Production of Sialylated Oligosaccharide in Host Cells
20230212628 · 2023-07-06
Inventors
- Joeri Beauprez (Zwijnaarde, BE)
- Pieter Coussement (Zwijnaarde, BE)
- Thomas Decoene (Zwijnaarde, BE)
- Annelies Vercauteren (Zwijnaarde, BE)
Cpc classification
C12P19/04
CHEMISTRY; METALLURGY
C12P19/18
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
C12P19/26
CHEMISTRY; METALLURGY
C12Y204/99001
CHEMISTRY; METALLURGY
C12N9/1081
CHEMISTRY; METALLURGY
C12P19/00
CHEMISTRY; METALLURGY
International classification
C12N15/70
CHEMISTRY; METALLURGY
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 host cells. The present invention describes a method of making sialylated oligosaccharide by fermentation with a genetically modified cell, as well as to the genetically modified cell used in the method. The genetically modified cell comprises at least one nucleic acid sequence coding for an enzyme involved in sialylated oligosaccharide synthesis and at least one nucleic acid expressing a membrane protein.
Claims
1-34. (canceled)
35. Method for the production of sialylated oligosaccharide by a genetically modified cell, comprising the steps of: providing a cell capable of producing sialylated oligosaccharide, said cell comprising at least one nucleic acid sequence coding for an enzyme for sialylated oligosaccharide synthesis, said cell genetically modified for i) overexpression of an endogenous membrane protein, ii) expression or overexpression of a homologous membrane protein, and/or iii) expression or overexpression of a heterologous membrane protein culturing the cell in a medium under conditions permissive for the production of sialylated oligosaccharide.
36. Method according to claim 35, wherein said cell is genetically modified for the production of sialylated oligosaccharide and wherein said genetically modified cell a) excretes sialylated oligosaccharide at a ratio of the supernatant concentration to whole broth concentration higher than 0.5 and/or b) has an enhanced production of sialylated oligosaccharide compared to a cell with the same genetic makeup but lacking the i) overexpression of the endogenous membrane protein, ii) expression or overexpression of the homologous membrane protein and/or iii) expression or overexpression of the heterologous membrane protein, respectively.
37. Method according to claim 35, wherein said membrane protein comprises i) an amino acid sequence encoding a siderophore exporter as part of any one of NOG families COG0477, 0ZVQG, 0ZPI7, 0ZVXV, 0XNN3, COG3182, 0ZW7F, 0XP7I, 0ZVCH, 0XQZX, 0XNQK, 0ZVYD, COG2271, 0XNNX, 0ZZWT, COG2814, 0ZITE, 0ZVC8, 0XT98, 0XNQ6, 0YAQV, 0ZVQA, COG2211, COG3104, 1269U, 0ZW8Z, COG1132, COG1173, COG0842, COG4615, COG0577, COG2274, COG4618, COG4172, COG5265, COG1136, 0XPIZ, COG0444, COG4779, COG4606, COG0601, COG1108, COG3182, COG4214, COG4605, COG2409, COG0841, COG3696, COG0845, COG1033, COG0534, 0Y3TF, COG2244, 0XPYW, COG2223 or bactNOG families 05E8G, 08HFG, 089VA, 07TNI, 05C0R, 07Y9F, 05CSH, 05QRD, 05EDF, 05C6X, 08NGX, 05C2C, 07FU4, 07U9Z, 080SS, 07SFI, 05EYM, 05C57, 08E7F, 07QF7, 05CSP, 07UZE, 07VHC, 08EFJ, 05CT4, 05FCD, 07YDJ, 08MMW, 08TKV, 07XMP, 05BZ1, 05IBP, 05CK8, 05IUH, 05D6C, 08E0J, 08116, 08JJA, 05FDX, 05EGG, 08JN3, 08N1B, 05IDI, 08ITX, 05TVJ, 05DHS, 05CM4, 07RUJ, 05EYF, 07R13, 05BZ5, 08IJF, 05UQX, 05C3S, 07U3M, 07R73, 07T1S, 07TJ5, 07XCD, 05DJC, 07RBJ, 05CXP; or ii) an amino acid sequence encoding an ABC transporter comprising a) a conserved domain GxSGxGKST (SEQ ID NO 94) and b) a conserved domain SGGQxQRxxxxRAxxxxPK (SEQ ID NO 95) wherein x can be any distinct amino acid; or iii) an amino acid sequence encoding an MFS transporter comprising a) a conserved domain [AGMS]x[FLMVY]x[DGKNQR]xx[EGST][PRTVY][KR]x[GILMV] (SEQ ID NO 96) and b) a conserved domain [LRST]xxx[AG][AFILV] (SEQ ID NO 97), wherein x can be any distinct amino acid; or iv) an amino acid sequence encoding a Sugar Efflux Transporter, preferably said membrane protein is an MFS transporter comprising the conserved domain L[FY]AxNR[HN]Y (SEQ ID NO 98), wherein x can be any distinct amino acid.
38. Method according to claim 35, wherein i) when said membrane protein is a siderophore exporter, said membrane protein is selected from SEQ ID NOs 9, 4, 6, 11, 13, 15, 20, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 or 122 or functional homolog or functional fragment of any one of the above membrane protein or a sequence having at least 80% sequence identity to any one of said SEQ ID NOs 9, 4, 6, 11, 13, 15, 20, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 or 122 and providing improved production and/or efflux of sialylated oligosaccharides; ii) when said membrane protein is an ABC transporter, said membrane protein is selected from oppF from Escherichia coli K12 MG1655 with SEQ ID NO 18, lmrA from Lactococcus lactis subsp. lactis bv. Diacetylactis with SEQ ID NO 15, Blon_2475 from B. longum subsp. Infantis (strain ATCC 15697) with SEQ ID NO 19 or gsiA from Escherichia coli K12 MG1655 with SEQ ID NO 63, or functional homolog or functional fragment of any one of the above transporter membrane protein or a sequence having at least 80% sequence identity to any one of said SEQ ID NOs 18, 15, 19 or 63 and providing improved production and/or efflux of sialylated oligosaccharides; iii) when said membrane protein is an MFS transporter, said membrane protein is selected from SEQ ID NOs 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 20, 21, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 100, 106, 107, 108, 111, 113, 116, 117, 118, 119, 121 or 122 and providing improved production and/or efflux of sialylated oligosaccharides or functional homolog or functional fragment of any one of the above transporter membrane protein or a sequence having at least 80% sequence identity to any one of said SEQ ID NOs 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 20, 21, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 100, 106, 107, 108, 111, 113, 116, 117, 118, 119, 121 or 122 and providing improved production and/or efflux of sialylated oligosaccharides; iv) when said membrane protein is a Sugar Efflux Transporter, said membrane protein is selected from SEQ ID NOs 2, 1, 3, 16, 17 or 62, or functional homolog or functional fragment of any one of the above transporter membrane protein or a sequence having at least 80% sequence identity to any one of said SEQ ID NOs 2, 1, 3, 16, 17 or 62 and providing improved production and/or efflux of sialylated oligosaccharides.
39. Method according to claim 35, the method further comprising at least one of the following steps: i) Adding to the culture medium a precursor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of precursor per litre of initial reactor volume wherein the total reactor volume ranges from 250 mL (millilitre) to 10.000 m3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture medium before the addition of said precursor feed; ii) Adding a precursor feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution; iii) Adding a precursor feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein the concentration of said precursor feeding solution is 50 g/L, preferably 75 g/L, more preferably 100 g/L, more preferably 125 g/L, more preferably 150 g/L, more preferably 175 g/L, more preferably 200 g/L, more preferably 225 g/L, more preferably 250 g/L, more preferably 275 g/L, more preferably 300 g/L, more preferably 325 g/L, more preferably 350 g/L, more preferably 375 g/L, more preferably, 400 g/L, more preferably 450 g/L, more preferably 500 g/L, even more preferably, 550 g/L, most preferably 600 g/L; and wherein preferably the pH of said solution is set between 3 and 7 and wherein preferably the temperature of said feed solution is kept between 20° C. and 80° C.; iv) Said method resulting in sialylated oligosaccharide concentration of at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L, more preferably at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L in the final volume of said culture medium.
40. Method according to claim 39, wherein the precursor feed is accomplished by adding precursor from the beginning of the cultivating in a concentration of at least 5 mM, preferably in a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably in a concentration >300 mM.
41. Method according to claim 39, wherein said precursor feed is accomplished by adding precursor to the cultivation medium in a concentration, such, that throughout the production phase of the cultivation a precursor concentration of at least 5 mM, preferably 10 mM or 30 mM is obtained.
42. Method according to claim 39, wherein the host cells are cultivated for at least about 60, 80, 100, or about 120 hours or in a continuous manner.
43. Method according to claim 35, wherein a precursor feed is added to the culture medium and wherein precursor is chosen from the group comprising lactose, lacto-N-biose (LNB), lacto-N-triose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), N-acetyl-lactosamine (LacNAc), lacto-N-pentaose (LNP), lacto-N-neopentaose, para lacto-N-pentaose, para lacto-N-neopentaose, lacto-N-novopentaose I, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), para lacto-N-neohexaose (pLNnH), para lacto-N-hexaose (pLNH), lacto-N-heptaose, lacto-N-neoheptaose, para lacto-N-neoheptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N-neooctaose, iso lacto-N-octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N-neooctaose, iso lacto-N-nonaose, novo lacto-N-nonaose, lacto-N-nonaose, lacto-N-decaose, iso lacto-N-decaose, novo lacto-N-decaose, lacto-N-neodecaose, galactosyllactose, a lactose extended with 1, 2, 3, 4, 5, or a multiple of N-acetyllactosamine units and/or 1, 2, 3, 4, 5, or a multiple of lacto-N-biose units, and oligosaccharide containing 1 or multiple N-acetyllactosamine units and/or 1 or multiple lacto-N-biose units or an intermediate into sialylated oligosaccharide, fucosylated and sialylated versions thereof.
44. Method according to claim 35, wherein a carbon and energy source, preferably sucrose, glucose, fructose, glycerol, maltose, maltodextrines, trehalose, polyols, starch, succinate, malate, pyruvate, lactate, ethanol, citrate, lactose, is also added, preferably continuously to the culture medium, preferably with the precursor.
45. Method according to claim 35, wherein a first phase of exponential cell growth is provided by adding a carbon-based substrate, preferably glucose or sucrose, to the culture medium before the lactose is added to the culture medium in a second phase.
46. Method according to claim 35, wherein said sialylated oligosaccharide is 6′-sialyllactose, 3′-sialyllactose, 3-fucosyl-3′-sialyllactose (3′-O-sialyl-3-0-fucosyllactose, FSL), 2′-fucosyl-3′-sialyllactose, 2′-fucosyl-6′-sialyllactose, 3,6-disialyllactose, 6,6′-disialyllactose, sialyllacto-N-tetraose a (LSTa), fucosyl-LSTa (FLSTa), sialyllacto-N-tetraose b (LSTb), fucosyl-LSTb (FLSTb), sialyllacto-N-neotetraose c (LSTc), fucosyl-LSTc (FLSTc), sialyllacto-N-neotetraose d (LSTd), fucosyl-LSTd (FLSTd), sialyl-lacto-N-hexaose (SLNH), sialyl-lacto-N-neohexaose I (SLNH-I), sialyl-lacto-N-neohexaose II (SLNH-II), disialyl-lacto-N-tetraose (DS-LNT), 6′-O-sialylated-lacto-N-neotetraose, 3′-O-sialylated-lacto-N-tetraose, 6′-sialylN-acetyllactosamine, 3′-sialylN-acetyllactosamine, 3-fucosyl-3′-sialylN-acetyllactosamine (3′-O-sialyl-3-O-fucosyl-N-acetyllactosamine), 3,6-disialylN-acetyllactosamine, 6,6′-disialyl-Nacetyllactosamine, 2′-fucosyl-3′-sialylN-acetyllactosamine, 2′-fucosyl-6′-sialyl-N-acetyllactosamine, 6′-sialyl-LactoNbiose, 3′-sialyl-LactoNbiose, 4-fucosyl-3′-sialyl-LactoNbiose (3′-O-sialyl-4-O-fucosyl-LactoNbiose), 3′,6′-disialyl-LactoNbiose, 6,6′-disialyl-LactoNbiose, 2′-fucosyl-3′-sialyl-LactoNbiose, 2′-fucosyl-6′-sialyl-LactoNbiose.
47. Method according to claim 35, wherein the method is producing a mixture of sialylated oligosaccharides.
48. Method according to claim 35, wherein said genetically modified cell is selected from the group consisting of microorganism, plant, or animal cells, preferably said microorganism is a bacterium, fungus or a yeast, preferably said plant is a rice, cotton, rapeseed, soy, maize or corn plant, preferably said animal is an insect, fish, bird or non-human mammal, preferably the cell is an Escherichia coli cell.
49. Host cell genetically modified for the production of sialylated oligosaccharide, wherein the host cell comprises at least one nucleic acid sequence coding for an enzyme for sialylated oligosaccharide synthesis and wherein said cell is genetically modified for i) overexpression of an endogenous membrane protein, ii) expression or overexpression of a homologous membrane protein, and/or iii) expression or overexpression of a heterologous membrane protein, wherein said membrane protein comprises i) an amino acid sequence encoding a siderophore exporter, preferably a siderophore exporter as part of any one of NOG families COG0477, 0ZVQG, 0ZPI7, 0ZVXV, 0XNN3, COG3182, 0ZW7F, 0XP7I, 0ZVCH, 0XQZX, 0XNQK, 0ZVYD, COG2271, 0XNNX, 0ZZWT, COG2814, 0ZITE, 0ZVC8, 0XT98, 0XNQ6, 0YAQV, 0ZVQA, COG2211, COG3104, 1269U, 0ZW8Z, COG1132, COG1173, COG0842, COG4615, COG0577, COG2274, COG4618, COG4172, COG5265, COG1136, 0XPIZ, COG0444, COG4779, COG4606, COG0601, COG1108, COG3182, COG4214, COG4605, COG2409, COG0841, COG3696, COG0845, COG1033, COG0534, 0Y3TF, COG2244, 0XPYW, COG2223 or bactNOG families 05E8G, 08HFG, 089VA, 07TNI, 05C0R, 07Y9F, 05CSH, 05QRD, 05EDF, 05C6X, 08NGX, 05C2C, 07FU4, 07U9Z, 080SS, 07SFI, 05EYM, 05C57, 08E7F, 07QF7, 05CSP, 07UZE, 07VHC, 08EFJ, 05CT4, 05FCD, 07YDJ, 08MMW, 08TKV, 07XMP, 05BZ1, 05IBP, 05CK8, 05IUH, 05D6C, 08E0J, 08116, 08JJA, 05FDX, 05EGG, 08JN3, 08N1B, 05IDI, 08ITX, 05TVJ, 05DHS, 05CM4, 07RUJ, 05EYF, 07R13, 05BZS, 08IJF, 05UQX, 05C3S, 07U3M, 07R73, 07T1S, 07TJ5, 07XCD, 05DJC, 07RBJ, 0500; or ii) an amino acid sequence encoding an ABC transporter comprising a) a conserved domain GxSGxGKST (SEQ ID NO 94) and b) a conserved domain SGGQxQRxxxxRAxxxxPK (SEQ ID NO 95) wherein x can be any distinct amino acid; or iii) an amino acid sequence encoding an MFS transporter comprising a) a conserved domain [AGMS]x[FLMVY]x[DGKNQR]xx[EGST][PRTVY][KR]x[GILMV] (SEQ ID NO 96) and b) a conserved domain [LRST]xxx[AG][AFILV] (SEQ ID NO 97), wherein x can be any distinct amino acid; or iv) an amino acid sequence encoding a Sugar Efflux Transporter, preferably said membrane protein is an MFS transporter comprising the conserved domain L[FY]AxNR[HN]Y (SEQ ID NO 98), wherein x can be any distinct amino acid.
50. Cell according to claim 49, wherein i) when said membrane protein is a siderophore exporter, said membrane protein is selected from SEQ ID NOs 9, 4, 6, 11, 13, 15, 20, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 or 122 or functional homolog or functional fragment of any one of the above membrane protein or a sequence having at least 80% sequence identity to any one of said SEQ ID NOs 9, 4, 6, 11, 13, 15, 20, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 or 122 and providing improved production and/or efflux of sialylated oligosaccharides; ii) when said membrane protein is an ABC transporter, said membrane protein is selected from oppF from Escherichia coli K12 MG1655 with SEQ ID NO 18, lmrA from Lactococcus lactis subsp. lactis bv. Diacetylactis with SEQ ID NO 15, Blon_2475 from B. longum subsp. Infantis (strain ATCC 15697) with SEQ ID NO 19 or gsiA from Escherichia coli K12 MG1655 with SEQ ID NO 63, or functional homolog or functional fragment of any one of the above transporter membrane protein or a sequence having at least 80% sequence identity to any one of said SEQ ID NOs 18, 15, 19 or 63 and providing improved production and/or efflux of sialylated oligosaccharides; iii) when said membrane protein is an MFS transporter, said membrane protein is selected from SEQ ID NOs 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 20, 21, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 100, 106, 107, 108, 111, 113, 116, 117, 118, 119, 121 or 122 or functional homolog or functional fragment of any one of the above transporter membrane protein or a sequence having at least 80% sequence identity to any one of said SEQ ID NOs 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 20, 21, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 100, 106, 107, 108, 111, 113, 116, 117, 118, 119, 121 or 122 and providing improved production and/or efflux of sialylated oligosaccharides; iv) when said membrane protein is a Sugar Efflux Transporter, said membrane protein is selected from SEQ ID NOs 2, 1, 3, 16, 17 or 62, or functional homolog or functional fragment of any one of the above transporter membrane protein or a sequence having at least 80% sequence identity to any one of said SEQ ID NOs 2, 1, 3, 16, 17 or 62 and providing improved production and/or efflux of sialylated oligosaccharides.
51. Cell according to claim 49, wherein said cell is selected from the group consisting of microorganism, plant, or animal cells, preferably said microorganism is a bacterium, fungus or a yeast, preferably said plant is a rice, cotton, rapeseed, soy, maize or corn plant, preferably said animal is an insect, fish, bird or non-human mammal; preferably the cell is an Escherichia coli cell.
52. Cell according to claim 49, wherein the cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for the synthesis of sialylated oligosaccharide.
53. Cell according to claim 49, wherein said sialylated oligosaccharide is 6′-sialyllactose, 3′-sialyllactose, 3-fucosyl-3′-sialyllactose (3′-O-sialyl-3-O-fucosyllactose, FSL), 2′-fucosyl-3′-sialyllactose, 2′-fucosyl-6′-sialyllactose, 3,6-disialyllactose, 6,6′-disialyllactose, sialyllacto-N-tetraose a (LSTa), fucosyl-LSTa (FLSTa), sialyllacto-N-tetraose b (LSTb), fucosyl-LSTb (FLSTb), sialyllacto-N-neotetraose c (LSTc), fucosyl-LSTc (FLSTc), sialyllacto-N-neotetraose d (LSTd), fucosyl-LSTd (FLSTd), sialyl-lacto-N-hexaose (SLNH), sialyl-lacto-N-neohexaose I (SLNH-I), sialyl-lacto-N-neohexaose II (SLNH-II), disialyl-lacto-N-tetraose (DS-LNT), 6′-O-sialylated-lacto-N-neotetraose, 3′-O-sialylated-lacto-N-tetraose, 6′-sialylN-acetyllactosamine, 3′-sialylN-acetyllactosamine, 3-fucosyl-3′-sialylN-acetyllactosamine (3′-O-sialyl-3-O-fucosyl-N-acetyllactosamine), 3,6-disialylN-acetyllactosamine, 6,6′-disialyl-Nacetyllactosamine, 2′-fucosyl-3′-sialylN-acetyllactosamine, 2′-fucosyl-6′-sialyl-N-acetyllactosamine, 6′-sialyl-LactoNbiose, 3′-sialyl-LactoNbiose, 4-fucosyl-3′-sialyl-LactoNbiose (3′-O-sialyl-4-O-fucosyl-LactoNbiose), 3′,6′-disialyl-LactoNbiose, 6,6′-disialyl-LactoNbiose, 2′-fucosyl-3′-sialyl-LactoNbiose, 2′-fucosyl-6′-sialyl-LactoNbiose.
54. Cell according to claim 49, characterized in that it is further transformed to comprise at least one nucleic acid sequence coding for a protein facilitating or promoting the import of substrate required for oligosaccharide synthesis, wherein the protein is selected from the group consisting of lactose transporter, fucose transporter, sialic acid transporter, galactose transporter, mannose transporter, N-acetylglucosamine transporter, N-acetylgalactosamine transporter, ABC-transporter, transporter for a nucleotide-activated sugar and transporter for a nucleobase, nucleoside or nucleotide.
55. Cell according to claim 49, characterized in that it is further transformed to comprise at least one nucleic acid sequence coding for a protein selected from the group consisting of nucleotidyltransferase, guanylyltransferase, uridylyltransferase, Fkp, L-fucose kinase, fucose-1-phosphate guanylyltransferase, CMP-sialic acid synthetase, galactose kinase, galactose-1-phosphate uridylyltransferase, glucose kinase, glucose-1-phosphate uridylyltransferase, mannose kinase, mannose-1-phosphate guanylyltransferase, GDP-4-keto-6-deoxy-D-mannose reductase, glucosamine kinase, glucosamine-phosphate acetyltransferase, N-acetyl-glucosamin-phosphate uridylyltransferase, UDP-N-acetylglucosamine 4-epimerase, UDP-N-acetyl-glucosamine 2-epimerase, cytidyltransferase, fructose-6-P-aminotransferase, glucosamine-6-P-aminotransferase, phosphatase, N-acetylglucosamine-2-epimerase, sialic acid synthase, ManNAc kinase, sialic acid synthetase, sialic acid phosphatase.
56. A bacterial cell for the production of sialyllactose, the cell being transformed to comprise at least one nucleic acid sequence coding for a sialyltransferase, characterized in that: the cell in addition is transformed to comprise at least one nucleic acid sequence coding for a membrane protein wherein said membrane protein comprises i) an amino acid sequence encoding a siderophore exporter, preferably a siderophore exporter as part of any one of NOG families COG0477, 0ZVQG, 0ZPI7, 0ZVXV, 0XNN3, COG3182, 0ZW7F, 0XP7I, 0ZVCH, 0XQZX, 0XNQK, 0ZVYD, COG2271, 0XNNX, 0ZZWT, COG2814, 0ZITE, 0ZVC8, 0XT98, 0XNQ6, 0YAQV, 0ZVQA, COG2211, COG3104, 1269U, 0ZW8Z, COG1132, COG1173, COG0842, COG4615, COG0577, COG2274, COG4618, COG4172, COG5265, COG1136, 0XPIZ, COG0444, COG4779, COG4606, COG0601, COG1108, COG3182, COG4214, COG4605, COG2409, COG0841, COG3696, COG0845, COG1033, COG0534, 0Y3TF, COG2244, 0XPYW, COG2223 or bactNOG families 05E8G, 08HFG, 089VA, 07TNI, 05C0R, 07Y9F, 05CSH, 05QRD, 05EDF, 05C6X, 08NGX, 05C2C, 07FU4, 07U9Z, 080SS, 07SFI, 05EYM, 05C57, 08E7F, 07QF7, 05CSP, 07UZE, 07VHC, 08EFJ, 05CT4, 05FCD, 07YDJ, 08MMW, 08TKV, 07XMP, 05BZ1, 05IBP, 05CK8, 05IUH, 05D6C, 08E0J, 08116, 08JJA, 05FDX, 05EGG, 08JN3, 08N1B, 051DI, 08ITX, 05TVJ, 05DHS, 05CM4, 07RUJ, 05EYF, 07R13, 05BZS, 08IJF, 05UQX, 05C3S, 07U3M, 07R73, 07T1S, 07TJ5, 07XCD, 05DJC, 07RBJ, 05CXP; or ii) an amino acid sequence encoding an ABC transporter comprising a) a conserved domain GxSGxGKST (SEQ ID NO 94) and b) a conserved domain SGGQxQRxxxxRAxxxxPK (SEQ ID NO 95) wherein x can be any distinct amino acid; or iii) an amino acid sequence encoding an MFS transporter comprising a) a conserved domain [AGMS]x[FLMVY]x[DGKNQR]xx[EGST][PRTVY][KR]x[GILMV] (SEQ ID NO 96) and b) a conserved domain [LRST]xxx[AG][AFILV] (SEQ ID NO 97), wherein x can be any distinct amino acid; or iv) an amino acid sequence encoding a Sugar Efflux Transporter, preferably said membrane protein is an MFS transporter comprising the conserved domain L[FY]AxNR[HN]Y (SEQ ID NO: 98), wherein x can be any distinct amino acid.
57. Bacterial cell according to claim 56, characterized in that the cell is an Escherichia coli cell.
Description
DESCRIPTION OF THE FIGURES
[0521]
[0522]
[0523]
[0524]
[0525]
[0526]
[0527]
[0528]
[0529]
[0530]
[0531]
[0532]
EXAMPLES
Example 1: Material and Methods
[0533] Material and Methods Escherichia coli
Media
[0534] Three different media were used, namely a rich Luria Broth (LB), a minimal medium for shake flask (MMsf) and a minimal medium for fermentation (MMf). Both minimal media use a trace element mix.
[0535] Trace element mix consisted of 3.6 g/L FeCl.sub.2.Math.4H.sub.2O, 5 g/L CaCl.sub.2.Math.2H.sub.2O, 1.3 g/L MnCl.sub.2.Math.2H.sub.2O, 0.38 g/L CuCl.sub.2.Math.2H.sub.2O, 0.5 g/L CoCl.sub.2.Math.6H.sub.2O, 0.94 g/L ZnCl.sub.2, 0.0311 g/L H.sub.3BO.sub.4, 0.4 g/L Na.sub.2EDTA.Math.2H.sub.2O and 1.01 g/L thiamine.Math.HCl. The molybdate solution contained 0.967 g/L NaMoO.sub.4.Math.2H.sub.2O. The selenium solution contained 42 g/L SeO.sub.2.
[0536] 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). Luria Broth agar (LBA) plates consisted of the LB media, with 12 g/L agar (Difco, Erembodegem, Belgium) added.
[0537] The minimal medium for the shake flasks (MMsf) experiments contained 2.00 g/L NH.sub.4Cl, 5.00 g/L (NH.sub.4).sub.2SO.sub.4, 2.993 g/L KH.sub.2PO.sub.4, 7.315 g/L K.sub.2H PO.sub.4, 8.372 g/L MOPS, 0.5 g/L NaCl, 0.5 g/L MgSO.sub.4.7H.sub.2O, 14.26 g/L sucrose or another carbon source when specified in the examples, 1 ml/L trace element mix, 100 μl/L molybdate solution, and 1 mL/L selenium solution. The medium was set to a pH of 7 with 1M KOH. Depending on the experiment lactose, LNB or LacNAc could be added as a precursor.
[0538] The minimal medium for fermentations (MMf) contained 6.75 g/L NH.sub.4Cl, 1.25 g/L (NH.sub.4).sub.2SO.sub.4, 2.93 g/L KH.sub.2PO.sub.4 and 7.31 g/L KH.sub.2PO.sub.4, 0.5 g/L NaCl, 0.5 g/L MgSO.sub.4.Math.7H2O, 14.26 g/L sucrose, 1 mL/L trace element mix, 100 μL/L molybdate solution, and 1 mL/L selenium solution with the same composition as described above.
[0539] Complex medium, e.g. LB, was sterilized by autoclaving (121° C., 21′) and minimal medium by filtration (0.22 μm Sartorius). When necessary, the medium was made selective by adding an antibiotic (e.g. ampicillin (100 mg/L), chloramphenicol (20 mg/L), carbenicillin (100 mg/L), spectinomycin (40 mg/L) and/or kanamycin (50 mg/L)).
Plasmids
[0540] 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).
[0541] Plasmids for membrane protein and for additional sialyltransferase expression from plasmid, were constructed in a pSC101 or a p15A ori containing backbone vector, respectively, using Golden Gate assembly. All membrane protein and sialyltransferase encoding genes were synthetically synthetized at Twist Biosciences (San Francisco, USA). Polynucleotide sequences of the membrane proteins and the corresponding membrane protein polypeptides are shown in SEQ ID NOs 01 to 21, 37 to 93 and 99 to 122 and enlisted in Table 1.
Transcription Units
[0542] Both membrane protein and sialyl transferase genes were expressed in different transcriptional units (TUs) using specific promoter, UTR and terminator combinations as enlisted in Table 2. The genes were expressed using promoters from Mutalik et al. (Nat. Methods 2013, No. 10, 354-360), as described herein as “PROM0005”, “PROM0010”, “PROM0012”, “PROM0025”, “PROM0032” and “PROM0050”, a promoter from De Mey et al. (BMC Biotechnology 2007, 7:34)), as described herein as “PROM0015” and a modified promoter of apFAB115 (as described by Mutalik et al. (Nat. Methods 2013, No. 10, 354-360), described herein as “PROM0171”. UTRs used as described herein as “UTR0003”, “UTR0011”, “UTR0013”, “UTR0014”, “UTR0029”, “UTR0038”, “UTR0051” and “UTR0055” were obtained from Mutalik et al. (Nat. Methods 2013, No. 10, 354-360). Terminators used in the examples are described as “TER0010” and “TER0020” as obtained from Dunn et al. (Nucleic Acids Res. 1980, 8(10), 2119-32) and “TER0002” are as obtained from Orosz et al. (Eur. J. Biochem. 1991, 201, 653-59). Table 2 shows the overview of the transcriptional units used in the examples by combination of the above promoter UTRs and terminators. 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.
TABLE-US-00001 TABLE 1 Country of origin of SEQ ID NO Name/ digital sequence (protein) TCDB group Organism Origin information 01 EcSetA E. coli K12 MG1655 Synthetic USA 02 EcSetB E. coli K12 MG1655 Synthetic USA 03 EcSetC E. coli K12 MG1655 Synthetic USA 04 EcMdfA E. coli K12 MG1655 Synthetic USA 05 EcYnfM E. coli K12 MG1655 Synthetic USA 06 EcIceT E. coli K12 MG1655 Synthetic USA 07 EcYhhs E. coli K12 MG1655 Synthetic USA 08 EcYdhC E. coli K12 MG1655 Synthetic USA 09 EcEntS/EcYbdA E. coli K12 MG1655 Synthetic USA 10 EcMhpT E. coli K12 MG1655 Synthetic USA 11 EcYebQ E. coli K12 MG1655 Synthetic USA 12 EcYjhB E. coli K12 MG1655 Synthetic USA 13 EcBcr E. coli K12 MG1655 Synthetic USA 14 EcFucP E. coli K12 MG1655 Synthetic USA 15 LlImrA Lactococcus lactis strain Synthetic South Korea SRCM103457 16 PcSetA_01 Pectobacterium carotovorum Synthetic South Korea (short version) 17 PcSetA_02 Pectobacterium carotovorum Synthetic South Korea (long version) 18 EcOppF E. coli strain K12 (MG1655) Synthetic USA 19 Blon_2475 B. longum subsp. Infantis Synthetic Germany (strain ATCC 15697) 20 Blon_0247 B. longum subsp. Infantis Synthetic Germany (strain ATCC 15697) 21 Blon_0245 B. longum subsp. Infantis Synthetic Germany (strain ATCC 15697) 37 CuNAm Curtobacterium sp. Synthetic USA 314Chir4.1 38 mdtD Citrobacter freundii MGH152 Synthetic USA 39 mdtD Citrobacter werkmanii NBRC Synthetic Belgium 105721 40 mdtD Citrobacter amalonaticus Synthetic USA 41 mdtD Klebsiella oxytoca Synthetic United Kingdom 42 mdtD Escherichia albertii B156 Synthetic USA 43 yegB Salmonella enterica subsp. Synthetic United Kingdom salamae 44 mdtD Klebsiella pneumoniae Synthetic USA 30684/NJST258_2 45 mdtD Klebsiella pneumoniae kneu Synthetic USA 46 mdtD Pseudocitrobacter faecalis Synthetic USA 47 YrMdfA Yokenella regensburgei Synthetic United Kingdom ATCC43003 48 CmMdfA Cronobacter muytjensii Synthetic USA 49 KoMdfA Klebsiella oxytoca Synthetic Sweden 50 CkMdfA Citrobacter koseri Synthetic USA 51 EmMdfA Escherichia marmotae Synthetic USA 52 Cmr Shigella flexneri Synthetic United Kingdom 53 SeMdfA Salmonella enterica subsp. Synthetic Denmark salamae 54 Cmr Citrobacter youngae ATCC Synthetic USA 29220 55 CfMdfA Citrobacter freundii Synthetic USA 56 EkMdfA Enterobacter kobei Synthetic USA 57 EnMdfA Enterobacter sp. Synthetic Australia 58 MdfA Lelliottia sp. WB101 Synthetic USA 59 ElMdfA Enterobacter ludwigii Synthetic USA EcWSU1 60 EsMdfA Enterobacter soli LF7a Synthetic Unknown 61 TIC76629 Bifidobacterium longum Synthetic USA subsp. infantis (strain Bi-26) 62 CnSetA Cedecea neteri Synthetic USA 63 EcgsiA E. coli K12 MG1655 Synthetic USA 64 EcGalP E. coli K12 MG1655 Synthetic USA 65 EcYdeA E. coli K12 MG1655 Synthetic USA 66 EcWzxE E. coli K12 MG1655 Synthetic USA 67 HpWzk Helicobacter pylori strain Synthetic United Kingdom ATCC 700392/26695 68 Blon0345 B. longum subsp. Infantis Synthetic Germany (strain ATCC 15697) 69 EcMsbA E. coli K12 MG1655 Synthetic USA 70 NcCDT2 Neurospora crassa OR74A Synthetic USA 71 AoCDT2 Aspergillus oryzae RIB40 Synthetic Japan 72 ChWzx Chitinophaga sp. CF118 Synthetic USA 73 EuWzx Eubacterium sp. CAG: 581 Synthetic Denmark 74 LrWzx Lactococcus raffinolactis Synthetic South Korea KACC 13441 75 PrWzx Prevotella ruminicola (AR32) Synthetic USA 76 PsTolC Candidatus Planktophila Synthetic Switzerland sulfonica 77 BhTolC Butyrivibrio hungatei Synthetic USA XBD2006 78 RiMsbA Roseburia intestinalis Synthetic Denmark CAG: 13 79 PgMsbA Pedobacter ginsengisoli Synthetic South Korea 80 VbMsbA Verrucomicrobia bacterium Synthetic USA CG1_02_43_26 81 AsNAm Actinobaculum suis DSM Synthetic Unknown 20639 82 RgNAm Ruminococcus gnavus Synthetic USA 83 EcmdIA E. coli K12 MG1655 Synthetic USA 84 EcmdIB E. coli K12 MG1655 Synthetic USA 85 EcgsiC E. coli K12 MG1655 Synthetic USA 86 EcgsiD E. coli K12 MG1655 Synthetic USA 87 EcoppB E. coli K12 MG1655 Synthetic USA 88 EcoppC E. coli K12 MG1655 Synthetic USA 89 EcoppD E. coli K12 MG1655 Synthetic USA 90 EcwzxC E. coli K12 MG1655 Synthetic USA 91 EcyihP E. coli K12 MG1655 Synthetic USA 92 Abaet Azospirillum brasiliense LMG Synthetic Belgium 04375 93 Bjnodj Bradyrhizobium japonicum Synthetic USA USDA 110 99 Blon2331 Bifidobacterium longum Synthetic Germany subsp. Infantis (strain ATCC 15697) 100 KpIceT Klebsiella pneumoniae Synthetic USA MB369 101 Abaet Azospirillum brasiliense LMG Synthetio Belgium 04375 102 EcAcrB Escherichia coli str. K-12 Synthetic USA substr. W3110 103 EcAmpG Escherichia coli str. K-12 Synthetic USA substr. W3110 104 EcEmrB Escherichia coli str. K-12 Synthetic USA substr. W3110 105 EcEmrY Escherichia coli str. K-12 Synthetic USA substr. W3110 106 EcEntS Escherichia coli Synthetic United Kingdom 107 EcHsrA Escherichia coli str. K-12 Synthetic USA substr. W3110 108 EcLgoT Escherichia coli str. K-12 Synthetic USA substr. W3110 109 EcMelB Escherichia coli str. K-12 Synthetic USA substr. W3110 110 EcProP Escherichia coli str. K-12 Synthetic USA substr. W3110 111 EcShiA Escherichia coli str. K-12 Synthetic USA substr. W3110 112 EcUidB Escherichia coli str. K-12 Synthetic USA substr. W3110 113 EcYdfJ Escherichia coli str. K-12 Synthetic USA substr. W3110 114 EcYhjE Escherichia coli str. K-12 Synthetic USA substr. W3110 115 EcYicJ Escherichia coli str. K-12 Synthetic USA substr. W3110 116 FtFsIB Francisella tularensis subsp. Synthetic USA tularensis SCHU S4 117 KaEntS Kluyvera ascorbata Synthetic USA 118 KiEntS Kluyvera intermedia strain Synthetic USA CAV1151 119 LaEntS Leclercia adecarboxylata Synthetic USA strain USDA-ARS-USMARC- 60222 120 SaSfaA Staphylococcus aureus Synthetic United Kingdom subsp. aureus NCTC 8325 121 SeEntS Salmonella enterica subsp. Synthetic USA arizonae 122 SeEntS Salmonella enterica subsp. Synthetic USA enterica serovar Derby
TABLE-US-00002 TABLE 2 TU number Promoter part UTR part Terminator part TU 01 PROM0015 UTR0003 TER0010 TU 02 PROM0005 UTR0014 TER0010 TU 03 PROM0050 UTR0014 TER0002 TU 04 PROM0012 UTR0003 TER0010 TU 05 PROM0171 UTR0003 TER0010 TU 06 PROM0015 UTR0014 TER0010 TU 07 PROM0005 UTR0014 TER0010 TU 08 PROM0012 UTR0014 TER0010 TU 09 PROM0171 UTR0014 TER0010 TU 10 PROM0012 UTR0029 TER0010 TU 11 PROM0171 UTR0029 TER0010 TU 12 PROM0015 UTR0051 TER0010 TU 13 PROM0032 UTR0014 TER0010 TU 14 PROM0005 UTR0055 TER0010 TU 15 PROM0025 UTR0014 TER0010 TU 16 PROM0012 UTR0055 TER0010 TU 17 PROM0005 UTR0014 TER0020 TU 18 PROM0005 UTR0038 TER0010 TU 19 PROM0032 UTR0038 TER0010 TU 20 PROM0032 UTR0055 TER0010 TU 21 PROM0012 UTR0038 TER0010 TU 22 PROM0005 UTR0038 TER0020 TU 23 PROM0015 UTR0003 TER0020 TU 24 PROM0032 UTR0014 TER0020 TU 25 PROM0005 UTR0011 TER0010 TU 26 PROM0005 UTR0011 TER0020 TU 27 PROM0005 UTR0055 TER0020 TU 28 PROM0010 UTR0013 TER0010 TU 29 PROM0032 UTR0038 TER0020
Strains and Mutations
[0543] Escherichia coli KU MG1655 [lambda.sup.−, F.sup.−, rph-1] was obtained from the Coli Genetic Stock Center (US), CGSC Strain #: 7740, in March 2007. Gene disruptions as well as gene introductions 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.
[0544] Transformants carrying a Red helper plasmid pKD46 were grown in 10 ml LB media with ampicillin, (100 mg/L) and L-arabinose (10 mM) at 30° C. to an OD.sub.600 nm 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 μl of ice-cold water. Electroporation was done with 50 μl of cells and 10-100 ng of linear double-stranded-DNA product by using a Gene Pulser™ (BioRad) (600Ω, 25 μFD, and 250 volts).
[0545] 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.
[0546] 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, repurified from an agarose gel, and suspended in elution buffer (5 mM Tris, pH 8.0).
[0547] The selected mutants (chloramphenicol or kanamycin resistant) 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 (Fw/Rv-gene-out).
[0548] A sialic acid producing base strain derived from E. coli KU MG1655 was created by knocking out the genes asl, IdhA, poxB, atpl-gidB and ackA-pta, and knocking out the operons lacZYA, nagAB and the genes nanA, nanE and nanK. Additionally, the E. coli lacY gene was introduced at the location of lacZYA. A fructose kinase gene (frk) originating from Zymomonas mobilis, an E. coli W sucrose transporter (cscB), a sucrose phosphorylase (SP) originating from Bifidobacterium adolescentis, an E. coli mutant fructose-6-P-aminotransferase (EcglmS*54, as described by Deng et al. (Biochimie 88, 419-29 (2006)), glucosamine-6-P-aminotransferase from Saccharomyces cerevisiae (ScGNA1), an N-acetylglucosamine-2-epimerase from Bacteroides ovatus (BoAGE) and a sialic acid synthase from Campylobacter jejuni (CjneuB) were knocked in into the genome.
[0549] To allow production of 6′-SL, the sialic acid base strain was further modified by introducing two constructs both expressing a CMP-sialic acid synthetase from Neisseria meningitidis (NmneuA, SEQ ID NO 31) and an α-2,6-sialyltransferase from Photobacterium damselae (PdbST, SEQ ID NO 32) into the genome.
[0550] To allow production of 3′-SL, the sialic acid base strain was further modified by introducing a construct expressing a CMP-sialic acid synthetase from Neisseria meningitidis (NmneuA, SEQ ID NO 31) and an α-2,3-sialyltransferase from Neisseria meningitidis (NmST, SEQ ID NO 33) which were knocked in into the genome.
[0551] To allow production of sialylated LacNAc (sLacNAc), the sialic acid base strain was further modified by a CMP-sialic acid synthetase from Neisseria meningitidis (NmneuA, SEQ ID NO 31) and a sialyltransferase which were knocked in into the genome. For 6′-sLacNAc, a sialyltransferase from Photobacterium damselae (PdbST, SEQ ID NO 32) was used and for 3′-sLacNAc, a sialyltransferase from Neisseria meningitidis (NmST, SEQ ID NO 33) was used.
[0552] To allow production of sialylated LNB (sLNB), the sialic acid base strain was further modified by introducing a CMP-sialic acid synthetase from Neisseria meningitidis (NmneuA, SEQ ID NO 31) and a sialyltransferase which were knocked in into the genome. For 6′-sLNB, a sialyltransferase from Photobacterium damselae (PdbST, SEQ ID NO 32) was used and for 3′-sLNB, a sialyltransferase from Neisseria meningitidis (NmST, SEQ ID NO 33) was used.
[0553] To allow production of LSTa and b, the sialic acid base strain was further modified by introducing a beta-1,3-GlcNAc transferase from Neisseria meningitidis (NmIgtA, SEQ ID NO 34), a beta-1,3-galactosyltransferase from E. coli 055:H7 (EcwbgO, SEQ ID NO 36), a CMP-sialic acid synthetase and an alpha-2,3-sialyltransferase or an alpha-2,6-sialyltransferase for production of LSTa or LSTb, respectively. Alternatively, sialic acid can be fed to an optimized lacto-N-tetraose producing strain with expression of a beta-1,3-GlcNAc transferase from Neisseria meningitidis (NmIgtA, SEQ ID NO 34) and a beta-1,3-galactosyltransferase from E. coli O55:H7 (EcwbgO, SEQ ID NO 36) (as described and demonstrated in Example 8 of WO18122225), and additional expression of a CMP-sialic acid synthetase and an α-2,3-sialyltransferase or an α-2,6-sialyltransferase to allow LSTa or LSTb production, respectively.
[0554] To allow production of LSTc and d, the sialic acid base strain was further modified by introducing a beta-1,3-GlcNAc transferase from Neisseria meningitidis (NmIgtA), a beta-1,4-galactosyltransferase from Neisseria meningitidis (NmIgtB), a CMP-sialic acid synthetase and an alpha-2,3-sialyltransferase or an alpha-2,6-sialyltransferase for production of LSTc or LSTd, respectively. Alternatively, sialic acid can be fed to an optimized lacto-N-neotetraose producing strain with expression of a beta-1,3-GlcNAc transferase from Neisseria meningitidis (NmIgtA) and a beta-1,4-galactosyltransferase from Neisseria meningitidis (NmIgtB) (as described and demonstrated in Example 8 of WO18122225), and additional expression of a CMP-sialic acid synthetase and an alpha-2,3-sialyltransferase or an alpha-2,6-sialyltransferase to allow LSTc or LSTd production, respectively.
[0555] All these genes were constitutively expressed with promoters originating from the promoter library described by De Mey et al. (BMC Biotechnology, 2007) or by Mutalik et al. (Nat. Methods 2013, No. 10, 354-360). UTRs originated from Mutalik et al. (Nat. Methods 2013, No. 10, 354-360) and terminators originated from Dunn et al. (Nucleic Acids Res. 1980, 8(10), 2119-32) and Orosz et al. (Eur. J. Biochem. 1991, 201, 653-59). These genetic modifications are also described in WO18122225.
[0556] For all of the above-mentioned strains, daughter strains could further be made by adding an additional production plasmid expressing a CMP-sialic acid synthetase and an alpha-2,6- or alpha-2,3-sialyltransferase.
[0557] All membrane protein genes were evaluated in these mutant strains derived from E. coli K12 MG1655. Membrane protein genes were evaluated by either genomic or plasmid-based expression.
[0558] All strains are stored in cryovials at −80° C. (overnight LB culture mixed in a 1:1 ratio with 70% glycerol).
Cultivation Conditions
[0559] A preculture of 96-well microtiter plate experiments was started from a cryovial, in 150 μL 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 μL MMsf 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 72 h, 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 the whole broth concentration (by boiling the culture broth for 15 min at 60° C. before spinning down the cells (=intra- and extracellular sugar concentrations together)).
[0560] Also, a dilution of the cultures was made to measure the optical density at 600 nm. The cell performance index or CPI is determined by dividing the sialylated 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 ⅓.sup.rd of the optical density measured at 600 nm. The sialylated oligosaccharide export ratio was determined by dividing the sialylated oligosaccharide concentrations measured in the supernatant by the sialylated oligosaccharide concentrations measured in the whole broth, in relative percentages compared to the reference strain.
[0561] A preculture for the bioreactor was started from an entire 1 mL cryovial of a certain strain, inoculated in 250 mL or 500 mL of MMsf 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 H.sub.2SO.sub.4 and 20% NH.sub.4OH. The exhaust gas was cooled. 10% solution of silicone antifoaming agent was added when foaming raised during the fermentation.
Material and Methods Bacillus subtilis
Media
[0562] Two different media are used, namely a rich Luria Broth (LB) and a minimal medium for shake flask (MMsf). The minimal medium uses a trace element mix.
[0563] Trace element mix consisted of 0.735 g/L CaCl2.Math.2H2O, 0.1 g/L MnC12.Math.2H2O, 0.033 g/L CuC12.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).
[0564] 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). Luria Broth agar (LBA) plates consisted of the LB media with 12 g/L agar (Difco, Erembodegem, Belgium) added.
[0565] The minimal medium for the shake flasks (MMfs) experiments contained 2.00 g/L (NH.sub.4)2SO.sub.4, 7.5 g/L KH.sub.2PO.sub.4, 17.5 g/L K.sub.2HPO.sub.4, 1.25 g/L Na-citrate, 0.25 g/L MgSO.sub.4.7H.sub.2O, 0.05 g/L tryptophan, from 10 up to 30 g/L glucose or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose when specified in the examples, 10 ml/L trace element mix and 10 ml/L Fe-citrate solution.
[0566] The medium was set to a pH of 7 with 1M KOH. Depending on the experiment lactose, LNB or LacNAc could be added as a precursor.
[0567] Complex medium, e.g. LB, was sterilized by autoclaving (121° C., 21′) and minimal medium by filtration (0.22 μm Sartorius). When necessary, the medium was made selective by adding an antibiotic (e.g. zeocin (20 mg/L)).
Strains
[0568] Bacillus subtilis 168, available at Bacillus Genetic Stock Center (Ohio, USA).
Plasmids for Gene Disruptions and Genomic Integrations
[0569] 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.
[0570] 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.
Heterologous and Homologous Expression
[0571] Genes that needed to be expressed, including the different exporters with SEQ ID NOs 01 to 21, 37 to 93 and 99 to 122, 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.
[0572] 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
[0573] A preculture of 96-well microtiter plate experiments was started from a cryovial or a single colony from an LB plate, in 150 μL 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 μL MMsf 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 72 h, 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).
[0574] 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 sialylated 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 ⅓.sup.rd of the optical density measured at 600 nm. The sialylated oligosaccharide export ratio was determined by dividing the sialylated oligosaccharide concentrations measured in the supernatant by the sialylated oligosaccharide concentrations measured in the whole broth, in relative percentages compared to the reference strain.
Material and Methods Saccharomyces cerevisiae
Media
[0575] Strains are grown on Synthetic Defined yeast medium with Complete Supplement Mixture (SD CSM) or on SD CSM drop-out medium containing 6.7 g/L Yeast Nitrogen Base without amino acids (YNB w/o AA, Difco), 20 g/L agar (Difco) (for solid cultures), 22 g/L glucose monohydrate or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose when specified in the examples and 0.79 g/L CSM or 0.77 g/L CSM drop-out mixture (MP Biomedicals). Depending on the experiment lactose, LNB or LacNAc could be added as a precursor.
Strains
[0576] Saccharomyces cerevisiae BY4742 created by Brachmann et al. (Yeast (1998) 14:115-32) was used available in the Euroscarf culture collection. All mutant strains were created by homologous recombination or plasmid transformation using the method of Gietz (Yeast 11:355-360, 1995). Kluyveromyces marxianus lactis is available at the LMG culture collection (Ghent, Belgium).
Plasmids and Gene Overexpression
[0577] Yeast expression plasmid p2a_2μ_exporter (Chan 2013 (Plasmid 70 (2013) 2-17)) was used for expression of foreign genes in Saccharomyces cerevisiae. This plasmid contains an ampicillin resistance gene and a bacterial origin of replication to allow for selection and maintenance in E. coli. The plasmid further contains the 2μ yeast ori and the URA3 selection marker for selection and maintenance in yeast. Finally, the plasmid can contain a beta-galactosidase expression cassette. All different exporters with SEQ ID NOs 01 to 21, 37 to 93 and 99 to 122 were cloned in the p2a_2μ_exporter plasmid. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation. All exporters are overexpressed using synthetic, constitutive promoters as described in Blazeck et al., 2012 (Biotechnology and Bioengineering, Vol. 109, No. 11) and Decoene et al., 2019 (PLoS ONE, 14(11)).
[0578] Plasmids were maintained in the host E. coli DH5alpha (F−, phi80dlacZdeltaM15, delta(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk−, mk+), phoA, supE44, lambda-, thi-1, gyrA96, relA1) bought from Invitrogen.
Gene Disruptions and Genomic Integrations For the construction of strains with gene knock-outs and for the introduction of genes in the yeast genome, knock-out and knock-in cassettes were PCR-amplified from template plasmids and transformed as linear DNA by the transformation technique of Gietz and Woods (2002). Template plasmids for knock-outs exist of a yeast auxotrophic marker (e.g. HISS, LEU2) flanked by 500 bp homologies of the target gene and are made in a pJET backbone. After integration, markers can be removed by the Cre/LoxP recombination system. Template plasmids for genomic knock-ins contain the different transcription units flanked by 500 bp homologies of the knock-in target site and are made in a pJET backbone. All genes are expressed using synthetic, constitutive promoters as described in Blazeck et al., 2012 (Biotechnology and Bioengineering, Vol. 109, No. 11) and Decoene et al., 2019 (PLoS ONE, 14(11)).
[0579] Plasmids were maintained in the host E. coli DH5alpha (F−, phi80dlacZdeltaM15, delta(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk−, mk+), phoA, supE44, lambda-, thi-1, gyrA96, relA1) bought from Invitrogen.
Heterologous and Homologous Expression
[0580] 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.
[0581] 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
[0582] A preculture of 96-well microtiter plate experiments was started from a cryovial or a single colony from a (selective) SD CSM plate, in 150 μL (selective SD CSM) and was incubated for 24 h at 30° C. on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96-well square microtiter plate, with 400 μL MMsf medium by diluting 150×. 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 30° C. on an orbital shaker at 800 rpm for 72 h, 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).
[0583] 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 sialylated 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 ⅓.sup.rd of the optical density measured at 600 nm. The sialylated oligosaccharide export ratio was determined by dividing the sialylated oligosaccharide concentrations measured in the supernatant by the sialylated oligosaccharide concentrations measured in the whole broth, in relative percentages compared to the reference strain.
Analytical Methods
Optical Density
[0584] 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).
Productivity
[0585] The specific productivity Qp is the specific production rate of the sialylated oligosaccharide product, typically expressed in mass units of product per mass unit of biomass per time unit (=g sialylated oligosaccharide /g biomass /h). The Qp value has been determined for each phase of the fermentation runs, i.e. Batch and Fed-Batch phase, by measuring both the amount of product and biomass formed at the end of each phase and the time frame each phase lasted.
[0586] The specific productivity Qs is the specific consumption rate of the substrate, e.g. sucrose, typically expressed in mass units of substrate per mass unit of biomass per time unit (=g sucrose /g biomass /h). The Qs value has been determined for each phase of the fermentation runs, i.e. Batch and Fed-Batch phase, by measuring both the total amount of sucrose consumed and biomass formed at the end of each phase and the time frame each phase lasted.
[0587] The yield on sucrose Ys is the fraction of product that is made from substrate and is typically expressed in mass unit of product per mass unit of substrate (=g sialylated oligosaccharide /g sucrose). The Ys has been determined for each phase of the fermentation runs, i.e. Batch and Fed-Batch phase, by measuring both the total amount of sialylated oligosaccharide produced and total amount of sucrose consumed at the end of each phase.
[0588] The yield on biomass Yx is the fraction of biomass that is made from substrate and is typically expressed in mass unit of biomass per mass unit of substrate (=g biomass /g sucrose). The Yp has been determined for each phase of the fermentation runs, i.e. Batch and Fed-Batch phase, by measuring both the total amount of biomass produced and total amount of sucrose consumed at the end of each phase.
[0589] The rate is the speed by which the product is made in a fermentation run, typically expressed in concentration of product made per time unit (=g sialylated oligosaccharide /L/ h). The rate is determined by measuring the concentration of sialylated oligosaccharide that has been made at the end of the Fed-Batch phase and dividing this concentration by the total fermentation time.
[0590] The lactose conversion rate is the speed by which lactose is consumed in a fermentation run, typically expressed in mass units of lactose per time unit (=g lactose consumed /h). The lactose conversion rate is determined by measurement of the total lactose that is consumed during a fermentation run, divided by the total fermentation time. Similar conversion rates can be calculated for other precursors such as Lacto-N-biose, N-acetyllactosamine, Lacto-N-tetraose, or Lacto-N-neotetraose.
Growth Rate/Speed Measurement
[0591] The maximal growth rate (μMax) was calculated based on the observed optical densities at 600 nm using the R package grofit.
Liquid Chromatography
[0592] Standards for 6′-sialyllactose, 3′-sialyllactose, LNT and LNnT were synthetized in house. Other standards such as but not limited to lactose, sucrose, glucose, glycerol, fructose were purchased from Sigma, and LST, LacNAc and LNB were purchased from Carbosynth. Carbohydrates were analyzed via an UPLC-RI (Waters, USA) method, whereby RI (Refractive Index) detects the change in the refraction index of a mobile phase when containing a sample. The sugars were separated in an isocratic flow using an Acquity BEH Amide column (Waters, USA) and a mobile phase containing 70% acetonitrile, 26% ammonium acetate buffer and 4% methanol. The column size was 2.1×100 mm with 1.7 μm particle size. The temperature of the column was set at 25° C. and the pump flow rate was 0.13 mL/min.
Normalization of the Data
[0593] For all types of cultivation conditions, data obtained from the mutant strains was normalized against data obtained in identical cultivation conditions with reference strains having an identical genetic background as the mutant strains but lacking the membrane protein expression cassettes. The dashed horizontal line on each plot that is shown in the examples indicates the setpoint to which all adaptations were normalized. All data is given in relative percentages to that setpoint.
Example 2: Membrane Proteins Identified that Enhance 6′-Sialyllactose (6′-SL) Production in an E. coli Host Cultivated 72 h in a Growth Experiment in Minimal Media Supplemented with 20 g/L Lactose
[0594] An experiment was set up to evaluate membrane proteins for their ability to enhance 6′-sialyllactose production of a host cell growing in minimal media supplemented with 20 g/L lactose. The membrane proteins with SEQ ID NOs 02, 03, 04, 06, 07, 09, 10, 11, 14, 15, 16, or 18 in TU 01, SEQ ID NO 10 in TU 03 or SEQ ID NOs 20 and 21 in their native transcriptional operon structure showed that they are able to enhance 6′-SL production that is being produced in a 6′-SL production host expressing a sialyllactose pathway with α-2,6-sialyltransferase ST1. Candidate genes were presented to the 6′-SL production hosts on a pSC101 plasmid. A growth experiment was performed according to the cultivation conditions provided in Example 1.
Example 3: Membrane Proteins Identified that Enhance 6′-SL Secretion in an E. coli Host Cultivated 72 h in a Growth Experiment in Minimal Media Supplemented with 20 g/L Lactose
[0595] An experiment was set up to evaluate membrane proteins for their ability to enhance 6′-sialyllactose secretion of a host cell growing in minimal media supplemented with 20 g/L lactose. The membrane proteins with SEQ ID NOs 02, 03, 04, 06, 07, 09, 10, 11, 12, 13, 14, 15, 16, 18 or 19 in TU 01, SEQ ID NO 19 in TU 02, SEQ ID NO 10 in TU 03 or SEQ ID 20 and 21 in their native transcriptional operon structure showed that they are able to enhance secretion of 6′-SL that is being produced intracellularly in a 6′-SL bacterial production host expressing a sialyllactose pathway with α-2,6-sialyltransferase ST1. Candidate genes were presented to the 6′-SL production hosts on a pSC101 plasmid. A growth experiment was performed according to the cultivation conditions provided in Example 1.
Example 4: Membrane Proteins Identified that Enhance Growth Speed in an E. coli Host Cultivated 72 h in a Growth Experiment in Minimal Media Supplemented with 20 g/L Lactose
[0596] An experiment was set up to evaluate membrane proteins for their ability to influence growth speed of a host cell growing in minimal media supplemented with 20 g/L lactose. Membrane proteins with SEQ ID NOs 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 15, 16, 17, or 18 in TU 01, SEQ ID NO 19 in TU 02 or SEQ ID NOs 20 and 21 in their native transcriptional operon structure showed to be able to enhance the growth speed of a 6′-SL production host expressing a sialyllactose pathway with α-2,6-sialyl transferase ST1 (SEQ ID NO 32). Candidate genes were presented to the 6′-SL production hosts on a pSC101 plasmid. A growth experiment was performed according to the cultivation conditions provided in Example 1.
Example 5: Membrane Proteins Identified that, when Integrated in the Host's Genome, Increase 6′-SL Secretion in an E. coli Host Cultivated 72 h in a Growth Experiment in Minimal Media Supplemented with 20 g/L Lactose
[0597] Another series of experiments was set up to evaluate the ability of membrane proteins integrated in the genome to increase 6′-sialyllactose secretion by a host cell cultivated for 72 h in minimal media supplemented with 20 g/L lactose. The membrane proteins with SEQ ID NOs 02, 04, 07, 09, 11, 16 or 18 in TU 01 or SEQ ID NOs 20 and 21 in their native transcriptional operon structure showed that they are able to enhance secretion of 6′-SL that is being produced intracellularly in a 6′-SL production host expressing a sialyllactose pathway with α-2,6-sialyltransferase ST1 (SEQ ID NO 32). The genes were presented to the genome of the 6′-SL production hosts as genomic knock-in. A growth experiment was performed according to the cultivation conditions provided in Example 1.
Example 6: The Membrane Protein EcEntS (SEQ ID NO 09), when Varied in Gene Expression Levels and Integrated in the Host's Genome, can Further Enhance the Production and/or Secretion of 6′-SL in an E. coli Host Cultivated 72 h in a Growth Experiment in Minimal Media Supplemented with 20 g/L Lactose
[0598] Another experiment was set up to evaluate the ability of the membrane protein with SEQ ID NO 09 when varied in gene expression and integrated in the genome, to enhance 6′-sialyllactose production and/or secretion of a host cell cultivated for 72 h in minimal media supplemented with 20 g/L lactose. The membrane protein with SEQ ID NO 09 was combined in transcription units TU 04, TU 05, TU 06, TU 07, TU 08, TU 09, TU 10, TU 11 or TU 12 and presented to the genome of the 6′-SL production hosts as genomic knock-in. These different transcriptional units with SEQ ID NO 09 showed that they were able to enhance 6′-SL production and/or secretion of 6′-SL that is being produced intracellularly in a 6′-SL production host expressing a sialyllactose pathway with α-2,6-sialyltransferase ST1 (SEQ ID NO 32). A growth experiment was performed according to the cultivation conditions provided in Example 1.
Example 7: The Membrane Protein EcEntS (SEQ ID NO 09), when Expressed on Plasmid, Enhances the Export Ratio of 6′-SL in an E. coli Host in 5 L Fermentation Runs
[0599] A 6′-SL producing E. coli host having the membrane protein gene with SEQ ID NO 09 expressed in TU 01 on a pSC101 plasmid and expressing the α-2,6-sialyltransferase ST1 (SEQ ID NO 32) from genome, or expressing the α-2,6-sialyltransferase ST1 (SEQ ID NO 32) from genome and plasmid, was evaluated for its productivity in bioreactor settings. For Ferm 03, an additional CMP-sialic acid synthetase and α-2,6-sialyltransferase ST1 were expressed from a p15A plasmid. Four fermentation runs were performed according to the conditions provided in Example 1. Also, a reference strain identical to the 6′-SL production host but lacking the membrane protein gene was analyzed in identical fermentation settings.
Example 8: Additional Expression of a Membrane Protein Enhances the Production and/or Secretion of 3′-SL in an E. coli Host Cultivated 72 h in a Growth Experiment in Minimal Media Supplemented with 20 g/L Lactose
[0600] A 3′-SL producing E. coli as described in Example 1 wherein membrane proteins with SEQ ID NOs 01 up to 21 are expressed from plasmid or from the genome was cultivated for 72 h in minimal media supplemented with 20 g/L lactose. Candidate genes were combined in transcriptional unit TU 01, TU 02, TU 03 or their native transcriptional operon structure for SEQ ID NOs 20 and 21. A growth experiment was performed according to the cultivation conditions provided in Example 1. Said membrane proteins showed that they are able to enhance the production and/or secretion of 3′-SL that is being produced in a 3′-SL production host expressing a sialyllactose pathway with α-2,3-sialyl transferase ST2 (SEQ ID NO 33).
Example 9: Additional Expression of a Membrane Protein Enhances the Production and/or Secretion of Sialylated LNB (sLNB) in an E. coli Host Cultivated 72 h in a Growth Experiment in Minimal Media Supplemented with 20 g/L LNB
[0601] An sLNB producing E. coli as described in Example 1 wherein membrane proteins with SEQ ID NOs 01 up to 21 are expressed from plasmid or from the genome was cultivated for 72 h in minimal media supplemented with 20 g/L LNB. Candidate genes were combined in transcriptional unit TU 01, TU 02, TU 03 or their native transcriptional operon structure for SEQ ID NOs 20 and 21. A growth experiment was performed according to the cultivation conditions provided in Example 1. Said membrane proteins showed that they are able to enhance the production and/or secretion of sLNB that is being produced in an sLNB production host expressing a sialyllactose pathway with an α-2,6-sialyl transferase ST1 in the case of 6′-sLNB or an α-2,3-sialyl transferase ST2 (SEQ ID NO 33) in the case of 3′-sLNB.
Example 10: Additional Expression of a Membrane Protein Enhances the Production and/or Secretion of Sialylated LacNAc (sLacNAc) in an E. coli Host Cultivated 72 h in a Growth Experiment in Minimal Media Supplemented with 20 g/L LacNAc
[0602] An sLacNAc producing E. coli as described in Example 1 wherein membrane proteins with SEQ ID NOs 01 up to 21 are expressed from plasmid or from the genome was cultivated for 72 h in minimal media supplemented with 20 g/L LacNAc. Candidate genes were combined in transcriptional unit TU 01, TU 02, TU 03 or their native transcriptional operon structure for SEQ ID NOs 20 and 21. A growth experiment was performed according to the cultivation conditions provided in Example 1. Said membrane proteins showed that they are able to enhance the production and/or secretion of sLacNAc that is being produced in an sLacNAc production host expressing a sialyllactose pathway with an α-2,6-sialyl transferase ST1 in the case of 6′-sLacNAc or an α-2,3-sialyl transferase ST2 (SEQ ID NO 33) in the case of 3′-sLacNAc.
Example 11: Additional Expression of a Membrane Protein Enhances the Production and/or Secretion of LSTa in an E. coli Host Cultivated 72 h in a Growth Experiment in Minimal Media
[0603] An LSTa producing E. coli as described in Example 1 wherein membrane proteins with SEQ ID NOs 01 up to 21 are expressed from plasmid or from the genome was cultivated for 72 h in minimal media supplemented with 20 g/L lactose. Candidate genes were combined in transcriptional unit TU 01, TU 02, TU 03 or their native transcriptional operon structure for SEQ ID NOs 20 and 21. A growth experiment was performed according to the cultivation conditions provided in Example 1. Said membrane proteins showed that they are able to enhance the production and/or secretion of LSTa that is being produced in an LSTa production host expressing an LNT pathway and a sialic acid pathway with an α-2,3-sialyl transferase ST2 (SEQ ID NO 33).
Example 12: Additional Expression of a Membrane Protein Enhances the Production and/or Secretion of LSTb in an E. coli Host Cultivated 72 h in a Growth Experiment in Minimal Media
[0604] An LSTb producing E. coli as described in Example 1 wherein membrane proteins with SEQ ID NOs 01 up to 21 are expressed from plasmid or from the genome was cultivated for 72 h in minimal media supplemented with 20 g/L lactose. Candidate genes were combined in transcriptional unit TU 01, TU 02, TU 03 or their native transcriptional operon structure for SEQ ID NOs 20 and 21. A growth experiment was performed according to the cultivation conditions provided in Example 1. Said membrane proteins showed that they are able to enhance the production and/or secretion of LSTb that is being produced in an LSTb production host expressing an LNT pathway and a sialic acid pathway with an α-2,6-sialyl transferase like ST6Gall or ST6GalII.
Example 13: Additional Expression of a Membrane Protein Enhances the Production and/or Secretion of LSTc in an E. coli Host Cultivated 72 h in a Growth Experiment in Minimal Media
[0605] An LSTc producing E. coli as described in Example 1 wherein membrane proteins with SEQ ID NOs 01 up to 21 are expressed from plasmid or from the genome was cultivated for 72 h in minimal media supplemented with 20 g/L lactose. Candidate genes were combined in transcriptional unit TU 01, TU 02, TU 03 or their native transcriptional operon structure for SEQ ID NOs 20 and 21. A growth experiment was performed according to the cultivation conditions provided in Example 1. Said membrane proteins showed that they are able to enhance the production and/or secretion of LSTc that is being produced in an LSTc production host expressing an LNnT pathway and a sialic acid pathway with an α-2,6-sialyl transferase ST1 (SEQ ID NO 32).
Example 14: Additional Expression of a Membrane Protein Enhances the Production and/or Secretion of LSTd in an E. coli Host Cultivated 72 h in a Growth Experiment in Minimal Media
[0606] An LSTd producing E. coli as described in Example 1 wherein membrane proteins with SEQ ID NOs 01 up to 21 are expressed from plasmid or from the genome was cultivated for 72 h in minimal media supplemented with 20 g/L lactose. Candidate genes were combined in transcriptional unit TU 01, TU 02, TU 03 or their native transcriptional operon structure for SEQ ID NOs 20 and 21. A growth experiment was performed according to the cultivation conditions provided in Example 1. Said membrane proteins showed that they are able to enhance the production and/or secretion of LSTd that is being produced in an LSTd production host expressing an LNnT pathway and a sialic acid pathway with an α-2,3-sialyl transferase ST2 (SEQ ID NO 33).
Example 15: Additional Expression of a Membrane Protein Enhances the Production and/or Secretion of 6′-SL or 3′-SL in a Bacillus subtilis Host
[0607] In another embodiment, these membrane proteins can be used to increase the production and/or secretion of 6′-SL or 3′-SL in another bacterial host like Bacillus subtilis. As described in WO1822225, a sialic acid producing B. subtilis strain is obtained by overexpressing the native fructose-6-P-aminotransferase (BsgImS) to enhance the intracellular glucosamine-6-phosphate pool. Further on, the enzymatic activities of the genes nagA, nagB and gamA were disrupted by genetic knockouts and a glucosamine-6-P-aminotransferase from S. cerevisiae (ScGNA1), an N-acetylglucosamine-2-epimerase from Bacteroides ovatus (BoAGE) and a sialic acid synthase from Campylobacter jejuni (CjneuB) were overexpressed on the genome. In addition, a lactose permease from E. coli (EclacY) was integrated in the genome to establish lactose uptake.
[0608] To allow production of 6′-SL, a CMP-sialic acid synthetase from Neisseria meningitidis (NmneuA, SEQ ID NO 31) and a sialyltransferase from Photobacterium damselae (PdbST, SEQ ID NO 32) were overexpressed.
[0609] To allow production of 3′-SL, a CMP-sialic acid synthetase from Neisseria meningitidis (NmneuA, SEQ ID NO 31) and a sialyltransferase from Neisseria meningitidis (NmST, SEQ ID NO 33) were overexpressed.
[0610] In these 6′-SL or 3′-SL producing B. subtilis host strains, membrane proteins with SEQ ID NOs 01 up to 21 are expressed from plasmid or from the genome and cultivated for 72 h in minimal media supplemented with 20 g/L lactose. Candidate genes were combined in transcriptional unit TU 01, TU 02, TU 03 or their native transcriptional operon structure for SEQ ID 20 and 21. A growth experiment was performed according to the cultivation conditions for B. subtilis as provided in Example 1. Said membrane proteins showed that they are able to enhance the production and/or secretion of 6′-SL that is being produced in a 6′-SL production B. subtilis host expressing a sialyllactose pathway with α-2,6-sialyl transferase ST1 or 3′-SL that is being produced in a 3′-SL production B. subtilis host expressing a sialyllactose pathway with α-2,3-sialyl transferase ST2 (SEQ ID NO 33).
[0611] In another embodiment, these membrane proteins could be used to increase the production and/or secretion of other sialylated oligosaccharides like but not limited to sLNB, sLacNAc, LSTa, LSTb, LSTc and LSTd in a Bacillus subtilis host strain.
Example 16: Additional Expression of a Membrane Protein Enhances the Production and/or Secretion of 6′-SL or 3′-SL in Saccharomyces cerevisiae
[0612] In another embodiment, these membrane proteins can be used to increase the production and/or secretion of 6′-SL or 3′-SL in a eukaryotic organism like Saccharomyces cerevisiae. A strain with increased flux towards N-acetylglucosamine-6-phosphate was made by overexpressing a fructose-6-P-aminotransferase mutant from E. coli (EcgImS*54, as described by Deng et al. (Biochimie 88, 419-29 (2006)), an N-acetylglucosamine-2-epimerase from Bacteroides ovatus (BoAGE) and a sialic acid synthase from Campylobacter jejuni (CjneuB). Also, a lactose permease from Kluyveromyces lactis (KILAC12, SEQ ID NO 23) was expressed to establish lactose import.
[0613] To allow production of 6′-SL, a CMP-sialic acid synthetase from Neisseria meningitidis (NmneuA) and a sialyltransferase from Photobacterium damselae (PdbST, SEQ ID NO 32) were overexpressed. To allow production of 3′-SL, a CMP-sialic acid synthetase from Neisseria meningitidis (NmneuA, SEQ ID NO 31) and a sialyltransferase from Neisseria meningitidis (NmST, SEQ ID NO 33) were overexpressed. The different gene modules were integrated in the yeast genome by homologous recombination; EcgImS*54 and BoAGE were introduced at the LEU2 locus, KILAC12 and CjneuB were introduced at the HIS3 locus, and NmneuA and PdbST or NmneuA and NmST were introduced at the LYS2 locus. All genes are expressed by synthetic, constitutive yeast promoters (Blazeck et al., 2012 (Biotechnology and Bioengineering, Vol. 109, No. 11) and Decoene et al., 2019 (PLoS ONE, 14(11))) as described in Example 1 and are introduced by the transformation technique of Gietz and Woods (2002).
[0614] In this 6′-SL or 3′-SL producing S. cerevisiae host strain, membrane proteins with SEQ ID NOs 01 up to 21 are expressed from a 2-micron plasmid containing a URA3 auxotrophic marker gene or from the genome and cultivated for 72 h in minimal media supplemented with 20 g/L lactose. Candidate genes were expressed using synthetic, constitutive yeast promoters (Blazeck et al., 2012 (Biotechnology and Bioengineering, Vol. 109, No. 11) and Decoene et al., 2019 (PLoS ONE, 14(11))). A growth experiment was performed according to the cultivation conditions for S. cerevisiae as provided in Example 1. Said membrane proteins showed that they are able to enhance the production and/or secretion of 6′-SL that is being produced in a 6′-SL production S. cerevisiae host expressing a sialyllactose pathway with α-2,6-sialyl transferase ST1, or 3′-SL that is being produced in a 3′-SL production S. cerevisiae host expressing a sialyllactose pathway with α-2,3-sialyl transferase ST2 (SEQ ID NO 33).
[0615] In another embodiment, these membrane proteins could be used to increase the production and/or secretion of other sialylated oligosaccharides like but not limited to sLNB, sLacNAc, LSTa, LSTb, LSTc and LSTd in a Saccharomyces cerevisiae host strain.
Example 17: Membrane Proteins Identified that Obtain Ratios for Supernatant Concentration Over Whole Broth Concentration of 6′-SL Higher than 0.65 in an E. coli Host Cultivated 72 h in a Growth Experiment in Minimal Media Supplemented with 20 g/L Lactose
[0616] An experiment was set up to evaluate membrane proteins for their ability to excrete 6′-sialyllactose by a host cell growing in minimal media supplemented with 20 g/L lactose and having a supernatant concentration over whole broth concentration ratio higher than 0.65. The membrane proteins with SEQ ID NOs 02, 03, 04, 06, 07, 09, 10, 11, 12, 13, 14, 15, 16, 18 or 19 in TU 01, SEQ ID NO 19 in TU 02, SEQ ID NO 10 in TU 03 or SEQ ID 20 and 21 in their native transcriptional operon structure showed that they had ratios of supernatant concentration over whole broth concentration of 6′-SL higher than 0.65 produced by a 6′-SL bacterial production host expressing a sialyllactose pathway with α-2,6-sialyltransferase ST1 (SEQ ID NO 32). Candidate genes were presented to the 6′-SL production hosts on a pSC101 plasmid. A growth experiment was performed according to the cultivation conditions provided in Example 1. Table 3 demonstrates the mean and standard deviation of supernatant over whole broth concentration ratios of 6′-SL in these strains and in a reference strain lacking any extra overexpressed membrane protein.
TABLE-US-00003 TABLE 3 SEQ ID NO TU NO mean ratio stdev ratio SEQ ID 02 TU 01 0.69 0.014 SEQ ID 03 TU 01 0.73 0.058 SEQ ID 04 TU 01 0.72 0.021 SEQ ID 06 TU 01 0.74 0.033 SEQ ID 07 TU 01 0.79 NA SEQ ID 09 TU 01 0.86 0.011 SEQ ID 10 TU 01 0.76 0.005 SEQ ID 10 TU 03 0.75 0.004 SEQ ID 11 TU 01 0.85 0.075 SEQ ID 12 TU 01 0.82 NA SEQ ID 13 TU 01 0.72 0.028 SEQ ID 14 TU 01 0.77 0.031 SEQ ID 15 TU 01 0.76 0.032 SEQ ID 16 TU 01 0.79 0.007 SEQ ID 18 TU 01 0.77 0.106 SEQ ID 19 TU 02 0.74 0.014 SEQ ID 19 TU 01 0.84 0.032 SEQ ID 20-21 nat. op. 0.74 0.061 NA - reference NA 0.65 0.022
Example 18: Ratios for Supernatant Concentration Over Whole Broth Concentration of 6′-SL, Produced by an E. coli Expressing Membrane Protein EcEntS (SEQ ID NO 09) on Plasmid and Grown in 5 L Fermentation Runs, are Increased Compared to a Reference Strain Lacking, the Overexpressed Membrane Protein Gene EcEntS and Cultivated in an Identical Fermentation Setting
[0617] A 6′-SL producing E. coli host having the membrane protein gene with SEQ ID NO 09 expressed in TU 01 on a pSC101 plasmid and expressing the α-2,6-sialyltransferase ST1 (SEQ ID NO 32) from genome, or expressing the α-2,6-sialyltransferase ST1 (SEQ ID NO 32) from genome and plasmid, was evaluated for the 6′-SL ratio of supernatant concentration over whole broth concentration during different time points in bioreactor settings. For Ferm 03, an additional CMP-sialic acid synthetase and α-2,6-sialyltransferase ST1 (SEQ ID NO 32) were expressed from a p15A plasmid. Four fermentation runs were performed according to the conditions provided in Example 1. Also, a reference strain identical to the 6′-SL production host but lacking the membrane protein gene was analyzed in identical fermentation settings. Table 4 demonstrates the enhanced ratio of supernatant over whole broth concentration of 6′-SL taken during different time points of 5 L fermentation runs from the strain overexpressing the membrane protein EcEntS with SEQ ID NO 09. From t1 onwards, all ratios are higher than the reference on that specific time point.
TABLE-US-00004 TABLE 4 Timepoint Reference Ferm 01 Ferm 02 Ferm 03 Ferm 04 t1 0.50 0.67 0.60 0.71 0.88 t2 0.67 0.75 1.00 0.90 0.92 t3 0.55 0.86 0.75 0.80 0.88 t4 0.47 0.77 1.00 0.80 0.89 t5 0.52 0.97 1.00 0.88 1.00 t6 0.55 1.00 0.98 0.90 0.97 t7 0.61 0.98 0.96 0.85 0.98 t8 0.56 0.96 1.00 0.91 0.98 t9 0.71 1.05 1.00 0.92 0.96 t10 0.72 1.05 NA 0.97 NA Mean ratio 0.58 0.89 0.92 0.86 0.94 Stdev ratio 0.087 0.173 0.145 0.074 0.048
Example 19: Example Identification of Siderophore Exporters in Neighborhood of Siderophore Biosynthesis Genes Using EFI-GNT
[0618] A first set of membrane proteins were found by identifying the EggNOG4.5.1 ortholog family members of the membrane proteins found in the neighborhood of siderophore biosynthesis genes. Protein identifiers belonging to dihydroxybenzoate-2, 3-dehydrogenase (cd05331), isochorismate pyruvate lyase (IPR019996), L-ornithine N5-monooxygenase (COG3486) and N(6)-hydroxylysine synthase (PF04183) were extracted from UniProtKB/trembl. These identifiers were used as input in https://efi.igb.illinois.edu/efi-gnt/. EFI-GNT allows exploration of the genome neighborhoods. A neighborhood window size of 10 was selected. Neighboring genes were classified based on Eggnog4.5.1 orthology using a stand-alone version of eggnog-mapperv1 (https://github.com/jhcepas/eggnog-mapper/releases). The most frequent observed putative siderophore transporter NOG and bactNOG orthology families are present near siderophore biosynthesis genes are shown in Table 5.
TABLE-US-00005 TABLE 5 Superfamily NOG bactNOG MFS COG0477, 0ZVQG, 0ZPI7, 0ZVXV, 0XNN3, 05E8G, 08HFG, 089VA, 07TNI, 05C0R, COG3182, 0ZW7F, 0XP7I, 0ZVCH, 0XQZX, 07Y9F, 05CSH, 05QRD, 05EDF, 05C6X, 0XNQK, 0ZVYD, COG2271, 0XNNX, 0ZZWT, 08NGX, 05C2C, 07FU4, 07U9Z, 080SS, COG2814, 0ZITE, 0ZVC8, 0XT98, 0XNQ6, 07SFI, 05EYM, 05C57, 08E7F, 07QF7, 0YAQV, 0ZVQA, COG2211, COG3104 05CSP, 07UZE, 07VHC, 08EFJ, 05CT4, 05FCD, 07YDJ, 08MMW, 08TKV ABC COG1132, COG1173, COG0842, COG4615, 05BZ1, 05IBP, 05CK8, 05IUH, 05D6C, COG0577, COG2274, COG4618, COG4172, 08E0J, 08JJ6, 08JJA, 05FDX, 05EGG, 08JN3, COG5265, COG1136, 0XPIZ, COG0444 08N1B, 05IDI, 08ITX RND COG2409, COG0841, COG3696, COG0845, 05EYF, 07R13, 05BZS, 08IJF COG1033 MOP COG0534, 0Y3TF, COG2244 05UQX, 05C3S, 07U3M, 07R73, 07T1S, 07TJ5, 07XCD
Example 20: Example Identification of Siderophore Exporters in Siderophore Biosynthesis Gene Clusters by Antismash
[0619] A second set of membrane proteins were found by identifying the EggNOG4.5.1 ortholog family members of the membrane proteins found in siderophore biosynthesis gene clusters by antiSMASH (https://antismash.secondarymetabolites.org/#!/download). AntiSMASH allows the rapid genome-wide identification, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genomes. Complete representative fungal and bacterial genome assemblies from ncbi (https://www.ncbi.nlm.nih.gov/assembly) were used as input in the stand-alone antiSMASH5.0 version. Siderophore biosynthesis clusters were annotated using a stand-alone version of eggnog-mapperv1 (https://github.com/jhcepas/eggnog-mapper/releases). The most frequent observed putative siderophore transporter NOG and bactNOG orthology families are present near siderophore biosynthesis genes are shown in Table 6.
TABLE-US-00006 TABLE 6 Superfamily NOG bactNOG MFS COG0477, 0ZW7F, 0ZVCH, 0ZPI7, 0XNN3, 05QRD, 07Y9F, 05C6X, 089VA, 05C0R, 0XNNX, 1269U, 0XP7I, 0ZW8Z 05C2C, 05CSH, 05E8G, 07SFI, 07VHC, 07XMP, 08E7F ABC COG1132, COG0842, 0XPIZ, COG4779, 05BZ1, 05EGG, 05TVJ, 05DHS, 05CM4, COG4606, COG0601, COG1108, COG3182, 07RUJ COG4214, COG4605 RND COG2409 MOP COG0534, 0XPYW 05C3S, 07XCD, 05DJC
Example 21: Membrane Proteins Identified that Enhance 6′-SL Production in an E. coli Host Cultivated 72 h in a Growth Experiment in Minimal Media Supplemented with 20 g/L Lactose
[0620] An experiment was set up to evaluate membrane proteins for their ability to enhance 6′-sialyllactose production of a host cell growing in minimal media supplemented with 20 g/L lactose. In addition, part of the membrane proteins was assembled in different transcription units to evaluate the effect of different expression levels on production. The membrane proteins with SEQ ID NO 19 in TU 02, SEQ ID NOs 66 and 68 in TU08, SEQ ID NOs 19 and 99 in TU 13, SEQ ID NOs 100, 19, 57, 60 and 74 in TU 14, SEQ ID NOs 102, 103, 105, 106, 108, 109, 110, 111, 114, 115, 117, 118, 119 and 121 in TU 15, SEQ ID NO 66 in TU 16, SEQ ID NO 71 in TU 17, SEQ ID NOs 47, 55 and 75 in TU 18, SEQ ID NOs 19 and 68 in TU 21, SEQ ID NO 80 in TU 22, SEQ ID NOs 70, 71, 72, 74 and 80 in TU 25, SEQ ID NOs 75 and 81 in TU 26 and SEQ ID NO 80 in TU 27 showed that they are able to enhance 6′-SL production that is being produced in a 6′-SL production host expressing a sialyllactose pathway with α-2,6-sialyltransferase ST1. Candidate genes were presented to the 6′-SL production hosts on a pSC101 plasmid. A growth experiment was performed according to the cultivation conditions provided in Example 1.
Example 22: Membrane Proteins Identified that Enhance 6′-SL Secretion in an E. coli Host Cultivated 72 h in a Growth Experiment in Minimal Media Supplemented with 20 g/L Lactose
[0621] An experiment was set up to evaluate membrane proteins for their ability to enhance 6′-sialyllactose secretion of a host cell growing in minimal media supplemented with 20 g/L lactose. In addition, part of the membrane proteins was assembled in different transcription units to evaluate the effect of different expression levels on secretion. The membrane proteins with SEQ ID NO 66 in TU 01, SEQ ID NO 19 in TU 02, SEQ ID NOs 19, 66, 67, 68 and 99 in TU 08, SEQ ID NOs 19, 66, 67 and 99 in TU 13, SEQ ID NOs 100, 19, 57, 59 and 74 in TU 14, SEQ ID NOs 102, 103, 104, 105, 106, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 and 122 in TU 15, SEQ ID NOs 19 and 66 in TU 16, SEQ ID NOs 66 and 72 in TU 17, SEQ ID NOs 67, 74 and 75 in TU 18, SEQ ID NOs 19 and 67 in TU 19 and TU 20, SEQ ID NOs 19, 67 and 68 in TU 21, SEQ ID NOs 19, 68, 79 and 80 in TU 22, SEQ ID NO 19 in TU 23, SEQ ID NO 68 in TU 24, SEQ ID NOs 71, 72, 74, 79 and 80 in TU 25, SEQ ID NOs 75, 78 and 81 in TU 26, SEQ ID NOs 72 and 80 in TU 27 and SEQ ID NO 68 in TU 29 showed that they are able to enhance secretion of 6′-SL that is being produced intracellularly in a 6′-SL bacterial production host expressing a sialyllactose pathway with α-2,6-sialyltransferase ST1. Candidate genes were presented to the 6′-SL production hosts on a pSC101 plasmid. The TUs used are enlisted in Table 2. A growth experiment was performed according to the cultivation conditions provided in Example 1.
Example 23: Membrane Proteins Identified that Enhance Growth Speed in an E. coli Host Cultivated 72 h in a Growth Experiment in Minimal Media Supplemented with 20 g/L Lactose
[0622] An experiment was set up to evaluate membrane proteins for their ability to influence growth speed of a host cell growing in minimal media supplemented with 20 g/L lactose. Membrane proteins with SEQ ID NO 66 in TU 01, SEQ ID NO 19 in TU 07, SEQ ID NOs 19, 66, 67 and 99 in TU 08 and TU 13, SEQ ID NOs 100, 19, 48, 57, 59, 60 and 74 in TU 14, SEQ ID NOs 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 and 121 in TU 15, SEQ ID NOs 19 and 66 in TU 16, SEQ ID NOs 66, 71 and 72 in TU 17, SEQ ID NOs 47, 55 and 67 in TU 18, SEQ ID NOs 19 and 67 in TU 19 and TU 20, SEQ ID NOs 19 and 68 in TU 21, SEQ ID NOs 19, 68 and 80 in TU 22, SEQ ID NO 19 in TU 23, SEQ ID NO 68 in TU 24, SEQ ID NOs 71, 72, 74 and 80 in TU 25, SEQ ID NOs 75 and 78 in TU 26, SEQ ID NO 80 in TU 27 and SEQ ID NO 101 in TU 28 showed to be able to enhance the growth speed of a 6′-SL production host expressing a sialyllactose pathway with α-2,6-sialyl transferase ST1 (SEQ ID NO 32). Candidate genes were presented to the 6′-SL production hosts on a pSC101 plasmid. The TUs used are enlisted in Table 2. A growth experiment was performed according to the cultivation conditions provided in Example 1.
Example 24: Additional Expression of a Membrane Protein Enhances the Production and/or Secretion of 3′-SL in an E. coli Host Cultivated 72 h in a Growth Experiment in Minimal Media Supplemented with 20 g/L Lactose
[0623] An experiment was set up to evaluate membrane proteins for their ability to enhance production and/or secretion of 3′-sialyllactose in a host cell growing in minimal media supplemented with 20 g/L lactose. Membrane proteins with SEQ ID NOs 02, 07, 11, 14, 16 and 18 in TU 01 and SEQ ID NOs 20 and 21 in their natural operon structure showed that they are able to enhance 3′-SL production that is being produced in a 3′-SL production host expressing a sialyllactose pathway with α-2,3-sialyl transferase ST2 (SEQ ID NO 33). Membrane proteins with SEQ ID NOs 02, 07, 09, 11, 14, 16 and 18 in TU 01 and SEQ ID NOs 20 and 21 in their natural operon structure showed that they are able to enhance secretion of 3′-SL that is being produced intracellularly in a 3′-SL production host expressing a sialyllactose pathway with α-2,3-sialyl transferase ST2 (SEQ ID NO 33). Candidate genes were presented to the 3′-SL production hosts on a pSC101 plasmid. A growth experiment was performed according to the cultivation conditions provided in Example 1.
Example 25: The Membrane Protein EcEntS (SEQ ID NO 09), when Expressed on Plasmid, Leads to Higher 6′-SL Titers in an E. coli Host in 5 L Fermentation Runs
[0624] A 6′-SL producing E. coli host having the membrane protein gene with SEQ ID NO 09 expressed in TU 01 on a pSC101 plasmid and expressing the α-2,6-sialyl transferase ST1 (SEQ ID NO 32) from genome, or expressing the α-2,6-sialyl transferase ST1 (SEQ ID NO 32) from genome and plasmid, was evaluated for its productivity in bioreactor settings (5 L fermenter). Four fermentation runs were performed according to the conditions provided in Example 1. Also, a reference strain identical to the 6′-SL production host but lacking the membrane protein gene was analyzed in identical fermentation settings. At the end of all fermentation runs, the 6′-SL titers measured in supernatant and whole broth samples varied between 50 g/L and 65 g/L for the strains expressing the membrane protein EcEntS from E. coli (SEQ ID NO 09). The reference strain had 6′-SL titers between 20 g/L and 40 g/L measured in supernatant and whole broth samples, which shows the positive effect of the membrane protein EcEntS (SEQ ID NO 09) on 6′-SL production in 5 L fermentation runs.