PRODUCTION OF 3-FUCOSYLLACTOSE AND LACTOSE CONVERTING ALPHA-1,3-FUCOSYLTRANSFERASE ENZYMES

Abstract

Methods for producing 3-fucosyllactose (3-FL) as well as novel fucosyltransferases, more specifically novel lactose binding alpha-1,3-fucosyltransferase polypeptides, and their applications. Furthermore, methods are provided for producing 3-fucosyllactose (3-FL) using the novel lactose binding alpha-1,3-fucosyltransferases.

Claims

1.-39. (canceled)

40. A method of producing α-1,3-fucosyllactose, the method comprising: contacting a polypeptide with a mixture comprising GDP-fucose as donor substrate, and lactose as acceptor substrate, under conditions wherein the polypeptide catalyzes the transfer of a fucose residue from the donor substrate to the acceptor substrate, wherein the polypeptide has α-1,3-fucosyltransferase activity and is able to use lactose as acceptor substrate, wherein the polypeptide comprises: i) an amino acid sequence comprising a conserved GDP-fucose binding domain [Y/W/L/H/F/M]X[T/S/C][E/Q/D/A][K/R] (SEQ ID NO: 33); ii) an amino acid sequence comprising a conserved [K/D][L/K/M]XXX[F/Y] domain (SEQ ID NO: 34), and iii) if ii) is DM[A/S]VSF (SEQ ID NO: 36), then a conserved motif [N/H]XDPAXLD (SEQ ID NO: 35) is present at the N-terminal region; wherein X can be any distinct amino acid; and wherein the C-terminus of the polypeptide has less than or equal to 100 amino acids starting from the first amino acid of the GDP-fucose binding domain; so as to thereby produce α-1,3-fucosyllactose.

41. The method according to claim 40, wherein the polypeptide is provided in a cell-free system.

42. The method according to claim 40, wherein the polypeptide is produced by a cell comprising a polynucleotide encoding the polypeptide.

43. The method according to claim 42, wherein GDP-fucose and/or lactose is provided by a cell producing the GDP-fucose and/or lactose.

44. The method according to claim 42, wherein the cell is genetically modified to produce α-1,3-fucosyllactose, and wherein the cell comprises at least one polynucleotide encoding an enzyme for α-1,3-fucosyllactose synthesis, wherein the cell has the ability to use lactose as acceptor substrate.

45. The method according to claim 42, wherein a cell is grown, which cell expresses the polypeptide with α-1,3-fucosvitransferase activity and with the ability to use lactose as acceptor substrate, under suitable nutrient conditions permissive for producing α-1,3-fucosyllactose, and also permissive for the expression of the polypeptide; and wherein, simultaneously or subsequently thereto, a donor substrate GDP-fucose and the acceptor substrate lactose is provided, in order for the α-1,3-fucosyltransferase polypeptide to catalyze the transfer of a fucose residue from GDP-fucose to lactose, thereby producing α-1,3-fucosyllactose.

46. The method according to claim 42, wherein the cell is transformed or transfected to express an exogenous polypeptide with α-1,3-fucosyltransferase activity and with the ability to use lactose as an acceptor substrate.

47. The method according to claim 42, wherein the GDP-fucose and/or lactose is provided by an enzyme simultaneously expressed in the cell or by the metabolism of the cell.

48. The method according to claim 40, farther comprising purifying α-1,3-fucosyllactose.

49. The method according to claim 40, wherein the polypeptide is selected from the group consisting of: i) any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 28, 30, or 32; ii) an amino acid sequence having 87% or more sequence identity to the full length amino acid sequence of SEQ ID NOS: 2, 20, or 22; iii) an amino acid sequence having 80% or more sequence identity to the full length amino acid sequence of any one of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 28, 30, or 32; iv) a fragment of an amino acid sequence of SEQ ID NOS: 2, 20, or 22, wherein the fragment comprises at least 45 contiguous amino acids thereof; and v) a fragment of an amino acid sequence of any one of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 28, 30, or 32, wherein the fragment comprises at least 10 contiguous amino acids thereof and has lactose binding alphα-1,3-fucosyltransferase activity; wherein, optionally, the polypeptide is modified by an N-terminal and/or C-terminal amino acid stretch.

50. The method according to claim 40, wherein the method comprises at least one of the following: i) adding to a culture medium a lactose feed comprising at least 150 gram of lactose per initial reactor volume, in a continuous manner, so that the final volume of the culture medium is not more than two-fold of the volume of culture medium before addition of the lactose feed; ii) adding a lactose feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, or 5 days by means of a feeding solution; iii) adding a lactose feed in a continuous manner to the culture medium over the course of 1 day, 2 days, 3 days, 4 days, or 5 days by means of a feeding solution, wherein the concentration of the lactose feeding solution is 600 g/L, wherein the pH of the solution is set between 3 and 7 and wherein the temperature of the feed solution is kept between 20° C. and 80° C.; or iv) wherein the method results in a 3-fucosyllactose concentration of at least 200 g/L in the final volume of the culture medium.

51. A cell comprising at least one polynucleotide encoding a polypeptide with α-1,3-fucosyltransferase activity and able to use lactose as acceptor substrate, wherein the polypeptide comprises: i) an amino acid sequence comprising a conserved GDP-fucose binding domain [Y/W/L/H/F/M]X[T/S/C][E/Q/D/A][K/R] (SEQ ID NO: 33); ii) an amino acid sequence comprising a conserved [K/D][L/K/M]XXX[F/Y] domain (SEQ ID NO: 34), and iii) if the domain of ii) is DM[A/S]VSF (SEQ ID NO: 36), then a conserved motif [N/H]XDPAXLD (SEQ ID NO: 35) is present at the N-terminal region of the polypeptide; wherein X can be any distinct amino acid; and wherein the C-terminus of the polypeptide has less than or equal to 100 amino acids starting from the first amino acid of the GDP-fucose binding domain.

52. The cell of claim 51, whereinthe cell comprises: i) a polynucleotide encoding the polypeptide with lactose binding α-1,3-fucosyltransferase activity, wherein the polynucleotide is foreign to the cell and wherein the polynucleotide is integrated into the cell's genome, or ii) a vector comprising a polynucleotide encoding the polypeptide, wherein the polynucleotide is operably linked to control sequences recognized by a cell transformed with the vector.

53. The cell of claim 51, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: i) any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 28, 30, or 32; ii) an amino acid sequence having 87% or more sequence identity to the full length amino acid sequence of SEQ ID NOS: 2, 20, or 22; iii) an amino acid sequence having 80% or more sequence identity to the full length amino acid sequence of any one of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 28, 30, or 32; iv) a fragment of an amino acid sequence of SEQ ID NOS: 2, 20, or 22, wherein the fragment comprises at least 45 contiguous amino acids thereof; and v) a fragment of an amino acid sequence of any one of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 28, 30 or 32, wherein the fragment comprises at least 10 contiguous amino acids thereof and has lactose binding alphα-1,3-fucosyltransferase activity; and wherein, optionally, the polypeptide is modified by an N-terminal and/or C-terminal amino acid stretch.

54. The method according to claim 42, wherein the cell is selected from the group consisting of a microorganism, plant cell, animal cell, bacterium, fungus, and yeast.

55. The cell of claim 51, wherein the cell is selected from the group consisting of a bacterium, an Escherichia coli strain, an Escherichia coli K12 strain, and Escherichia coli MG1655.

56. The cell of claim 51, wherein the cell is a yeast cell.

57. The cell of claim 51, wherein the polynucleotide is adapted to the codon usage of the respective cell.

58. A method of using the cell of claim 51 to produce α-1,3 fucosyllactose, the method comprising: a) cultivating the cell in a medium under conditions permissive for producing α-1.sub.23-fucosyltransferase, and b) optionally, separating the α-1,3-fucosyltransferase from the cultivation.

59. A microorganism that heterologously expresses a lactose binding α-1,3-fucosyltransferase polypeptide, wherein the polypeptide comprises: i) an amino acid sequence comprising a conserved GDP-fucose binding domain [Y/W/L/H/F/M]X[T/S/C][E/Q/D/A][K/R] (SEQ ID NO: 33); ii) an amino acid sequence comprising a conserved [K/D][L/K/M]XXX[F/Y] domain (SEQ ID NO: 34), and iii) if ii) is DM[A/S]VSF (SEQ ID NO: 36), then a conserved motif [N/H]XDPAXLD (SEQ ID NO: 35) is present at the N-terminal region; wherein X can be any distinct amino acid; and wherein the C-terminus of the polypeptide has less than or equal to 100 amino acids starting from the first amino acid of the GDP-fucose binding domain.

60. The microorganism of claim 59, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: i) any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 28, 30, or 32; ii) an amino acid sequence having 87% or more sequence identity to the full length amino acid sequence of SEQ ID NOS: 2, 20, or 22; iii) an amino acid sequence having 80% or more sequence identity to the full length amino acid sequence of any one of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 28, 30, or 32; iv) a fragment of an amino acid sequence of SEQ ID NOS: 2, 20, or 22, wherein the fragment comprises at least 45 contiguous amino acids thereof; v) a fragment of an amino acid sequence of any one of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 28, 30, or 32, wherein the fragment comprises at least 10 contiguous amino acids thereof and has lactose binding alphα-1,3-fucosyltransferase activity; and wherein, optionally, the polypeptide is modified by an N-terminal and/or C-terminal amino acid stretch.

61. A method of producing α-1,3-fucosyllactose, the method comprising: utilizing the microorganism of claim 59 to produce alphα-1,3-fucosyllactose. 62, (New) The method according to claim 40, further comprising: separating the α-1,3-fucosyllactose from a cell or a medium of its growth.

63. The method according to claim 62, wherein the separation comprises at least one of the following: clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated charcoal or carbon treatment, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, or ligand exchange chromatography.

64. The method according to claim 40, further comprising: purifying α-1,3-fucosyllactose.

65. The method according to claim 64, wherein purifying α-1,3-fucosyllactose comprises at least one of the following: use of activated charcoal or carbon, use of charcoal, nanofiltration, ultrafiltration or ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying, or lyophilization.

66. The method according to claim 42, wherein the polypeptide is produced in a cell selected from the group consisting of a fungal cell, yeast, bacterial, insect, animal, or plant expression system 67, (New) The method according to claim 66, wherein the cell is a bacterium, Escherichia coli, an Escherichia coli K12 strain, or Escherichia coli MG1655.

68. The method according to claim 66, wherein the cell is a yeast cell.

69. The method according to claim 40, wherein the lactose concentration in culture medium ranges from 50 to 150 g/L.

70. The method according to claim 40, wherein the final concentration of 3-fucosyllactose ranges between 70 g/L to 200 g/L.

71. The method according to claim 40, wherein the production results in a lactose concentration to 3-fucosyllactose concentration ratio of less than 1:5 at the end of fermentation.

72. The method according to claim 40, wherein the 3-fucosyllactose thus produced has a purity of 80% or more.

73. The method according to claim 40, wherein the catalysis results in a lactose concentration to 3-fucosyllactose concentration ratio of less than 1:5 at the end of fermentation.

74. The method according to claim 40, wherein the catalysis results in a 3-fucosyllactose purity of 80% or more at the end of fermentation.

75. The method according to claim 50, resulting in a 3-fucosyllactose purity of 80% or more in the final volume of the culture.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0266] FIG. 1 shows an alignment of the polypeptide sequences of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16.

[0267] FIG. 2 shows normalized production of 3-fucosyllactose in a growth experiment.

[0268] FIG. 3 shows normalized production of 3-fucosyllactose in a growth experiment with low to high amounts of lactose in the medium.

[0269] FIG. 4 shows normalized production of 3-fucosyllactose in a growth experiment with low amounts of lactose in the medium.

[0270] FIG. 5 shows the percentage of lactose that is converted to 3-FL of one of the identified lactose binding alphα-1,3-fucosyltransferases.

[0271] FIG. 6 shows the percentage of lactose that is converted to 3-FL of different of the identified lactose binding alphα-1,3-fucosyltransferases driven by different promoters.

[0272] FIG. 7 shows the normalized production of 3-fucosyllactose of a further experiment.

[0273] FIG. 8 shows the normalized production of 3-fucosyllactose of a subset of the identified lactose binding alphα-1,3-fucosyltransferases driven by different promoters.

[0274] FIG. 9 shows the normalized production of 3-fucosyllactose of strains expressing H. pylori fucT (SEQ ID NO: 18) from 2 different promoters.

[0275] FIG. 10 shows the normalized production of 3-fucosyllactose of strains expressing polypeptides with the DM[AS]VSF consensus motif

[0276] FIGS. 11A-11C show an alignment of the polypeptide sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 32. The consensus motifs [Y/W/L/H/F/M]X[T/S/C][E/Q/D/A][K/R], [K/D][L/K/M]XXX[F/Y] and [FW]W, wherein X can be any distinct amino acid, are marked with a box.

[0277] FIG. 12 shows an alignment of the polypeptide sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 26. The consensus motifs DM[A/S]VSF and [N/H]XDPAXLD, wherein X can be any distinct amino acid (and unrelated motifs) are marked with a box.

[0278] FIG. 13 shows an alignment of the polypeptide sequences of SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 32. The consensus motifs K[I/V]F[F/L]XGEN (SEQ ID NO: 41) and RFPLW (SEQ ID NO: 42), wherein X can be any distinct amino acid, are marked with a box.

DETAILED DESCRIPTION

EXAMPLES

Example 1

[0279] Materials and Methods Escherichia Coli Media

[0280] The Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). The medium for the shake flasks experiments contained 2.00 g/L NH.sub.4Cl, 5.00 g/L (NH.sub.4)2SO.sub.4, 2.993 g/L KH.sub.2PO.sub.4, 7.315 g/L K.sub.2HPO.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 vitamin solution, 100 μl/L molybdate solution, and 1 mL/L selenium solution. The medium was set to a pH of 7 with 1 M KOH. Vitamin solution consisted of 3.6 g/L FeCl.sub.2.4H.sub.2O, 5 g/L CaCl.sub.2.2H.sub.2O, 1.3 g/L MnCl.sub.2.2H.sub.2O, 0.38 g/L CuCl.sub.2.2H.sub.2O, 0.5 g/L CoCl.sub.2.6H.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.2H.sub.2O and 1.01 g/L thiamine.HCl. The molybdate solution contained 0.967 g/L NaMoO.sub.4.2H.sub.2O. The selenium solution contained 42 g/L SeO.sub.2.

[0281] The minimal medium for fermentations 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.4.7H.sub.2O, 14.26 g/L sucrose, 1 mL/L vitamin solution, 100 μL/L molybdate solution, and 1 mL/L selenium solution with the same composition as described above.

[0282] Complex medium was sterilized by autoclaving (121° C., 21 minutes) and minimal medium by filtration (0.22 μm Sartorius). When necessary, the medium was made selective by adding an antibiotic (e.g., chloramphenicol (20 mg/L), carbenicillin (100 mg/L), spectinomycin (40 mg/L) and/or kanamycin (50 mg/L)).

Plasmids

[0283] 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).

[0284] Plasmids for alphα-1,3-fucosyltransferase expression were constructed in a pMB1 ori vector using Golden Gate assembly. The genes were expressed using promoters apFAB305 (“PROM0012”), apFAB146 (“PROM0032”) (both as described by Mutalik et al. (Nat. Methods 2013, No. 10, 354-360)), and p14 (“PROM0016” in combination with “UTR0019”) (as described by De Mey et al. (BMC Biotechnology 2007)) and UTRs Gene10-LeuAB-BCD2 (“UTR0002”) (as described by Mutalik et al. (Nat. Methods 2013, No. 10, 354-360)).

[0285] Plasmids were maintained in the host E. coli DH5alpha (F.sup.−, phi80dlacZdeltaM15, delta(lacZYA-argF) U169, deoR, recA1, endA1, hsdR17(rk.sup.−, mk.sup.+), phoA, supE44, lambda, thi-1, gyrA96, relA1) bought from Invitrogen.

Strains and Mutations

[0286] Escherichia coli K12 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.

[0287] 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 OD600 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) (600 Ω, 25 μED, and 250 volts).

[0288] After electroporation, cells were added to 1 ml LB media incubated 1 hour 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.

[0289] 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 Dpn1, repurified from an agarose gel, and suspended in elution buffer (5 mM Tris, pH 8.0).

[0290] 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).

[0291] A mutant strain derived from E. coli K12 MG1655 was created by knocking out the genes lacZ, lacY lacA, glgC, agp, pfkA, pflth, pgi, arcA, iclR, wcaf, pgi, ion and thyA. Additionally, the E. coli lacY gene, a fructose kinase gene (frk) originating from Zymomonas mobilis and a sucrose phosphorylase (SP) originating from Bifidobacterium adolescentis were knocked in into the genome and expressed constitutively. The constitutive promoters originate from the promoter library described by De Mey et al. (BMC Biotechnology, 2007). These genetic modifications are also described in WO2016075243 and WO2012007481.

[0292] All constructed plasmids with the hypothetical alphα-1,3-fucosyltransferase genes were evaluated in this mutant strain derived from E. coli K12 MG1655. All strains are stored in cryovials at −80° C. (overnight LB culture mixed in a 1:1 ratio with 70% glycerol). A list of all successful lactose binding alphα-1,3-fucosyltransferases (SEQ ID NOS: 1 to 16, 19 to 22 and 27 to 32) together with a prior art alphα-1,3-fucosyltransferase (SEQ ID NOS: 17-18) and two non-functional alphα-1,3-fucosyltransferases (SEQ ID NOS: 23 to 26) is provided in Table 3.

TABLE-US-00003 TABLE 3 SEQ ID Organism Country origin SEQ ID NOS: 1-2 Azospirillum oryzae A2P Japan SEQ ID NOS: 3-4 Azospirillum lipoferum B510 Japan SEQ ID NOS: 5-6 Basilea psittacipulmonis Switzerland SEQ ID NOS: 7-8 Planctopirus limnophila (strain ATCC 43296/DSM 3776/ Germany IFAM 1008/290) (Planctomyces limnophilus) SEQ ID NOS: 9-10 Pedobacter glucosidilyticus Germany SEQ ID NOS: 11-12 Porphyromonas catoniae (WGS, in genbank: United States JDFF01000001 till JDFF010000025) SEQ ID NOS: 13-14 Porphyromonas sp. COT-239 OH1446 (contig_18; United Kingdom NZ_JRA001000018.1) SEQ ID NOS: 15-16 Selenomonas infelix ATCC 43532 Unknown SEQ ID NOS: 17-18 Helicobacter pylori Australia SEQ ID NOS: 19-20 Azospirillum sp. TSH64 Japan SEQ ID NOS: 21-22 Azospirillum sp. TSH100 Japan SEQ ID NOS: 23-24 Azospirillum brasilense Unknown SEQ ID NOS: 25-26 Azospirillum sp. B510 Japan SEQ ID NOS: 27-28 Butyrivibrio sp. TB Unknown SEQ ID NOS: 29-30 Porphyromonas catoniae F0037 Unknown SEQ ID NOS: 31-32 Butyrivibrio fibrisolvens DSM 3071 Unknown

Heterologous and Homologous Expression

[0293] All potential alphα-1,3-fucosyltransferase genes that needed to be expressed, be it for a plasmid or for the genomic insertion, were synthetically synthetized at Twist Biosciences (San Francisco, USA). 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

[0294] 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×. These final 96-well culture plates were then incubated at 37° C. on an orbital shaker at 800 rpm for 72 hours, or shorter, or longer. At the end of the cultivation experiment samples were taken from each well to measure sugar concentrations in the broth supernatant (extracellular sugar concentrations, after spinning down the cells), or by boiling the culture broth for 15 minutes at 90° C. before spinning down the cells (=whole broth measurements, average of intra- and extracellular sugar concentrations).

[0295] 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 hours 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 conditions 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 H2S04 and 20% NH4OH. The exhaust gas was cooled. 10% solution of silicone antifoaming agent was added when foaming raised during the fermentation.

Optical Density

[0296] Cell density of the cultures was frequently monitored by measuring optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium or with a Spark 10 M microplate reader, Tecan, Switzerland).

Liquid Chromatography

[0297] Standards for 3-fucosyllactose were synthetized in house. Other standards such as but not limited to lactose, sucrose, glucose, fructose were purchased from Sigma.

[0298] Carbohydrates were analyzed via a HPLC-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 X-Bridge column (Waters X-bridge HPLC column, USA) and a mobile phase containing 75 ml acetonitrile and 25 ml Ultrapure water and 0.15 ml triethylamine. The column size was 4.6×150 mm with 3.5 μm particle size. The temperature of the column was set at 35° C. and the pump flow rate was 1 mL/minute.

Example 2

Evaluation of Different Lactose Binding alphα-1,3-Fucosyltransferase Enzymes Incorporated in Escherichia Coli

[0299] An experiment was set up to evaluate a number of genes coding for potential alphα-1,3-fucosyltransferase enzymes that are able to produce 3-fucosyllactose (3-FL) from GDP-fucose and lactose. A growth experiment was performed according to the cultivation conditions provided in Example 1.

[0300] FIG. 2 shows the normalized production of 3-fucosyllactose obtained in a growth experiment of the strains successfully expressing various lactose binding alphα-1,3-fucosyltransferases using two different promoters (PROM0012 and PROM0016) with 20 g/L lactose in the production medium. Each datapoint corresponds to data from one well. The dashed horizontal line indicates the setpoint to which all datapoints were normalized.

[0301] The experiment identified the following polypeptides with lactose binding 3-fucosyltransferase activity: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14 having similar to better lactose binding α-1,3-fucosyltransferase activity compared to a strain containing SEQ ID NO: 18 with previously confirmed lactose binding α-1,3-fucosyltransferase activity. The polypeptide of SEQ ID NO: 4 has 90.8% global sequence identity to SEQ ID NO: 2, herewith showing that also sequences that have 87% or more sequence identity to SEQ ID NO: 2 have lactose binding α-1,3-fucosyltransferase activity.

Example 3

Evaluation of a Lactose Binding alphα-1,3-Fucosyltransferase Enzyme Incorporated in Escherichia Coli for its Ability to Produce 3-FL with Low to High Lactose Concentrations in Minimal Media

[0302] A gene coding for SEQ ID NO: 6 (and combined with PROM0016) is evaluated on its ability to produce 3-FL in minimal media with various concentrations of lactose. A growth experiment was performed according to the cultivation conditions provided in Example 1. Strains with SEQ ID NO: 6 and SEQ ID NO: 18 (driven by PROM0016) were grown in multiple wells of a 96-well plate as described above. SEQ ID NO: 18 has previously confirmed alphα-1,3-fucosyltransferase activity on lactose.

[0303] FIG. 3 shows the normalized production of 3-fucosyllactose with six different concentrations of lactose as a precursor for 3-FL (90 g/L and a 1:2 dilution series thereof, until 2.8 g/L, as indicated in the figure). Each datapoint corresponds to data from one well. The dashed horizontal line indicates the setpoint to which all datapoints were normalized.

[0304] The experiment identified the polypeptide of SEQ ID NO: 6 to have better lactose binding α-1,3-fucosyltransferase activity at all lactose concentrations compared to a strain expressing SEQ ID NO: 18, a polypeptide with previously confirmed lactose binding alphα-1.3-fucosyltransferase activity.

Example 4

Evaluation of Various Lactose Binding alphα-1,3-Fucosyltransferase Enzymes Incorporated in Escherichia Coli for Their Ability to Produce 3-FL at Low Concentrations of Lactose in Minimal Media

[0305] Several of the above identified strains with genes coding for SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 12 and SEQ ID NO: 14 were evaluated on their ability to produce 3-fucosyllactose from GDP-fucose and lactose in a growth experiment at low concentrations of lactose. A growth experiment was performed according to the cultivation conditions provided in Example 1.

[0306] FIG. 4 shows the normalized production of 3-fucosyllactose with strains expressing various alphα-1,3-fucosyltransferases (using two different promoters PROM0012 and PROM0016) and grown in a medium with low amounts of lactose (2.8 g/L lactose). Each datapoint corresponds to data from one well. The dashed horizontal line indicates the setpoint to which all datapoints were normalized.

[0307] The experiment identified the following polypeptides with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 12 and SEQ ID NO: 14 to having similar to better lactose binding alphα-1,3-fucosyltransferase activity when provided with low concentrations of lactose compared to a strain containing SEQ ID NO: 18 with previously confirmed lactose binding alpha-1,3-fucosyltransferase activity.

Example 5

Evaluation of Enzyme Activity of the Polypeptide of SEQ ID NO: 6 Incorporated in Escherichia Coli on Two Low Concentrations of Lactose

[0308] A gene coding for SEQ ID NO: 6 (and combined with PROM0016) was evaluated for its ability to convert lactose into 3-fucosyllactose in a strain producing GDP-fucose in a growth experiment providing 2.8 g/L or 5.62 g/L of lactose and sucrose at 30 g/L. A growth experiment was performed according to the cultivation conditions provided in Example 1.

[0309] FIG. 5 shows the percentage of lactose that is converted to 3-FL, calculated by dividing the measured amount of 3-FL by the amount that could theoretically be obtained based on the input concentration of lactose. Theoretically, if all lactose is converted, a value of 100% is obtained. Each datapoint corresponds to data from one well.

[0310] The strain expressing polypeptide as shown in SEQ ID NO: 6 is compared to a strain expressing the polypeptide as shown in SEQ ID NO: 18 (driven by PROM0016), which is previously confirmed to have alphα-1,3-fucosyltransferase activity on lactose. At both concentrations of lactose the strain expressing the polypeptide as shown in SEQ ID NO: 6 is able to convert much more lactose to 3-FL than the strain expressing the polypeptide as shown in SEQ ID NO: 18 for a given amount of carbon source (30 g/L of sucrose).

Example 6

Evaluation of Enzyme Activity of Various Lactose Binding alphα-1,3-Fucosyltransferase Enzymes Incorporated in Escherichia Coli at Limited Concentrations of Lactose and Sucrose

[0311] Genes coding for the above identified polypeptides SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 12 and SEQ ID NO: 14 were evaluated on their ability to convert lactose into 3-fucosyllactose in a strain producing GDP-fucose in a growth experiment at low concentrations of lactose (2 g/L) and sucrose (7.5 g/L). A growth experiment was performed according to the cultivation conditions provided in Example 1.

[0312] FIG. 6 shows the percentage of lactose that is converted to 3-FL, calculated by dividing the measured amount of 3-FL by the amount that could theoretically be obtained based on the input concentration of lactose. Each datapoint corresponds to data from one well.

[0313] The strains expressing the polypeptides with SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 12 or SEQ ID NO: 14 are able to convert more lactose to 3-FL than the strain expressing the polypeptide with SEQ ID NO: 18 for a given amount of carbon source (7.5 g/L sucrose).

Example 7

Evaluation of Escherichia Coli Strains Expressing Various Lactose Binding alphα-1,3-Fucosyltransferase Enzymes in a Batch Fermentation

[0314] Batch fermentations at bioreactor scale were performed to evaluate strains, derived from the mutant E. coli K12 MG1655 strain background as described in Example 1, expressing various alphα-1,3-fucosyltransferase enzymes with SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 12 and SEQ ID NO: 14. The bioreactor runs were performed as described in Example 1. In these examples, sucrose was used as a carbon source. Lactose was added in the batch medium at 90 g/L as a precursor for 3-FL formation.

[0315] FIG. 7 shows the normalized production of 3-fucosyllactose obtained in batch fermentations with strains successfully expressing various lactose binding alphα-1,3-fucosyltransferases with lactose in the production medium as a precursor. Each datapoint corresponds to data from one fermentation run. The dashed horizontal line indicates the setpoint to which all datapoints were normalized.

[0316] The experiment shows that mutant E. coli strains expressing the lactose binding alphα-1,3-fucosyltransferase genes with SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 12 or SEQ ID NO: 14 produce higher amounts of 3-FL compared to the strain expressing the polypeptide with SEQ ID NO: 18.

Example 8

Evaluation of Different Lactose Binding alphα-1,3-Fucosyltransferase Enzymes Incorporated in Escherichia Coli

[0317] A further experiment was set up with strains expressing the enzymes with SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16 and evaluated whether these are able to produce 3-fucosyllactose from lactose in a strain producing GDP-fucose. A growth experiment was performed according to the cultivation conditions provided in Example 1.

[0318] FIG. 8 shows normalized production of 3-fucosyllactose with strains successfully expressing various lactose binding alphα-1,3-fucosyltransferases (using three different promoters PROM0012, PROM0016 and PROM0026) with 20 g/L lactose in the production medium. Each datapoint corresponds to data from one well. The dashed horizontal line indicates the setpoint to which all datapoints were normalized.

[0319] The experiment confirmed the results from Example 2 for the strains expressing polypeptides with SEQ ID NO: 12, SEQ ID NO: 6, SEQ ID NO: 12 and SEQ ID NO: 14, and identified the polypeptide with SEQ ID NO: 16 to also have better lactose binding alphα-1,3-fucosyltransferase activity compared to a strain containing SEQ ID NO: 18 with previously confirmed lactose binding alphα-1,3-fucosyltransferase activity.

Example 9

Material and Methods Saccharomyces Cerevisiae Media

[0320] Strains are grown on Synthetic Defined yeast medium with Complete Supplement Mixture (SD CSM) or CSM drop-out (SD CSM-Ura) containing 6.7 g/L Yeast Nitrogen Base without amino acids (YNB w/o AA, Difco), 20 g/L agar (Difco) (solid cultures), 22 g/L glucose monohydrate or 20 g/L lactose and 0.79 g/L CSM or 0.77 g/L CSM-Ura (MP Biomedicals).

Strains

[0321] Saccharomyces cerevisiae BY4742 created by Bachmann 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

[0322] Yeast expression plasmid p2a_2 μ_sia_GFA1 (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 2 μ yeast ori and the Ura3 selection marker for selection and maintenance in yeast. Next, this plasmid can be modified to p2_a2 μ_ff to contain a lactose permease (for example, LAC12 from Kluyveromyces lactis), a GDP-mannose 4,6-dehydratase (such as Gmd from E. coli) and a GDP-L-fucose synthase (such as fcl from E. coli).

[0323] Yeast expression plasmids p2a 2 μ_fl_3ft is based on p2a 2.sub.11. ft but modified in a way that also SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18 are expressed. Preferably but not necessarily, the fucosyltransferase proteins are N-terminally fused to a SUMOstar tag (e.g., obtained from pYSUMOstar, Life Sensors, Malvern, Pa.) to enhance the solubility of the fucosyltransferase enzymes.

[0324] Plasmids were maintained in the host E. coli DH5alpha (F.sup.−, phi80diacZdeltaM15, delta(/acZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk.sup.+, mk.sup.+), phoA, supE44, lambda.sup.−, thi-1, gyrA96, rel A2) bought from Invitrogen.

Gene Expression Promoters

[0325] Genes are expressed using synthetic constitutive promoters, as described in by Blazeck (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012).

Heterologous and Homologous Expression

[0326] 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 or IDT.

[0327] 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.

Cultivations Conditions

[0328] In general, yeast strains were initially grown on SD CSM plates to obtain single colonies. These plates were grown for 2-3 days at 30° C.

[0329] Starting from a single colony, a preculture was grown over night in 5 mL at 30° C., shaking at 200 rpm. Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture, in 25 mL media. These shake flasks were incubated at 30° C. with an orbital shaking of 200 rpm. The use of an inducer is not required as all genes are constitutively expressed.

Example 10

Production of 3-Fucosyllactose in Saccharomyces Cerevisiae Using Various Lactose Binding alphα-1,3-Fucosyltransferase Enzymes

[0330] Another example provides use of a eukaryotic organism, in the form of Saccharomyces cerevisiae, for the disclosure. Using the strains, plasmids and methods as described in Example 9, strains are created that express SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18.

[0331] On top of that, further modifications are made in order to produce 3-fucosyllactose. These modifications comprise the addition of a lactose permease, a GDP-mannose 4,6-dehydratase and a GDP-L-fucose synthase. The preferred lactose permease is the KlLAC12 gene from Kluyveromyces lactis (WO 2016/075243). The preferred GDP-mannose 4,6-dehydratase and the GDP-L-fucose synthase are respectively gmd and fcl from Escherichia coli.

[0332] These strains are capable of growing on glucose or glycerol as carbon source, converting the carbon source into GDP-L-fucose, taking up lactose, and producing 3-fucosyllactose using GDP-L-fucose and lactose as substrates for the enzymes represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18, with SEQ ID NO: 18 as reference.

[0333] Preculture of the strains are made in 5 mL of the synthetic defined medium SD-CSM containing 22 g/L glucose and grown at 30° C. as described in Example 9. These precultures are inoculated in 25 mL medium in a shake flask with 10 g/L sucrose as sole carbon source and grown at 30° C. Regular samples are taken and the production of 3-fucosyllactose is measured as described in Example 1.

Example 11

Enzymatic Production of 3-Fucosyllactose

[0334] Another example provides the use of an enzyme with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16 of the present disclosure. These enzymes are produced in a cell-free expression system such as but not limited to the PURExpress system (NEB), or in a host organism such as but not limited to Escherichia coli or Saccharomyces cerevisiae, after which the above-listed enzymes can be isolated and optionally further purified.

[0335] Each of the above enzyme extracts or purified enzymes are added to a reaction mixture together with GDP-fucose, lactose and a buffering component such as Tris-HCl or HEPES. The reaction mixture is then incubated at a certain temperature (for example, 37° C.) for a certain amount of time (for example, 24 hours), during which the lactose will be converted to 3-fucosyllactose by the enzyme using GDP-fucose. The 3-fucosyllactose is then separated from the reaction mixture by methods known in the art. Further purification of the 3-FL can be performed if preferred. At the end of the reaction or after separation and/or purification, the production of 3-fucosyllactose is measured as described in Example 1.

Example 12

3-Fucosyllactose Production with Different Lactose Concentrations

[0336] A fermentation process as described in Examples 1 and 7, wherein the lactose concentration in the culture medium ranges from 50 to 150 g/L. The lactose is converted during the process into 3-fucosyllactose until minor amounts of lactose is left. The final ratio lactose to 3-fucosyllactose may be manipulated during this process by stopping the process earlier (higher lactose to 3-fucosyllactose ratio) or later (lower lactose to 3-fucosyllactose ratio) The lactose concentration may be increased in the vessel by feeding high concentrations of lactose solution with or without another carbon source to the bioreactor. The lactose feed contains lactose concentrations between 100 and 700 g/L and is kept at a temperature so that the lactose is kept soluble at a pH below or equal to 6 to avoid lactulose formation during the process, a standard method used in the dairy industry. The final concentrations of 3-fucosyllactose reached in such a production process ranges between 70 g/L when lower lactose concentrations are used and 200 g/L or higher when high lactose concentrations are used in the process as described above.

Example 13

Evaluation of the Helicobacter Pylori alphα-1,3-Fucosyltransferase fucT (SEQ ID NO: 18) Expressed from Various Promoters

[0337] The gene coding for the H. pylori alphα-1,3-fucosyltransferase fucT (SEQ ID NO: 18) was cloned in an expression vector under control of promoters PROM0012 or PROM0016, and the resulting plasmids were transformed to the E. coli mutant strain as described in Example 1. These strains were then evaluated in a growth experiment for their ability to produce 3-FL. Both strains were grown in multiple wells of a 96-well plate.

[0338] FIG. 9 shows the normalized production of 3-fucosyllactose produced by the strains. Each datapoint corresponds to data from one well. The dashed horizontal line indicates the setpoint to which all datapoints were normalized.

[0339] The experiment shows that the 3-FL production in a strain expressing H. pylori FucT using promoter PROM0012 drops to ±30% of the levels observed for a similar strain expressing the fucosyltransferase from promoter PROM0016.

[0340] By extrapolation of the data provided in Examples 2, 4 and 8, we can conclude that all strains containing any of the SEQ ID NOS: 2-16 show a significantly higher production compared to the control strain with α-1,3-fucosyltransferase fucT (SEQ ID NO: 18) when the fucosyltransferase is expressed from the same promoter (PROM0012 OR PROM0016), except for the strain with SEQ ID NO: 10, which shows a similar production as the control strain.

Example 14

Evaluation of Strains Expressing Polypeptides with the DMIASIVSF Consensus Motif for the Production of 3-Fucosyllactose

[0341] Mutant E. coli strains containing an expression construct for either SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 26 were evaluated for their 3-FL production in a growth experiment as described in Example 1. As indicated in FIG. 12, all polypeptide sequences contain the consensus domain DM[AS]VSF (SEQ ID NO: 36), but only SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 20 and SEQ ID NO: 22 additionally contain the consensus motif [NH]XDPAXLD (SEQ ID NO: 35) in the N-terminal region of the protein. The strain containing the H. pylori alphα-1,3-fucosyltransferase fucT (SEQ ID NO: 18) was taken along as a positive control. All strains were grown in multiple wells of a 96-well plate and tested in standard medium with 30 g/L sucrose and 20 g/L lactose.

[0342] FIG. 10 shows the normalized production of 3-fucosyllactose produced by the strains. Each datapoint corresponds to data from one well. The dashed horizontal line indicates the setpoint to which all datapoints were normalized.

[0343] The experiment shows that only the strains containing polypeptides with both consensus motifs [NH]xDPAxLD and DM[AS]VSF: i.e., SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 20 or SEQ ID NO: 22, are able to produce 3-FL, while the strains with polypeptides with DM[AS]VSF but lacking [NH]xDPAxLD: i.e., SEQ ID NO: 24 and SEQ ID NO: 26, do not produce any 3-FL. Based on this data, we can conclude that the presence of the [NH]xDPAxLD (SEQ ID NO: 35) consensus motif at the N-terminal region of polypeptides with the DM[AS]VSF (SEQ ID NO: 36) domain is crucial for the enzyme to have lactose binding alphα-1,3-fucosyltransferase activity.

[0344] Moreover, the polypeptide of SEQ ID NO: 22 has 92% global sequence identity to SEQ ID NO: 2, herewith showing that also sequences that have 87% or more sequence identity to SEQ ID NO: 2 have lactose binding alphα-1,3-fucosyltransferase activity.

Example 15

Evaluation of Strains Expressing Polypeptides with SEQ ID NO: 28, SEQ ID NO: 30 or SEQ ID NO: 32

[0345] Mutant E. colistrains containing an expression construct for either SEQ ID NO: 28, SEQ ID NO: 30 or SEQ ID NO: 32 can be evaluated for their 3-FL production in a growth experiment as described in Example 1. At the end of the growth experiment, the production of 3-fucosyllactose can be observed in the culture broth.

Example 16

Evaluation of the 3FL Purity at the End of a Fed-Batch Fermentation

[0346] Fed-batch fermentations at bioreactor scale were performed to evaluate strains, derived from the mutant E. coli K12 MG1655 strain background as described in Example 1, expressing various alphα-1,3-fucosyltransferase enzymes with SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 18. The bioreactor runs were performed as described in Example 1. In these examples, sucrose was used as a carbon source. Lactose was added in the batch medium at 90 g/L as a precursor for 3-FL formation, and a concentrated sucrose solution was fed during the fed-batch. For each strain, three independent fermentations were performed.

[0347] At the end of the fermentation, the broth was analyzed for the presence of lactose and 3-FL and the 3-FL purity was calculated using the formula 3FL (g/L)/(3FL (g/L)+lactose (g/L)). For strains containing SEQ ID NO: 18, an average purity of 85% was obtained, while for strains containing SEQ ID NO: 2 or 6 an average purity of over 98% and over 99% was obtained respectively.

[0348] The experiment shows that mutant E. coli strains expressing the lactose binding alphα-1,3-fucosyltransferase genes with SEQ ID NO: 2 or SEQ ID NO: 6 produce, in fed-batch fermentations at bioreactor scale, a broth with a higher 3-FL purity than similar strains containing SEQ ID NO: 18.