild-type CscB with SEQ ID NO 01.

    Example 4. Evaluation of Growth of E. coli 3-FL Production Strains Expressing Various Sucrose Permeases when Cultivated in Medium with Decreasing Sucrose Concentrations

    [0421] An E. coli strain modified for GDP-fucose production as described in Example 1 was adapted for growth on sucrose by genomic knock-ins of constitutive transcriptional units for the fructose kinase (Frk) from Z. mobilis with SEQ ID NO 63, the sucrose phosphorylase (SP) from B. adolescentis with SEQ ID NO 64 and either the wild-type E. coli W cscB sucrose permease with SEQ ID NO 01 or a variant thereof differing by a single mutation at position 316 (V316c), position 327 (C327d) or position 371 (F371e) wherein c, d and e were replaced by any amino acid residue possible as specified in SEQ ID NOs 04, 05 and 06, respectively. In a next step, the strains were further transformed with an expression plasmid having a constitutive transcriptional unit to express the a1,3-fucosyltransferase from B. psittacipulmonis with SEQ ID NO 69. The novel strains were evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium comprised 30, 8 or 4 g/L sucrose. The strains were grown in three biological replicates in a 96-well plate. Based on the absorbance measurements at 600 nm, the maximal growth rate (Max) was calculated and normalized to the average Max observed for the reference strain having the wild-type CscB gene from E. coli W with SEQ ID NO 01.

    [0422] FIG. 5 shows that the normalized maximal growth rate (Max in %) of the strains expressing a sucrose permease CscB differing from SEQ ID NO 01 by a C327A (SEQ ID NO 100), F371E (SEQ ID NO 102), F371N (SEQ ID NO 121), F371M (SEQ ID NO 120), F371G (SEQ ID NO 119), V316F (SEQ ID NO 99) or V316C (SEQ ID NO 98) mutation was comparable to the normalized growth rate of the reference strain expressing SEQ ID NO 01 on all media tested. The normalized maximal growth rate (Max in %) of the strains expressing a sucrose permease CscB differing from SEQ ID NO 01 by a C327G (SEQ ID NO 36), F371A (SEQ ID NO 105), F371C (SEQ ID NO 107), F371Q (SEQ ID NO 103), F371L (SEQ ID NO 104), F371T (SEQ ID NO 106), F371S (SEQ ID NO 108), F3711 (SEQ ID NO 109), F371V (SEQ ID NO 37) or V316A (SEQ ID NO 35) mutation was significantly increased compared to the normalized growth rate of the reference strain expressing SEQ ID NO 01 when grown on medium comprising 4 or 8 g/L sucrose.

    Example 5. Evaluation of Growth of E. coli 3-FL Production Strains Expressing Various Sucrose Permeases when Cultivated in Medium with a Low Sucrose Concentration

    [0423] An E. coli strain modified for GDP-fucose production as described in Example 1 was adapted for growth on sucrose by genomic knock-ins of constitutive transcriptional units for the fructose kinase (Frk) from Z. mobilis with SEQ ID NO 63, the sucrose phosphorylase (SP) from B. adolescentis with SEQ ID NO 64 and either the wild-type E. coli W cscB sucrose permease with SEQ ID NO 01 or a variant thereof chosen from the list comprising SEQ ID NOs 110, 111, 112, 113 and 114, and having three single point mutations at three positions (V316, C327 and F371) compared to SEQ ID NO 01. In a next step, the strains were further transformed with an expression plasmid having a constitutive transcriptional unit to express the a1,3-fucosyltransferase from B. psittacipulmonis with SEQ ID NO 69. The novel strains were evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium comprised 30 or 5 g/L sucrose. The strains were grown in three biological replicates in a 96-well plate. Based on the absorbance measurements at 600 nm, the maximal growth rate (Max) was calculated and normalized to the average Max observed for the reference strain having the wild-type CscB gene from E. coli W with SEQ ID NO 01.

    [0424] FIG. 6 shows that the normalized maximal growth rate (Max in %) of all novel strains expressing a sucrose permease CscB differing from SEQ ID NO 01 by three single point mutations at positions V316, C327 and F371 was significantly increased compared to the normalized maximal growth rate of the reference strain expressing SEQ ID NO 01, when grown on medium comprising 5 g/L sucrose. The normalized maximal growth rate of the strains expressing a sucrose permease with SEQ ID NO 111 (differing from SEQ ID NO 01 by a V316M, C327G and F371G mutation), SEQ ID NO 112 (differing from SEQ ID NO 01 by a V316L, C327G and F371S mutation), SEQ ID NO 113 (differing from SEQ ID NO 01 by a V316F, C327G and F371V mutation) or SEQ ID NO 114 (differing from SEQ ID NO 01 by a V3161, C327G and F371C mutation) was even twice as high as compared to the normalized maximal growth rate of the reference strain expressing SEQ ID NO 01, when grown on medium comprising 5 g/L sucrose.

    Example 6. Evaluation of Growth of E. coli 3-FL Production Strains Expressing Variant Sucrose Permeases when Cultivated in Medium with a Low Sucrose Concentration

    [0425] An E. coli strain modified for GDP-fucose production as described in Example 1 was adapted for growth on sucrose by genomic knock-ins of constitutive transcriptional units for the fructose kinase (Frk) from Z. mobilis with SEQ ID NO 63, the sucrose phosphorylase (SP) from B. adolescentis with SEQ ID NO 64 and either the wild-type E. coli W cscB sucrose permease with SEQ ID NO 01 or a variant thereof chosen of the list comprising SEQ ID NOs 40, 115, 116, 117 and 118. Herein, SEQ ID NOs 115 and 116 were different from SEQ ID NO 01 by a C-terminal truncation of 12 or 7 amino acids, respectively. These truncated variants were made by insertion of an in-frame stop codon directly after glutamate 403 (E403*) or glutamate 408 (E408*), respectively. SEQ ID NO 40 differed from SEQ ID NO 01 by a C327G and a F371V mutation. SEQ ID NOs 117 and 118 were different from SEQ ID NO 01 by a C327G and a F371V mutation and an additional C-terminal truncation of 12 or 7 amino acids, respectively. In a next step, the strains were further transformed with an expression plasmid having a constitutive transcriptional unit to express the a1,3-fucosyltransferase from B. psittacipulmonis with SEQ ID NO 69. The novel strains were evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium comprised 30 or 5 g/L sucrose. The strains were grown in three biological replicates in a 96-well plate. Based on the absorbance measurements at 600 nm, the maximal growth rate (Max) was calculated and normalized to the average Max observed for the reference strain having the wild-type CscB gene from E. coli W with SEQ ID NO 01.

    [0426] FIG. 7 shows that the normalized maximal growth rate (Max in %) of all novel strains expressing a variant sucrose permease CscB with SEQ ID NO 115, 116, 40, 117 or 118 was significantly increased compared to the normalized maximal growth rate of the reference strain expressing SEQ ID NO 01, when grown on medium comprising 5 g/L sucrose. Thus, a strain expressing a variant sucrose permease with a C-terminal truncation of 12 or 7 amino acid residues compared to the wild-type polypeptide sequence of CscB from E. coli W with SEQ ID NO 01 grew faster than a strain expressing the natural sucrose permease with SEQ ID NO 01 on medium with low (here 5 g/L) sucrose concentrations.

    Example 7. Evaluation of Growth of E. coli 3-FL Production Strains Expressing Variant Sucrose Permeases Expressed from the Genome or from a Plasmid

    [0427] An E. coli strain modified for GDP-fucose production as described in Example 1 was further adapted by genomic knock-ins of constitutive transcriptional units for the fructose kinase (Frk) from Z. mobilis with SEQ ID NO 63 and the sucrose phosphorylase (SP) from B. adolescentis with SEQ ID NO 64. Next, the strain was modified to contain either (1) a genomic knock-in of a constitutive transcriptional unit for the wild-type CscB sucrose permease from E. coli W with SEQ ID NO 01 or for the variant sucrose permease with SEQ ID NO 40 or (2) an expression plasmid with a constitutive transcriptional unit for the wild-type CscB sucrose permease from E. coli W with SEQ ID NO 01 or for a variant sucrose permease with SEQ ID NO 36 or SEQ ID NO 40. In a final step, the novel strains were further transformed with an expression plasmid having a constitutive transcriptional unit to express the a1,3-fucosyltransferase from B. psittacipulmonis with SEQ ID NO 69. The novel strains were evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium comprised 30 or 5 g/L sucrose. The strains were grown in three biological replicates in a 96-well plate. Based on the absorbance measurements at 600 nm, the maximal growth rate (Max) was calculated and normalized to the average Max observed for the reference strain having the wild-type CscB gene from E. coli W with SEQ ID NO 01.

    [0428] FIG. 8 shows that the normalized maximal growth rate (Max in %) of the novel strain expressing the wild-type CscB sucrose permease with SEQ ID NO 01 from plasmid was higher compared to the normalized maximal growth rate of the reference strain expressing SEQ ID NO 01 from its genome, when grown on medium comprising 5 g/L sucrose. FIG. 10 also shows that the normalized maximal growth rate of the strains expressing a variant sucrose permease (SEQ ID NO 36 or 40) from the genome of from a plasmid was increased compared to the normalized maximal growth rate of the reference strain expressing SEQ ID NO 01 from its genome, when grown on medium comprising 5 g/L sucrose. As is shown with the strain expressing the variant sucrose permease with SEQ ID NO 40, the effect caused by the mutated CscB variant in the novel strains (i.e. increased growth rate of the strains on low concentrations of sucrose) is independent from the location of the expression module for the sucrose permease variant.

    Example 8. Evaluation of 2-Fucosyllactose Production in a Fermentation Process Using E. coli Strains Expressing a Sucrose Permease

    [0429] An E. coli strain modified for GDP-fucose production as described in Example 1 was adapted for growth on sucrose by genomic knock-ins of constitutive transcriptional units for the fructose kinase (Frk) from Z. mobilis with SEQ ID NO 63, the sucrose phosphorylase (SP) from B. adolescentis with SEQ ID NO 64 and either the wild-type E. coli W cscB sucrose permease with SEQ ID NO 01 or a variant sucrose permease with SEQ ID NO 36 differing from SEQ ID NO 01 by a C327G mutation. In a next step, the novel strains were further transformed with an expression plasmid having a constitutive transcriptional unit to express the a1,2-fucosyltransferase from Helicobacter sp. MIT 01-6242 with SEQ ID NO 66.

    [0430] Both novel strains were evaluated in a fed-batch fermentation process as described in Example 1 wherein the lactose concentration in the culture medium was set at 170 g/L and the sucrose concentration at 60 g/L. At the end of the batch, when all sucrose was consumed, a sucrose-limited feed was applied. The same process was performed twice with both strains. At the end of the fermentation, significant amounts of 2FL were produced with both strains, but the yield of 2FL on sucrose (gram TEL produced per gram sucrose consumed) was 80% higher for the strain expressing the sucrose permease variant with SEQ ID NO 36 compared to wild-type sucrose permease from E. coli W with SEQ ID NO 01.

    Example 9. Evaluation of Growth of E. coli 2FL Production Strains Expressing Various Sucrose Permeases

    [0431] An E. coli strain modified for GDP-fucose production as described in Example 1 was adapted for growth on sucrose by genomic knock-ins of constitutive transcriptional units for the fructose kinase (Frk) from Z. mobilis with SEQ ID NO 63, the sucrose phosphorylase (SP) from B. adolescentis with SEQ ID NO 64 and either the wild-type E. coli W cscB sucrose permease with SEQ ID NO 01 or a variant chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121. In a next step, the strains were further transformed with an expression plasmid having a constitutive transcriptional unit to express an a1,2-fucosyltransferase with either SEQ ID NO 65 from H. pylori UA1234, SEQ ID NO 66 from Helicobacter sp. MIT 01-6242, SEQ ID NO 67 from P. catoniae or SEQ ID NO 68 from A. muciniphila. When evaluated in a growth experiment according to the culture conditions provided in Example 1 in which the culture medium comprises 5 g/L sucrose, the maximal growth rate of the novel strains expressing a variant sucrose permease is higher compared to the growth rate of a reference strain, expressing wild-type CscB gene from E. coli W with SEQ ID NO 01.

    Example 10. Evaluation of 3-Fucosyllactose (3-FL) Production in a Fermentation Process Using E. coli Strains Expressing a Sucrose Permease

    [0432] An E. coli strain modified for GDP-fucose production as described in Example 1 was adapted for growth on sucrose by genomic knock-ins of constitutive transcriptional units for the fructose kinase (Frk) from Z. mobilis with SEQ ID NO 63, the sucrose phosphorylase (SP) from B. adolescentis with SEQ ID NO 64 and either the wild-type E. coli W cscB sucrose permease with SEQ ID NO 01 or a variant chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121. In a next step, the novel strains were further transformed with an expression plasmid having a constitutive transcriptional unit to express an alpha-1,3-fucosyltransferase with either SEQ ID NO 69 from B. psittacipulmonis JF4266 or SEQ ID NO 70 from A. oryzae A2P. When evaluated in a fed-batch fermentation process as described in Example 1 wherein the lactose concentration in the culture medium is set at 170 g/L and the sucrose concentration at 60 g/L and wherein a sucrose-limited feed is applied at the end of the batch, the novel strains expressing a variant sucrose permease produce 3-FL whereby the yield of 3-FL on sucrose (gram 3-FL produced per gram sucrose consumed) is higher compared to the yield of 3-FL on sucrose obtained by the reference strain expressing wild-type CscB gene from E. coli W with SEQ ID NO 01.

    Example 11. Evaluation of Growth of E. coli LNT and LNnT Production Strains Expressing Various Sucrose Permeases

    [0433] An E. coli strain modified for LN3 production as described in Example 1 was adapted for growth on sucrose by genomic knock-ins of constitutive transcriptional units for the fructose kinase (Frk) from Z. mobilis with SEQ ID NO 63, the sucrose phosphorylase (SP) from B. adolescentis with SEQ ID NO 64 and either the wild-type E. coli W cscB sucrose permease with SEQ ID NO 01 or a variant chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121. In a next step, the strains were further transformed with an expression plasmid having a constitutive transcriptional unit to express the N-acetylglucosamine beta-1,3-galactosyltransferase (WbgO) from E. coli 055:H7 with SEQ ID NO 92 for LNT production or the N-acetylglucosamine beta-1,4-galactosyltransferase (LgtB) from N. meningitidis with SEQ ID NO 93 for LNnT production. When evaluated in a growth experiment according to the culture conditions provided in Example 1 in which the culture medium comprises 5 g/L sucrose, the maximal growth rate of the novel strains expressing a variant sucrose permease is higher compared to the growth rate of a reference strain, expressing wild-type CscB gene from E. coli W with SEQ ID NO 01.

    Example 12. Evaluation of Growth of E. coli 3SL and 6SL Production Strains Expressing Various Sucrose Permeases

    [0434] An E. coli strain modified for sialic acid production as described in Example 1 was adapted for growth on sucrose by genomic knock-ins of constitutive transcriptional units for the fructose kinase (Frk) from Z. mobilis with SEQ ID NO 63, the sucrose phosphorylase (SP) from B. adolescentis with SEQ ID NO 64 and either the wild-type E. coli W cscB sucrose permease with SEQ ID NO 01 or a variant chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121. In a next step, the strains were further transformed with an expression plasmid having a constitutive transcriptional unit to express the N-acylneuraminate cytidylyltransferase (NeuA) from P. multocida with SEQ ID NO 86, combined with a beta-galactoside alpha-2,3-sialyltransferase with either SEQ ID NO 87 from P. multocida or with SEQ ID NO 88 from N. meningitidis to produce 3SL, or combined with a beta-galactoside alpha-2,6-sialyltransferase with either SEQ ID NO 89 from P. damselae or with SEQ ID NO 90 from Photobacterium sp. JT-ISH-224 to produce 6SL. When evaluated in a growth experiment according to the culture conditions provided in Example 1 in which the culture medium comprises 5 g/L sucrose, the maximal growth rate of the novel strains expressing a variant sucrose permease is higher compared to the growth rate of a reference strain, expressing wild-type CscB gene from E. coli W with SEQ ID NO 01.

    Example 13. Materials and Methods Bacillus subtilis

    Media

    [0435] Two media are used to cultivate B. subtilis: i.e. a rich Luria Broth (LB) and a minimal medium for shake flask cultures. The LB medium consisted of 1% tryptone peptone (Difco), 0.5% yeast extract (Difco) and sodium chloride (VWR). Luria Broth agar (LBA) plates consisted of the LB media, with 12 g/L agar (Difco) added. The minimal medium contained 2.00 g/L (NH4)2SO4, 7.5 g/L KH2PO4, 17.5 g/L K2HPO4, 1.25 g/L Na-citrate, 0.25 g/L MgSO4.Math.7H2O, 0.05 g/L tryptophan, from 10 up to 30 g/L sucrose, 10 mL/L trace element mix and 10 mL/L Fe-citrate solution. The medium was set to a pH of 7 with 1 M KOH. Depending on the experiment lactose is added as a precursor. The trace element mix consisted of 0.735 g/L CaCl2.Math.2H2O, 0.1 g/L MnCl2.Math.2H2O, 0.033 g/L CuCl2.Math.2H2O, 0.06 g/L CoCl2.Math.6H2O, 0.17 g/L ZnCl2, 0.0311 g/L H3BO4, 0.4 g/L Na2EDTA.Math.2H2O and 0.06 g/L Na2MoO4. The Fe-citrate solution contained 0.135 g/L FeCl3.Math.6H2O, 1 g/L Na-citrate (Hoch 1973 PMC1212887).

    [0436] Complex medium, e.g. LB, was sterilized by autoclaving (121 C., 21) and minimal medium by filtration (0.22 m Sartorius). When necessary, the medium was made selective by adding an antibiotic (e.g. zeocin (20 mg/L)).

    Strains, Plasmids and Mutations

    [0437] Bacillus subtilis 168 is used as available at the Bacillus Genetic Stock Center (Ohio, USA).

    [0438] Plasmids for gene deletion via Cre/lox are constructed as described by Yan et al. (Appl & Environm microbial, September 2008, p 5556-5562). Gene disruption is done via homologous recombination with linear DNA and transformation via the electroporation as described by Xue et al. (J. microb. Meth. 34 (1999) 183-191). The method of gene knockouts is described by Liu et al. (Metab. Engine. 24 (2014) 61-69). This method uses 1000 bp homologies up- and downstream of the target gene.

    [0439] Integrative vectors as described by Popp et al. (Sci. Rep., 2017, 7, 15158) are used as expression vector and could be further used for genomic integrations if necessary. A suitable promoter for expression can be derived from the part repository (iGem): sequence id: BBa_K143012, BBa_K823000, BBa_K823002 or BBa_K823003. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.

    [0440] For GDP-fucose production, the mutant strain was derived from B. subtilis comprising knockouts of the B. subtilis thyA gene and genomic knock-ins of constitutive transcriptional units containing a sucrose permease chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121, a fructose kinase (Frk) originating from Zymomonas mobilis with SEQ ID NO 63 and a sucrose phosphorylase (SP) originating from Bifidobacterium adolescentis with SEQ ID NO 64, the E. coli genes manA with SEQ ID NO 72, manB with SEQ ID NO 73, manCwith SEQ ID NO 74, gmd with SEQ ID NO 75 and fcl with SEQ ID NO 76. For production of fucosylated oligosaccharides, the mutant GDP-fucose production strain was additionally modified with expression plasmids comprising constitutive transcriptional units for an alpha-1,2-fucosyltransferase chosen from the list comprising SEQ ID NO 65 (H. pylori), SEQ ID NO 66 (Helicobacter sp.), SEQ ID NO 67 (Porphyromonas catoniae ATCC 51270), and SEQ ID NO 68 (Akkermansia muciniphila) and/or an alpha-1,3-fucosyltransferase chosen from the list comprising SEQ ID NO 69 (Basilea psittacipulmonis JF4266) and SEQ ID NO 70 (Azospirillum oryzae A2P) and with a constitutive transcriptional unit for the E. coli thyA with SEQ ID NO 71 as selective marker.

    [0441] For LN3 production, the mutant strain was derived from B. subtilis comprising knockouts of the B. subtilis nagB, glmS and gamA genes and genomic knock-ins of constitutive transcriptional units containing a sucrose permease chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121, a fructose kinase (Frk) originating from Zymomonas mobilis with SEQ ID NO 63 and a sucrose phosphorylase (SP) originating from Bifidobacterium adolescentis with SEQ ID NO 64, the mutant glmS*54 from E. coli with SEQ ID NO 83, a galactoside beta-1,3-N-acetylglucosaminyltransferase (IgtA) with SEQ ID NO 91 from N. meningitidis and a lactose permease (LacY) with SEQ ID NO 79 from E. coli. For LNT or LNnT production, the LN3 producing strain was further transformed with constitutive transcriptional units for either an N-acetylglucosamine beta-1,3-galactosyltransferase (wbgO) with SEQ ID NO 92 from E. coli 055:H7 or an N-acetylglucosamine beta-1,4-galactosyltransferase (IgtB) with SEQ ID NO 93 from N. meningitidis, respectively.

    [0442] For sialic acid (Neu5Ac) production, the mutant strain was derived from B. subtilis comprising knockouts of the B. subtilis nagA, nagB, glmS and gamA genes and genomic knock-ins of constitutive transcriptional units containing a sucrose permease chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121, a fructose kinase (Frk) originating from Zymomonas mobilis with SEQ ID NO 63 and a sucrose phosphorylase (SP) originating from Bifidobacterium adolescends with SEQ ID NO 64, the mutant glmS*54 from E. coli with SEQ ID NO 83, a glucosamine 6-phosphate N-acetyltransferase (GNA1) from S. cerevisiae with SEQ ID NO 80, the phosphatase YqaB from E. coli with SEQ ID NO 84, an N-acetylglucosamine 2-epimerase (AGE) from B. ovatus with SEQ ID NO 81 and an N-acetylneuraminate (Neu5Ac) synthase (neuB) from N. meningitidis with SEQ ID NO 82. For sialylated oligosaccharide production, the sialic acid production strains further need to express an N-acylneuraminate cytidylyltransferase (neuA) from P. multocida with SEQ ID NO 86, and a beta-galactoside alpha-2,3-sialyltransferase comprising SEQ ID NO 87 from P. multocida and/or SEQ ID NO 88 from N. meningitidis, and/or a beta-galactoside alpha-2,6-sialyltransferase comprising SEQ ID NO 89 from P. damselae and/or SEQ ID NO 90 from Photobacterium sp. JT-ISH-224. Constitutive transcriptional units of neuA and the sialyltransferases can be delivered to the mutant strain either via genomic knock-in or via expression plasmids. If the mutant strains producing sialic acid and CMP-sialic acid were intended to make sialylated lactose structures, the strains were additionally modified with a genomic knock-in of a constitutive transcriptional unit for the E. coli LacY with SEQ ID NO 79.

    Heterologous and Homologous Expression

    [0443] Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: DNA2.0, Gen9, Twist Biosciences or IDT. Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Genes were optimized using the tools of the supplier.

    Cultivation Conditions

    [0444] A preculture of 96-well microtiter plate experiments was started from a cryovial or a single colony from an LB plate, in 150 L LB and was incubated overnight at 37 C. on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96-well square microtiter plate, with 400 L minimal medium by diluting 400. Each strain was grown in multiple wells of the 96-well plate as biological replicates. These final 96-well culture plates were then incubated at 37 C. on an orbital shaker at 800 rpm for 72h, or shorter, or longer. At the end of the cultivation experiment samples were taken from each well to measure the supernatant concentration (extracellular sugar concentrations, after 5 min. spinning down the cells), or by boiling the culture broth for 15 min at 90 C. or for 60 min at 60 C. before spinning down the cells (=whole broth concentration, intra- and extracellular sugar concentrations, as defined herein).

    [0445] Also, a dilution of the cultures was made to measure the optical density at 600 nm. The cell performance index or CPI was determined by dividing the oligosaccharide concentrations by the biomass, in relative percentages compared to a reference strain. The biomass is empirically determined to be approximately rd of the optical density measured at 600 nm.

    Optical Density, pH and Analytical Analysis

    [0446] The determination of the optical density and the pH of the bacterial cultures as well as the analytical analysis were performed as described in Example 1.

    Example 14. Evaluation of B. subtilis 3-FL Production Strains Expressing Various Sucrose Permeases

    [0447] A B. subtilis strain was first modified by a genomic knock-out of the thyA gene and genomic knock-ins of constitutive transcriptional units for the fructose kinase (Frk) from Z. mobilis with SEQ ID NO 63, the sucrose phosphorylase (SP) from B. adolescentis with SEQ ID NO 64 and either the wild-type E. coli W cscB sucrose permease with SEQ ID NO 01 or a variant chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121, the E. coli genes LacY with SEQ ID NO 79, manA with SEQ ID NO 72, manB with SEQ ID NO 73, manC with SEQ ID NO 74, gmd with SEQ ID NO 75 and fcl with SEQ ID NO 76. In a next step, the mutant strains were transformed with an expression plasmid containing constitutive transcriptional units for the alpha-1,3-fucosyltransferase from B. psittacipulmonis JF4266 with SEQ ID NO 69 and the E. coli thyA gene with SEQ ID NO 71 as selective marker. When evaluated in a growth experiment according to the culture conditions provided in Example 13 in which the culture medium comprises sucrose and lactose, the novel strains produce 3-FL.

    Example 15. Evaluation of B. subtilis LNT and LNnT Production Strains Expressing Various Sucrose Permeases

    [0448] A B. subtilis strain was first modified by a genomic knock-out of the nagB, glmS, gamA and thyA genes and genomic knock-ins of constitutive transcriptional units for the fructose kinase (Frk) from Z. mobilis with SEQ ID NO 63, the sucrose phosphorylase (SP) from B. adolescentis with SEQ ID NO 64 and either the wild-type E. coli W cscB sucrose permease with SEQ ID NO 01 or a variant chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121, the mutant glmS*54 from E. coli with SEQ ID NO 83, a galactoside beta-1,3-N-acetylglucosaminyltransferase (IgtA) with SEQ ID NO 91 from N. meningitidis and a lactose permease (LacY) with SEQ ID NO 79 from E. coli. In a next step, the mutant strains were further transformed with an expression plasmid containing constitutive transcriptional units for E. coli thyA with SEQ ID NO 71 as selective marker and either an N-acetylglucosamine beta-1,3-galactosyltransferase (wbgO) with SEQ ID NO 92 from E. coli 055:H7 to produce LNT or an N-acetylglucosamine beta-1,4-galactosyltransferase (IgtB) with SEQ ID NO 93 from N. meningitidis to produce LNnT. When evaluated in a growth experiment according to the culture conditions provided in Example 13 in which the culture medium comprises sucrose and lactose, the novel strains expressing the N-acetylglucosamine beta-1,3-galactosyltransferase produce LN3 and LNT whereas the novel strains expressing the N-acetylglucosamine beta-1,4-galactosyltransferase produce LN3 and LNnT.

    Example 16. Evaluation of B. subtilis 3SL Production Strains Expressing Various Sucrose Permeases

    [0449] A B. subtilis strain was first modified by a genomic knock-out of the nagA, nagB, glmS, gamA and thyA genes and genomic knock-ins of constitutive transcriptional units for the fructose kinase (Frk) from Z. mobilis with SEQ ID NO 63, the sucrose phosphorylase (SP) from B. adolescentis with SEQ ID NO 64 and either the wild-type E. coli W cscB sucrose permease with SEQ ID NO 01 or a variant chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121, the mutant glmS*54 from E. coli with SEQ ID NO 83, a glucosamine 6-phosphate N-acetyltransferase (GNA1) from S. cerevisiae with SEQ ID NO 80, the phosphatase YqaB from E. coli with SEQ ID NO 84, an N-acetylglucosamine 2-epimerase (AGE) from B. ovatus with SEQ ID NO 81, an N-acetylneuraminate (Neu5Ac) synthase (neuB) from N. meningitidis with SEQ ID NO 82 and the E. coli LacY with SEQ ID NO 79. In a next step, the mutant strains were further transformed with an expression plasmid containing constitutive transcriptional units for the E. coli thyA with SEQ ID NO 71 as selective marker, an N-acylneuraminate cytidylyltransferase (neuA) from P. multocida with SEQ ID NO 86, and a beta-galactoside alpha-2,3-sialyltransferase comprising SEQ ID NO 87 from P. multocida. When evaluated in a growth experiment according to the culture conditions provided in Example 13 in which the culture medium comprises sucrose and lactose, the novel strains produce 3SL.

    Example 17. Materials and Methods Corynebacterium glutamicum

    Media

    [0450] Two different media are used to cultivate C. glutamicum: i.e. a rich tryptone-yeast extract (TY) medium and a minimal medium. The TY medium consisted of 1.6% tryptone (Difco), 1% yeast extract (Difco) and 0.5% sodium chloride (VWR). TY agar (TYA) plates consisted of the TY media, with 12 g/L agar (Difco) added. The minimal medium for the shake flask experiments contained 20 g/L (NH4)2SO4, 5 g/L urea, 1 g/L KH2PO4, 1 g/L K2HPO4, 0.25 g/L MgSO4.Math.7H2O, 42 g/L MOPS, from 10 up to 30 g/L sucrose (or another carbon source including but not limited to glucose, fructose, maltose, glycerol and maltotriose) and 1 mL/L trace element mix. Depending on the experiment lactose is added as a precursor. The trace element mix consisted of 10 g/L CaCl.sub.2), 10 g/L FeSO4.Math.7H2O, 10 g/L MnSO4.Math.H2O, 1 g/L ZnSO4.Math.7H2O, 0.2 g/L CuSO4, 0.02 g/L NiCl2.Math.6H2O, 0.2 g/L biotin (pH 7.0) and 0.03 g/L protocatechuic acid.

    [0451] Complex medium, e.g. TY, was sterilized by autoclaving (121 C., 21) and minimal medium by filtration (0.22 m Sartorius). When necessary, the medium was made selective by adding an antibiotic (e.g. kanamycin, ampicillin).

    Strains and Mutations

    [0452] Corynebacterium glutamicum ATCC 13032 was used as available at the American Type Culture Collection. Integrative plasmid vectors based on the Cre/loxP technique as described by Suzuki et al. (Appl. Microbiol. Biotechnol., 2005 April, 67(2):225-33) and temperature-sensitive shuttle vectors as described by Okibe et al. (J. Microbiol. Meth. 85, 2011, 155-163) are constructed for gene deletions, mutations and insertions. Suitable promoters for (heterologous) gene expression can be derived from Yim et al. (Biotechnol. Bioeng., 2013 November, 110(11):2959-69). Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.

    [0453] For Neu5Ac production, the mutant strain was derived from C. glutamicum comprising knockouts of the C. glutamicum Idh, cgl2645, nagB, glmS and nanA genes and genomic knock-ins of constitutive transcriptional units for the fructose kinase (Frk) from Z. mobilis with SEQ ID NO 63, the sucrose phosphorylase (SP) from B. adolescentis with SEQ ID NO 64 and either the wild-type E. coli W cscB sucrose permease with SEQ ID NO 01 or a variant chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121, mutant glmS*54 from E. coli with SEQ ID NO 83, a glucosamine 6-phosphate N-acetyltransferase (GNA1) from S. cerevisiae with SEQ ID NO 80, an N-acetylglucosamine 2-epimerase (AGE) from B. ovatus with SEQ ID NO 81 and an N-acetylneuraminate (Neu5Ac) synthase (neuB) from N. meningitidis with SEQ ID NO 82. For sialylated oligosaccharide production, the sialic acid production strains further need to express an N-acylneuraminate cytidylyltransferase (neuA) from P. multocida with SEQ ID NO 86, and a beta-galactoside alpha-2,3-sialyltransferase comprising SEQ ID NO 87 from P. multocida and/or SEQ ID NO 88 from N. meningitidis, and/or a beta-galactoside alpha-2,6-sialyltransferase comprising SEQ ID NO 89 from P. damselae and/or SEQ ID NO 90 from Photobacterium sp. JT-ISH-224. Constitutive transcriptional units of neuA and the sialyltransferases can be delivered to the mutant strain either via genomic knock-in or via expression plasmids. If the mutant strains producing sialic acid and CMP-sialic acid were intended to make sialylated lactose structures, the strains were additionally modified with a genomic knock-in of a constitutive transcriptional unit for the E. coli LacY with SEQ ID NO 79.

    [0454] For LN3 production, the mutant strain was derived from C. glutamicum comprising knockouts of the C. glutamicum Idh, cg12645, nagB and glmS genes and genomic knock-ins of constitutive transcriptional units for the fructose kinase (Frk) from Z. mobilis with SEQ ID NO 63, the sucrose phosphorylase (SP) from B. adolescentis with SEQ ID NO 64 and either the wild-type E. coli W cscB sucrose permease with SEQ ID NO 01 or a variant chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121, mutant glmS*54 from E. coli with SEQ ID NO 83, a galactoside beta-1,3-N-acetylglucosaminyltransferase (IgtA) with SEQ ID NO 91 from N. meningitidis and a lactose permease (LacY) with SEQ ID NO 79 from E. coli. For LNT or LNnT production, the LNT3 producing strain was further transformed with constitutive transcriptional units for either an N-acetylglucosamine beta-1,3-galactosyltransferase (wbgO) with SEQ ID NO 92 from E. coli 055:H7 or an N-acetylglucosamine beta-1,4-galactosyltransferase (IgtB) with SEQ ID NO 93 from N. meningitidis, respectively.

    Heterologous and Homologous Expression

    [0455] Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: Twist Biosciences or IDT.

    [0456] Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Genes were optimized using the tools of the supplier.

    Cultivation Conditions

    [0457] A preculture of 96-well microtiter plate experiments was started from a cryovial or a single colony from a TY plate, in 150 L TY and was incubated overnight at 37 C. on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96-well square microtiter plate, with 400 L minimal medium by diluting 400. Each strain was grown in multiple wells of the 96-well plate as biological replicates. These final 96-well culture plates were then incubated at 37 C. on an orbital shaker at 800 rpm for 72h, or shorter, or longer. At the end of the cultivation experiment samples were taken from each well to measure the supernatant concentration (extracellular sugar concentrations, after 5 min. spinning down the cells), or by boiling the culture broth for 15 min at 60 C. before spinning down the cells (=whole broth concentration, intra- and extracellular sugar concentrations, as defined herein).

    [0458] Also, a dilution of the cultures was made to measure the optical density at 600 nm. The cell performance index was determined by dividing the oligosaccharide concentrations, e.g. sialyllactose concentrations, measured in the whole broth by the biomass, in relative percentages compared to the reference strain. The biomass is empirically determined to be approximately rd of the optical density measured at 600 nm.

    Optical Density, pH and Analytical Analysis

    [0459] The determination of the optical density and the pH of the bacterial cultures as well as the analytical analysis were performed as described in Example 1.

    Example 18. Evaluation of C. glutamicum 3SL or 6SL Production Strains Expressing Various Sucrose Permeases

    [0460] A wild-type C. glutamicum strain is first modified with genomic knockouts of the C. glutamicum genes ldh, cg12645, nagB, glmS and nanA, together with genomic knock-ins of constitutive transcriptional units for the fructose kinase (Frk) from Z. mobilis with SEQ ID NO 63, the sucrose phosphorylase (SP) from B. adolescentis with SEQ ID NO 64 and either the wild-type E. coli W cscB sucrose permease with SEQ ID NO 01 or a variant chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121, the mutant glmS*54 from E. coli with SEQ ID NO 83, GNA1 with SEQ ID NO 80 from S. cerevisiae, AGE with SEQ ID NO 81 from B. ovatus, neuB with SEQ ID NO 82 from N. meningitidis and LacY with SEQ ID NO 79 from E. coli. In a next step, the novel strain is transformed with an expression plasmid comprising constitutive transcriptional units for neuA with SEQ ID NO 86 from P. multocida and either an alpha-2,3-sialyltransferase (like SEQ ID NO 87 from P. multocida or SEQ ID NO 88 from N. meningitidis) or an alpha-2,6-sialyltransferase (like SEQ ID NO 89 from P. damselae or SEQ ID NO 90 from Photobacterium sp. JT-ISH-224). When evaluated in a 3-days growth experiment according to the culture conditions provided in Example 17 using appropriate selective medium comprising sucrose and lactose, the novel strains expressing an alpha-2,3-sialyltransferase synthesize 3SL whereas the novel strains expressing an alpha-2,6-sialyltransferase synthesize 6SL.

    Example 19. Evaluation of C. glutamicum LNT or LNnT Production Strains Expressing Various Sucrose Permeases

    [0461] A wild-type C. glutamicum strain is first modified with genomic knockouts of the C. glutamicum genes ldh, cgl2645, nagB and glmS together with genomic knock-ins of constitutive transcriptional units for the fructose kinase (Frk) from Z. mobilis with SEQ ID NO 63, the sucrose phosphorylase (SP) from B. adolescentis with SEQ ID NO 64 and either the wild-type E. coli W cscB sucrose permease with SEQ ID NO 01 or a variant chosen from the list comprising SEQ ID NOs 01 to 62 and 98 to 121, the mutant glmS*54 from E. coli with SEQ ID NO 83, the lactose permease LacY with SEQ ID NO 79 from E. coli and IgtA from N. meningitidis with SEQ ID NO 91. When evaluated in a 3-days growth experiment according to the culture conditions provided in Example 17 using appropriate selective medium comprising sucrose and lactose, the novel strain synthesizes LN3.

    [0462] In a next step for LNT or LNnT production, the LN3 producing C. glutamicum strain is further modified with constitutive transcriptional units for either wbgO from E. coli 055:H7 with SEQ ID NO 92 or IgtB from N. meningitidis with SEQ ID NO 93, respectively. When evaluated in a 3-days growth experiment according to the culture conditions provided in Example 17 using appropriate selective medium comprising sucrose and lactose, the novel strains expressing the N-acetylglucosamine beta-1,3-galactosyltransferase wbgO produce LN3 and LNT whereas the novel strains expressing the N-acetylglucosamine beta-1,4-galactosyltransferase IgtB produce LN3 and LNnT.