METHODS FOR PRODUCTION OF FUCOSYLATED OLIGOSACCHARIDES IN RECOMBINANT CELL CULTURE

Abstract

Methods for producing oligosaccharide products such as difucosylated oligosaccharides are disclosed. The methods include culturing recombinant cells in a cell culture medium comprising L-fucose, an oligosaccharide acceptor, and a carbon source. The cells are cultured under conditions in which a first fucosyltransferase polypeptide having a first substrate selectivity (e.g., an 1-2-fucosyltransferase polypeptide), a second fucosyltransferase polypeptide having a second substrate specificity (e.g., an 1-3-fucosyltransferase polypeptide), a nucleotide sugar pyrophosphorylase polypeptide, a lactose transporter polypeptide, and an L-fucose transporter polypeptide are expressed, and in which the oligosaccharide acceptor is converted to the difucosylated oligosaccharide. Recombinant cells for use in the methods are also described.

Claims

1. A recombinant cell for production of an difucosylated oligosaccharide product, the recombinant cell comprising: a polynucleotide encoding an 1-2-fucosyltransferase polypeptide, and a polynucleotide encoding an 1-3-fucosyltransferase polypeptide.

2. The recombinant cell of claim 1, further comprising one or more polynucleotides selected from the group consisting of: a polynucleotide encoding a nucleotide sugar pyrophosphorylase polypeptide, a polynucleotide encoding a lactose transporter polypeptide, and a polynucleotide encoding an L-fucose transporter polypeptide.

3. The recombinant cell of claim 1, further comprising: a polynucleotide encoding a nucleotide sugar pyrophosphorylase polypeptide, a polynucleotide encoding a lactose transporter polypeptide, and a polynucleotide encoding an L-fucose transporter polypeptide.

4. The recombinant cell of claim 1, wherein the 1-2-fucosyltransferase polypeptide is an E. coli O126 1-2-fucosyltransferase WbgL polypeptide.

5. The recombinant cell of claim 1, wherein the 1-3-fucosyltransferase polypeptide is a truncated 1-3-fucosyltransferase polypeptide.

6. The recombinant cell of claim 1, wherein the 1-3-fucosyltransferase polypeptide is an H. pylori UA948 1-3/4-fucosyltransferase (Hp3/4FT) polypeptide.

7. The recombinant cell of claim 2, wherein the nucleotide sugar pyrophosphorylase polypeptide is a bifunctional glycokinase and nucleotide sugar pyrophosphorylase polypeptide.

8. The recombinant cell of claim 2, wherein the nucleotide sugar pyrophosphorylase polypeptide is a B. fragilis bifunctional L-fucokinase/GDP-L-fucose pyrophosphorylase (Fkp) polypeptide.

9. The recombinant cell of claim 2, wherein the lactose transporter polypeptide is an E. coli LacY polypeptide.

10. The recombinant cell of claim 2, wherein the L-fucose transporter polypeptide is an E. coli FucP polypeptide.

11. The recombinant cell of claim 1, which is modified to eliminate or reduce expression of an L-fucose mutarotase.

12. The recombinant cell of claim 11, wherein the L-fucose mutarotase is E. coli fucU.

13. The recombinant cell of claim 1, which is modified to eliminate or reduce expression of a -galactosidase.

14. The recombinant cell of claim 13, wherein the -galactosidase is E. coli LacZ.

15. The recombinant cell of claim 1, further comprising an polynucleotide encoding an additional transporter polypeptide.

16. The recombinant cell of claim 15, wherein the additional transporter polypeptide is a Bifidobacterium fucosyllactose transporter polypeptide.

17. The recombinant cell claim 1, which is an E. coli cell, a B. subtilis cell, a C. glutamicum cell, or an S. cerevisiae cell.

18. A method for producing an oligosaccharide product comprising two or more fucose moieties, the method comprising culturing a recombinant cell according to any one of claim 1 in a cell culture medium comprising L-fucose, an oligosaccharide acceptor, and a carbon source; wherein the cell is cultured under conditions in which the 1-2-fucosyltransferase polypeptide and the 1-3-fucosyltransferase polypeptide are expressed, and wherein the oligosaccharide acceptor is converted to the difucosylated oligosaccharide.

19. The method of claim 18, wherein the oligosaccharide acceptor is lactose and the oligosaccharide product is lactodifucotetraose (LDFT).

20. The method of claim 18, wherein expression of the 1-2-fucosyltransferase polypeptide is induced at a level corresponding to 30-40% of maximum, and wherein expression of the 1-3-fucosyltransferase polypeptide is induced at a maximum level.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 shows a summary of various pathway design aspects for LDFT production in E. coli according to the present disclosure. L-Fucose (triangles) and lactose (glucose moiety, diagonal striped circles; galactose moiety, filled circles) are transported into the cytosol through sugar transporters FucP and LacY, respectively. The fucU and lacZ genes are deleted to prevent substrate assimilation into central carbon metabolism. Fucose is converted to donor substrate GDP-fucose by Fkp. WbgL glycosylates lactose at the C2 position with GDP-fucose to form 2-FL. Hp3/4FT glycosylates 2-FL at the C3 position with GDP-fucose to form LDFT. G-6-P: glucose-6-phosphate, DHAP: dihydroxyacetone phosphate, PTS: phosphotransferase system, SetA: sugar efflux transporter A.

[0013] FIG. 2A shows cell growth and protein expression resulting from modifications in E. coli B-strains for LDFT production. Growth of BL21 Star (DE3) and lacZ mutant (Strain 2, Table 1) in M9 minimal media with or without 1 g/L L-fucose or D-lactose.

[0014] FIG. 2B shows expression of P.sub.T7:sfgfp in BL21 Star (DE3) (Strain 3) and lacZ mutant (Strain 4, Table 1) in LB-media. Cultures were induced with or without 1 mM IPTG, respectively. A indicates that the gene was removed from the genome. Error bars indicate s.d. (n=3 biological replicates).

[0015] FIG. 3A shows cell growth and protein expression after installation of the T7 RNAP expression system into E. coli K-12-derivative strains. GFP fluorescence assay in K-12 derivative strains, BW25113 Z1 (Strain 7) and MG1655 Z1 (Strain 8, Table 1) with ss9::P.sub.lacuv5:T7rnap. Cultures were induced with or without 1 mM IPTG at 37 C. for 24 h.

[0016] FIG. 3B shows a growth assay of MG1655 Z1 (Strain 6) and Strain 10 (Strain 6 with fucU lacZ, Table 1) in M9-minimal media with or without 1 g/L L-fucose or D-lactose at 37 C. for 24 h.

[0017] FIG. 3C shows a fluorescence assay to evaluate GFP expression from P.sub.T7 in Strain 8 and Strain 12 (Strain 8 with fucU lacZ, Table 1). Cultures were grown in LB-media and induced with or without 1 mM IPTG at 37 C. for 24 h.

[0018] FIG. 3D shows a growth assay of MG1655 Z1 and Strain 15 (MG1655 Z1 with fucU lacZ, Table 1) in M9-minimal media with 1 g/L L-fucose or D-lactose. A indicates gene was removed from the genome. Error bars indicate s.d. (n=3 biological replicates).

[0019] FIG. 4A, taken with FIG. 4B, shows GFP expression under lac-promoter variants in K-12 derivative strains. Cultures were grown in LB-media and induced with or without 1 mM IPTG at 37 C. for 24 h. GFP expression under promoter P.sub.L1acO1 in Strain 17 (MG1655 Z1), Strain 18 (MG1655 Z1 with fucU lacZ) and Strain 19 (Strain 18 with ss9::P.sub.lacUV5:T7rnap, Table 1).

[0020] FIG. 4B shows GFP expression under promoter P.sub.lacUV5 in Strain 20 (MG1655 Z1), Strain 21 (Strain 20 with fucU) and Strain 22 (Strain 20 with fucU lacZ, Table 1). A indicates gene was removed from the genome. Error bars indicate s.d. (n=3 biological replicates).

[0021] FIG. 5 shows the effects of carbon sources on LDFT production. Strain 23 (MG1655 Z1 fucU lacZ with the LDFT production plasmids, Table 1) was grown in M9P with 5 g/L glucose or 10 g/L glycerol at 30 C. for 24 h. Cultures were supplemented with 1 g/L lactose and with or without 1 g/L L-fucose and induced with 1 mM IPTG and 100 ng/mL aTc. L-Fucose concentration (diagonal), lactose concentration (checkered), monofucosides (2-FL/3-FL) concentration (zigzag) and LDFT concentration (filled) were measured at 24 h. Error bars indicate s.d. (n=3 biological replicates).

[0022] FIG. 6 shows the additional expression of lactose and L-fucose permease genes to enhance lactose and L-fucose availabilities. Strain 14 (MG1655 Z1 fucU lacZ, Table 1) was used as a host strain. Strain 23 (Strain 14 with the LDFT production plasmids), Strain 24 (Strain 14 with the LDFT production plasmids with lacY), Strain 25 (Strain 14 with the LDFT production plasmids with fucU), and Strain 26 (Strain 14 with the LDFT production plasmids with lacY and fucU) were grown in M9P with 10 g/L glycerol at 30 C. for 24 h. Cultures were supplemented with 1 g/L lactose and 1 g/L L-fucose and induced with 1 mM IPTG and 100 ng/mL aTc. Growth and production of monofucosides (2-FL/3-FL, zigzag) and LDFT (filled) were determined at 24 h. + indicates the corresponding gene was expressed from the genome and +++ indicates the corresponding gene was additionally expressed from a plasmid. Error bars indicate s.d. (n=3 biological replicates).

[0023] FIG. 7A, taken with FIG. 7B, shows the effects of IPTG and aTc concentrations on LDFT production. Strain 26 (MG1655 Z1 fucU lacZ with the LDFT production plasmids with lacY and fucU) was grown in M9P with 10 g/L glycerol at 30 C. for 24 h. Cultures were supplemented with 1 g/L lactose and 1 g/L fucose and induced with 100 ng/mL aTc and various concentrations of IPTG (0, 25, 50, 100 and 1000 M).

[0024] FIG. 7B shows results for Strain 26, grown as described for FIG. 7A except with 50 M IPTG and various concentrations of aTc (0, 25, 50, 100 ng/mL). OD.sub.600, L-Fucose concentration (diagonal), lactose concentration (checkered), monofucosides (2-FL/3-FL) concentration (zigzag) and LDFT concentration (filled) were measured at 24 h. Error bars indicate s.d. (n3 biological replicates).

[0025] FIG. 8A shows growth (OD.sub.600) of strain 26 (MG1655 z1 fucU lacZ with the LDFT production plasmids with lacY and fucU) in M9P with 20 g/L glycerol at 30 C. for 12 h. Cultures were supplemented with 1 g/L lactose and 1 g/L L-fucose and induced with 50 M IPTG and 100 ng/mL aTc. Error bars indicate s.d. (n=3 biological replicates).

[0026] FIG. 8B shows glycerol concentration (cross), fucose concentration (diamond), lactose concentration (triangle), monofucosides (2-FL/3-FL) concentration (square) and LDFT concentration (circle) were monitored during growth of Strain 26 as described for FIG. 8A. Error bars indicate s.d. (n=3 biological replicates).

[0027] FIG. 9 shows sugar levels in cultures upon delayed expression of Hp3/4 ft in Strain 26. Strain 26 (MG1655 Z1 fucU lacZ with the LDFT production plasmids with lacY and fucU) was grown in M9P with 10 g/L glycerol at 30 C. for 24 h. Cultures were supplemented with 1 g/L lactose and 1 g/L L-fucose and induced with 50 M IPTG at 0 h. 100 ng/mL aTc was added to cultures at 0, 2, 4, and 6 h. OD.sub.600, monofucosides (2-FL/3-FL) concentration (zigzag) and LDFT concentration (filled) were measured at 24 h. Error bars indicate s.d. (n=3 biological replicates).

[0028] FIG. 10 shows LDFT production with 2-FL feeding. The wbgL gene was removed from pAL2029, generating pAL2059 (Table 2). Strain 27 (MG1655 Z1 fucU lacZ harboring pAL1760 (Hp3/4 ft) and pAL2059 (fkp, lacY, and fucU), Table 1) was grown in M9P with 10 g/L glycerol at 30 C. for 24 h. Cultures were supplemented with 1.4 g/L 2-FL (mole equivalent to 1 g/L lactose) and 0.5 g/L L-fucose and induced with 50 M IPTG and 100 ng/mL aTc. Glycerol concentration (cross), L-fucose concentration (diamond), 2-FL concentration (square) and LDFT concentration (circle) were monitored during the experiment. Error bars indicate s.d. (n=3 biological replicates).

[0029] FIG. 11 shows LDFT production in Strain 26 with various concentrations of lactose and fucose. Strain 26 (MG1655 Z1 fucU lacZ with the LDFT production plasmids with lacY and fucU) was grown in M9P with 20 g/L glycerol at 30 C. for 24 h. Cultures were supplemented with lactose and L-fucose and induced with 50 M IPTG and 100 ng/mL aTc. OD.sub.600, L-fucose concentration (diagonal), lactose concentration (dotted), 2-FL concentration (wave) and LDFT concentration (filled) were measured at 24 h. Error bars indicate s.d. (n3 biological replicates).

[0030] FIG. 12 shows fucosyltransferases and other enzymes for production of oligosaccharide products in recombination host cells.

DETAILED DESCRIPTION OF THE INVENTION

[0031] Provided herein are methods for producing oligosaccharide products, such as tetrasaccharide lactodifucotetraose (LDFT) and other difucosylated oligosaccharides, in recombinant hosts such as E. coli. The present invention is based, in part, on the pairing of glycosyltransferases with complementary substrate specificities, e.g., pairing of 1-2-fucosyltransferases with high activity towards lactose and 1-3-fucosyltransferases with higher activity towards 2-fucosyllactose (2-FL) than lactose. The selectivity of the 1-3-fucosyltransferase provides for minimal production of 3-fucosyllactose (3-FL) as a side product, resulting in the production of difucosylated oligosaccharides such as difucosylated tetrasaccharide lactodifucotetraose (LDFT) in high yield.

[0032] The use of bacterial fucosyltransferases with narrow acceptor selectivity can drive the sequential fucosylation of acceptors such as lactose and intermediates such as 2-fucosyllactose (2-FL) to produce LDFT and other fucosylated products. Deletion of substrate degradation pathways that decouple cellular growth from product fucosylation can enhance expression of native substrate transporters, and modular induction of the genes in relevant biosynthetic pathways allows for complete conversion of acceptors such as lactose into products such as LDFT with only minor quantities of side products such as 3-fucosyllactose (3-FL). In certain embodiments, for example, 5.1 g/L of LDFT can be produced from 3 g/L lactose and 3 g/L L-fucose in 24 h. The results described herein demonstrate promising applications of microbial biocatalysts for the production of multi-fucosylated HMOs.

[0033] LDFT can be synthesized from lactose and L-fucose in a two-step fucosylation process using an 1-2-fucosyltransferase and an 1-3-fucosyltransferase. While monofucosylation of lactose with a single fucosyltransferase for the microbial production of 2-FL and 3-FL has been studied, the effects of implementing an 1-2-fucosyltransferase and an 1-3-fucosyltransferase together in a cellular system to produce a difucosylated HMO has not been reported. As lactose is a suitable acceptor substrate for both fucosyltransferases, both 2-FL and 3-FL can be produced as mono-fucosylated products in the first fucosylation step of the system with the presence of both fucosyltransferases. It was shown previously that while an 1-3/4-fucosyltransferase from Helicobacter pylori (Hp3/4FT) can use both non-fucosylated and 1-2-fucosylated galactosyl oligosaccharides as substrates (McArthur et al., 2019; Yu et al., 2017), 1-2-fucosyltransferases from Escherichia coli 0126 (WbgL) (Engels and Elling, 2014; McArthur et al., 2019) and Thermosynechococcus elongates (Zhao et al., 2016) are selective towards lactose and other non-fucosylated galactosyl oligosaccharide acceptor substrates.

[0034] An E. coli-based system according to the present disclosure, for example, employs two fucosyltransferases that preferentially fucosylates lactose to form a 2-FL intermediate that is further fucosylated to produce the target LDFT. Various promoter expression systems were assessed to establish heterologous expression of the desired biosynthetic pathway. LDFT production was decoupled from bacterial growth by removing catabolic pathways of starting substrates and by maintaining cell density with glycerol, an inexpensive carbon source that does not activate carbon catabolite repression of lactose and L-fucose transporters (Kopp et al., 2017; Paulsen et al., 1998). To enhance intracellular availability of substrates, the lactose and L-fucose transporter genes, lacY and fucP, were additionally expressed from plasmids. With additional fine-tuning of the expression levels of individual glycosyltransferase genes, the strain produced 5.1 g/L of LDFT from 3 g/L lactose, achieving 910% of the theoretical maximum yield of LDFT in 24 h.

I. Recombinant Host Cells for Production of Oligosaccharides

[0035] Provided herein are recombinant cells for the production of oligosaccharide products. The cells include: [0036] a polynucleotide encoding a first glycosyltransferase polypeptide having a first substrate selectivity, and [0037] a polynucleotide encoding a second glycosyltransferase polypeptide having a second substrate selectivity.

[0038] Glycosyltransferases and other enzymes suitable for use in the methods described herein include, but are not limited to, those summarized in FIG. 12. In some embodiments, the first glycosyltransferase is a fucosyltransferase having a first substrate selectivity and the second glycosyltransferase is a fucosyltransferase polypeptide having a second substrate selectivity.

[0039] A. 1-2-fucosyltransferase

[0040] Fucosyltransferases are inverting glycosyltransferases and are classified into eight glycosyltransferase (GT) families in the Carbohydrate-Active enZYmes (CAZy) database: GT10, GT11, GT23, GT3, GT56, GT65, GT68 and GT74 (see, cazy.org; Drula, et al. Nucleic Acids Research, 2022, Vol. 50, D571-D577; and references cited therein).

[0041] In some embodiments, the first fucosyltransferase polypeptide is an 1-2-fucosyltransferase polypeptide classified by Enzyme Commission number 2.4.1.69. WbgL, according to SEQ ID NO:1, and other GT11 family fucosyltransferases are thought to be GT-B fold glycosyltransferases containing two separate Rossmann domains (characterized by a six-stranded parallel -sheet with a 321456 topology) with a connecting linker region and a catalytic site between the domains. See, Engels et al. (Glycobiology 2014, 24(2): 170-178) and Breton et al. (Glycobiology 2006, 16(2): 29R-37R). A high degree of conservation has been observed between protein members of the GT-B family, especially in the nucleotide-binding domain at the C-terminus. A glutamate residue and glycine-rich loops are thought to interact with the ribose and phosphate moieties of the nucleotide. The 1-2-fucosyltransferase may be a GT11 family fucosyltransferase having one or more conserved motifs corresponding to residues 8-16 (motif IV), 158-167 (motif I), 201-207 (motif II), and 234-273 (motif III) of SEQ ID NO:1. In some embodiments, the 1-2-fucosyltransferase includes from one to four amino acid sequences having at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to motif IV, motif I, motif, II, and/or motif III in SEQ ID NO:1. Highly conserved motif I is likely involved in GDP-fucose binding. Residues corresponding to R.sup.161 and D.sup.164 have been indicated to play roles in donor binding and enzyme activity (see, Li, et al. Biochemistry 2008, 47, 11590-11597). In addition to amino acid sequences corresponding to motifs I, II, III, and/or IV, the 1-2-fucosyltransferase may also include on or more acid sequences having at least 70% identity residues 1-7, 17-157, 168-200, 208-233, and/or 274-297 of SEQ ID NO:1.

[0042] Percentage of sequence identity can be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence (e.g., a peptide of the invention) in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0043] Identical and identity, in the context of two or more polypeptide sequences or nucleic acid sequences, refer to two or more sequences or subsequences that are the same. Sequences are substantially identical to each other if they have a specified percentage of nucleotides or amino acid residues that are the same (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.

[0044] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available at the National Center for Biotechnology Information website, ncbi.nlm.nih.gov. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0045] Examples of 1-2-fucosyltransferases include, but are not limited to, E. coli O126 1-2-fucosyltransferase (WbgL; GenBank: ABE98421.1; SEQ ID NO:1), H. mustelae 12198 1-2-fucosyltransferase (Hm2FT; GenBank: CBG40460; SEQ ID NO:8), E. coli 0128:B12 1-2-fucosyltransferase (WbsJ; GenBank: AA037698.1; SEQ ID NO:9), H. pylori UA1234 1-2-fucosyltransferase (Hp2FTa; GenBank: AAD29863.1; SEQ ID NO:10), and H. pylori UA802 1-2-fucosyltransferase (Hp2FTb; GenBank: AAC99764.1; SEQ ID NO:11). In some embodiments, the 1-2-fucosyltransferase polypeptide comprises an amino acid sequence having at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to WbgL (SEQ ID NO: 1), Hm2FT (SEQ ID NO:8), WbsJ (SEQ ID NO:9), Hp2FTa (SEQ ID NO:10), or Hp2FTb (SEQ ID NO:11).

[0046] In some embodiments, the 1-2-fucosyltransferase polypeptide is an E. coli 0126 1-2-fucosyltransferase WbgL polypeptide.

[0047] B. 1-3-fucosyltransferase

[0048] In some embodiments, the second fucosyltransferase polypeptide is an 1-3-fucosyltransferase polypeptide having, for example, -LacNac -1,3-L-fucosyltransferase activity (EC 2.4.4.1), galactoside -1,3/1,4-L-fucosyltransferase activity (EC 2.4.1.65), or galactoside -1,3-L-fucosyltransferase activity (EC 2.4.1.152). The 1-3-fucosyltransferase may be a GT10 family fucosyltransferase or a GT11 family fucosyltransferase. In some embodiments, the GT10 fucosyltransferase has a glycosyltransferase B (GT-B) fold containing two separated Rossmann domains as described, for example, by Breton et al. supra.

[0049] Examples of 1-3-fucosyltransferases include, but are not limited to H. pylori UA948 1-3/4-fucosyltransferase (Hp3/4FT; GenBank: AAF35291.2; SEQ ID NO:3), H. pylori ATCC43504 1-3-fucosyltransferase (Hp43504 3FT; GenBank: AAB93985; SEQ ID NO:12), H. pylori J99 1-3-fucosyltransferase (HpJ99 3FT; GenBank: AAD06169.1, AAD06573.1; SEQ ID NOS:13-14), H. pylori NCTC11637 1-3-fucosyltransferase (Hp11637 3FT; GenBank: AAB93985; SEQ ID NO:15), B. fragilis NCTC 9343 1-3/1-4-fucosyltransferase polypeptide (Bf3/4FT; GenBank: CAH09495.1; SEQ ID NO:16), and H. hepaticus ATCC 51449 Hh0072 (Hh0072; GenBank: AAP76669.1; SEQ ID NO:17). In some embodiments, the 1-2-fucosyltransferase polypeptide comprises an amino acid sequence having at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to Hp3/4FT (SEQ ID NO:3), Hp43504 3FT (SEQ ID NO:12), HpJ99 3FT (SEQ ID NOS:13 and/or 14), Hp11637 3FT (SEQ ID NO:15), Bf3/4FT (SEQ ID NO:16), or Hh0072 (SEQ ID NO:17).

[0050] In some embodiments, the 1-3-fucosyltransferase polypeptide is a truncated 1-3-fucosyltransferase polypeptide, e.g., residues 1-428 of SEQ ID NO:2, or a polypeptide having at least 70% identity to residues 1-428 of SEQ ID NO:2.

[0051] In some embodiments, the cells further include one or more polynucleotides selected from the group consisting of: [0052] a polynucleotide encoding a nucleotide sugar pyrophosphorylase, [0053] a polynucleotide encoding a monosaccharide transporter such as a fucose transporter, and [0054] a polynucleotide encoding an oligosaccharide transporter such as a lactose transporter.

[0055] C. Kinase/pyrophosphorylases

[0056] In some embodiments, the nucleotide sugar pyrophosphorylase polypeptide is a bifunctional glycokinase and nucleotide sugar pyrophosphorylase polypeptide. In some embodiments, the bifunctional enzyme is an L-fucokinase/GDP-fucose pyrophosphorylase (Fkp). Fkps are a class of enzymes that catalyze two steps of the L-fucose salvage pathway for the geeneration of activated GDP-L-fucose via a fucose-1-phosphate intermediate. Fkps have been observed to adopt a tetrameric formation, with each monomer containing an N-terminal GDP-fucose pyrophosphorylase domain, an intermediate linking domain, and a C-terminal fucokinase domain. The pyrophosphorylase domain contains a Rossmann fold and a left-handed -helix, and the fucokinase contains a GHMP sugar kinase fold. The linker between the two domains contains -helices. Examples of Fkps include, but are not limited to Bacteroides fragilis bifunctional L fucokinase/GDP-L-fucose pyrophosphorylase (BfFKP; GenBank: CAH08307.1; SEQ ID NO:3) and Arabidopsis thaliana bifunctional fucokinase/fucose pyrophosphorylase (AtFKGP; UniProt: Q9LNJ9; SEQ ID NO:18).

[0057] D. Glycotransporters

[0058] In some embodiments, the monosaccharide transporter is a fucose transporter, and the oligosaccharide transporter is a lactose transporter. Many such transporters belong to the major facilitator superfamily (MFS), which shuttle substrates across cell membranes by leveraging electrochemical potential. MFS transporters such as E. coli LacY are composed of 12 transmembrane helices, with the six N-terminal and the six C-terminal helices forming distinct helical bundles connected by a loop. The two bundles have the same topology and exhibit pseudo-two-fold symmetry around an axis perpendicular to the membrane bilayer. A hydrophilic cavity is defined by helices 1, 2, 4, and 5 in the N-terminal bundle and helices 7, 8, 9, and 11 in the C-terminal bundle, while helices 3, 6, 9, and 12 are largely embedded in the membrane. In some embodiments, the lactose transporter polypeptide is an E. coli LacY polypeptide. Similar lactose transporters have been identified in Citrobacter spp., Cronobacter spp., Enterobacter spp., Klebsiella spp., Salmonella spp., and Shigella spp., and may also be incorporated in the recombinant host cells. In some embodiments, the lactose transporter polypeptide comprises an amino acid sequencing having at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the E. coli str. K-12 substr. MG1655 LacY set forth in SEQ ID NO:4.

[0059] In some embodiments, the L-fucose transporter polypeptide is an E. coli FucP polypeptide. Similar fucose transporters have been identified in species including, but not limited to, Chryseobacterium mucoviscidosis, Enterobacter hormaechei, Escherichia albertii, Klebsiella pneumoniae, Salmonella enterica, and Shigella flexneri, and may also be incorporated into the recombinant host cells. In some embodiments, the lactose transporter polypeptide comprises an amino acid sequencing having at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the E. coli str. K-12 substr. MG1655 FucP set forth in SEQ ID NO:5.

[0060] In some embodiments, the cell further includes a polynucleotide encoding an additional transporter polypeptide. In some embodiments, the additional transporter polypeptide is a Bifidobacterium fucosyllactose transporter polypeptide, e.g., those including the domains set forth in SEQ ID NOS:19-21 and/or SEQ ID NOS:22-24.

[0061] Suitable microbial hosts include, but are not limited to, members of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces. Preferred hosts include: Escherichia coli, Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis and Saccharomyces cerevisiae. In some embodiments, the cell is an E. coli cell, a B. subtilis cell, a C. glutamicum cell, or an S. cerevisiae cell. In some embodiments, the cell is an E. coli BW25113 Z1 cell or an E. coli MG1655 Z1 cell.

[0062] Recombinant organisms containing the genes encoding glycosyltransferases and other enzymes for the production of human milk oligosaccharides and other oligosaccharide products can be constructed using techniques well known in the art. Polynucleotide sequences may be obtained from various organisms as described above, e.g., from a bacterial genome. For example, if the sequence of the gene is known, suitable genomic libraries may be created by restriction endonuclease digestion and may be screened with probes complementary to the desired gene sequence. Once the sequence is isolated, the DNA may be amplified using standard primer-directed amplification methods such as polymerase chain reaction to obtain amounts of DNA suitable for transformation using appropriate vectors. Tools for codon optimization for expression in a heterologous host are readily available.

[0063] Once the relevant pathway genes are identified and isolated they may be transformed into suitable expression hosts by means well known in the art. Vectors or cassettes useful for the transformation of a variety of host cells are common and commercially available from companies such as Thermo Fisher Scientific (Waltham, MA), MilliporeSigma (La Jolla, CA), and New England Biolabs, Inc. (Burlington, MA). Typically the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Such vectors may include a region upstream of the gene which harbors transcriptional initiation controls and a region downstream of the gene which controls transcriptional termination. Both control regions may be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions may also be derived from genes that are not native to the specific species chosen as a production host.

[0064] Initiation control regions or promoters, which are useful to drive expression of the relevant pathway coding regions in the desired host cell are numerous and familiar to those skilled in the art. Promoters capable of driving these genetic elements include, but are not limited to, CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, CUP1, FBA, GPD, and GPM (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, ara, tet, trp, IPL, IPR, T7, tac, and trc (useful for expression in Escherichia coli, Alcaligenes, and Pseudomonas); the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus subtilis, Bacillus licheniformis, and Paenibacillus macerans; nisA (useful for expression Gram-positive bacteria, Eichenbaum et al. Appl. Environ. Microbiol. 64(8):2763-2769 (1998)); and the synthetic P11 promoter (useful for expression in Lactobacillus plantarum, Rud et al., Microbiology 152:1011-1019 (2006)). Termination control regions, if present, may also be derived from various genes native to the preferred hosts.

[0065] In some embodiments, the cell is transformed with a first expression vector comprising: [0066] the polynucleotide encoding the first fucosyltransferase polypeptide, [0067] the polynucleotide encoding the nucleotide sugar pyrophosphorylase polypeptide, [0068] the polynucleotide encoding the lactose transporter polypeptide, and [0069] the polynucleotide encoding the L-fucose transporter polypeptide.

[0070] In some embodiments, the polynucleotide encoding the first fucosyltransferase polypeptide (e.g., WbgL) and the polynucleotide encoding the nucleotide sugar pyrophosphorylase polypeptide (e.g., Fkp) are operably linked to a first inducible promoter. In some embodiments, the first inducible promoter is a P.sub.L1acO1 promoter.

[0071] In some embodiments, the polynucleotide encoding the second fucosyltransferase polypeptide (e.g., Hp3/4FT) is operably linked to a second inducible promoter. In some embodiments, the second inducible promoter is a P.sub.LtetO1 promoter.

[0072] In some embodiments, the polynucleotide encoding the lactose transporter polypeptide and the polynucleotide encoding the L-fucose transporter polypeptide are operably linked to a constitutive promoter.

[0073] In some embodiments, the cell is modified to eliminate or reduce expression of an L-fucose mutarotase and/or a -galactosidase. In some embodiments, the L-fucose mutarotase is an E. coli fucU, as set forth in SEQ ID NO:6, or a polypeptide having at least 70% identity thereto. In some embodiments, the -galactosidase is an E. coli LacZ; as set forth in SEQ ID NO:6, or a polypeptide having at least 70% identity thereto. Knockout of L-fucose mutarotases and -galactosidases can be conducted as described in more detail below. Other CRISPR/Cas9-based strategies, e.g., as described by Zhao et al. (Microb Cell Fact 2016, 15: 205) or Knig et al (Bio Protoc. 2018, 8(2): e2688), may be employed, as well as methods employing phage Red recombinase and/or FLP recombinase (see, Datsenko and Wanner. PNAS, 2000, 97 (12): 6640-6645; Baba, et al. Molecular Systems Biology 2006, 2:2006.0008)

II. Production of Oligosaccharides

[0074] Also provided herein are methods for producing oligosaccharide products. In some embodiments, the oligosaccharide product includes two or more fucose moieties, and the method comprising culturing a recombinant cell as described herein in a cell culture medium comprising L-fucose, an oligosaccharide acceptor, and a carbon source. The cell is cultured under conditions in which a nucleotide sugar pyrophosphorylase polypeptide, a first fucosyltransferase polypeptide, a second fucosyltransferase polypeptide, a lactose transporter polypeptide, and/or an L-fucose transporter polypeptide are expressed and the oligosaccharide acceptor is converted to a fucosylated oligosaccharide.

[0075] In some embodiments, the acceptor is selected from the group consisting of lactose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT); lacto-N-hexaose (LNH); lacto-N-neohexaose (LNnH); para-lacto-N-hexaose (pLNH); and para-lacto-N-octaose (pLNO). Oligosaccharide products include, but are not limited to lactodifucotetraose (LDFT), difucosyl lacto-N-tetraose (DF-LNT), trifucosyl lacto-N-tetraose (TriF-LNT), trifucosyl para-lacto-N-hexaose (TriF-pLNH), and trifucosyl para-lacto-N-octaose (Tetra-F-pLNO)

[0076] In some embodiments, the oligosaccharide acceptor is lactose and the oligosaccharide product is lactodifucotetraose (LDFT).

[0077] Cell culture media generally contain a carbon source. Suitable substrates include, but are not limited to, monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, comsteep liquor, sugar beet molasses, and barley malt. Additionally the carbon substrate may also be one-carbon substrates such as carbon dioxide or methanol. In addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine, and a variety of amino acids for metabolic activity. For example, methylotrophic yeast are known to utilize the carbon from methylamine to form trehalose or glycerol. In some embodiments, the carbon source comprises glucose, glycerol, or a combination thereof.

[0078] In addition to an appropriate carbon source, fermentation media will typically contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway for production of the desired oligosaccharide.

[0079] Typically, recombinant host cells are grown at a temperature in the range of about 20 C. to about 40 C. in an appropriate medium such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast medium (YM) broth. Other defined or synthetic growth media may also be used, and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science. The use of agents known to modulate catabolite repression directly or indirectly, e.g., cyclic adenosine 2:3-monophosphate, may also be incorporated into the fermentation medium.

[0080] Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is preferred as the initial condition. Oligosaccharide production may be conducted under aerobic or anaerobic conditions, including microaerobic conditions.

[0081] In some embodiments, expression of the nucleotide sugar pyrophosphorylase polypeptide and the first fucosyltransferase polypeptide is induced at a level corresponding to 30-40% of maximum level (e.g., with isopropyl -D-1-thiogalactopyranoside in an amount around 50 M). In some embodiments, expression of the second fucosyltransferase polypeptide is induced at a maximum level (e.g., with anhydrotetracycline at around 100 ng/mL).

[0082] The terms about and around, as used herein to modify a numerical value, indicate a close range surrounding that explicit value. If X were the value, about X or around X would indicate a value from 0.8 X to 1.2 X, preferably a value from 0.9 X to 1.1 X, and, more preferably, a value from 0.95 X to 1.05 X. Any reference to about X or around X specifically indicates at least the values X, 0.9 X, 0.91 X, 0.92 X, 0.93 X, 0.94 X, 0.95 X, 0.96 X, 0.97 X, 0.98 X, 0.99 X, 1.01 X, 1.02 X, 1.03 X, 1.04 X, 1.05 X, 1.06 X, 1.07 X, 1.08 X, 1.09 X, and 1.10 X. Thus, about X and around X are intended to teach and provide written description support for a claim limitation of, e.g., 0.98 X.

[0083] The amount of oligosaccharide produced in the cell culture medium can be determined using a number of methods known in the art, for example, high performance liquid chromatography (HPLC) or thin-layer chromatography (TLC).

[0084] Oligosacharides may be produced in a batch fashion or continuous fashion. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Within batch cultures, cells may moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate. A variation on the standard batch system is the fed-batch system. Fed-batch fermentation processes typically include incremental addition of an oligosaccharide acceptor or other substrate as the fermentation progresses.

[0085] Continuous fermentation typically involves an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, a limiting nutrient such as the carbon source may be maintained at a fixed rate while all other parameters may be allowed to vary. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Batch, fed-batch, and continuous fermentation systems are described, for example, by Bull et al. (Eds.) (Manual of Industrial Microbiology and Biotechnology, Third Edition (2010) ASM Press, Washington DC.) which is incorporated herein by reference.

III. Examples

Methods

[0086] Reagents

[0087] All enzymes involved in the molecular cloning experiments were purchased from New England Biolabs (NEB). All synthetic oligonucleotides were synthesized by Integrated DNA Technologies. Sanger sequencing was provided by Genewiz. D-Lactose was purchased from Sigma-Aldrich. L-Fucose was purchased from V-Labs, Inc. An analytical standard of 2-FL was purchased from Carbosynth.

[0088] For synthesizing 3-FL, 8 mg lactose, L-fucose (1.3 equiv.), adenosine 5-triphosphate (ATP, 1.3 equiv.), and guanidine 5-triphosphate (GTP, 1.3 equiv.) were dissolved in 2.3 mL of 100 mM Tris-HCl buffer (pH 7.5) containing 20 mM MgCl.sub.2, 0.35 mg Bacteroides fragilis bifunctional L-fucokinase/GDP-L-fucose pyrophosphorylase (BfFKP) (Yi et al., 2009), 0.15 mg Pasteurella multocida inorganic pyrophosphatase (PmPpA) (Yu et al., 2010), and 0.3 mg Hp3/4FT. The reaction mixture was incubated at 30 C. at 100 rpm for 16 h. The product formation was monitored by liquid chromatography-mass spectrometry (LCMS) (Shimadzu). When all lactose was converted to 3-FL, the reaction was stopped by adding an equivalent volume of ice-cold ethanol. The mixture was kept at 4 C. for 30 min then centrifuged at 6,900 g for 30 min. The precipitates were removed and the supernatant was concentrated with a rotary evaporator and then passed through a Dowex 18 ion exchange column. The partially purified product was obtained by elution with water. The eluate was concentrated, passed through a Bio-Gel P-2 gel filtration column, and eluted with water. The fractions containing the pure 3-FT product were collected and lyophilized.

[0089] To synthesize the LDFT standard, 8 mg lactose, L-fucose (1.2 equiv.), ATP (1.2 equiv.), and GTP (1.2 equiv.) were dissolved in 2.3 mL of 100 mM Tris-HCl buffer (pH 7.5) containing 20 mM MgCl.sub.2, 0.3 mg BfFKP, 0.1 mg PmPpA, and 0.2 mg Helicobacter mustelae 1-2-fucosyltransferase (Hm2FT) (Ye et al., 2019). The reaction mixture was incubated at 30 C. at 100 rpm for 16 h. The product formation was monitored by LCMS. When all lactose was converted to 2-FL, the reaction mixture was concentrated and applied to the next fucosylation step without purification. In the second step, the reaction mixture containing 10 mM 2-FL formed from the previous step, L-fucose (1.2 equiv.), ATP (1.2 equiv.), and GTP (1.2 equiv.) in 2.3 mL of 100 mM Tris-HCl buffer (pH 7.5) containing 20 mM MgCl.sub.2, 0.35 mg BfFKP, 0.15 mg PmPpA, and 0.3 mg Hp3/4FT. The reaction mixture was incubated at 30 C. at 100 rpm for 16 h. When all 2-FL was converted to LDFT as monitored by LCMS, the reaction was stopped by adding an equal volume of ice-cold ethanol. The mixture was kept at 4 C. for 30 min and then centrifuged at 6,900 g for 30 min. The precipitates were removed and the supernatant was concentrated with a rotary evaporator and then passed through a Dowex 18 ion exchange column. The partially purified product was obtained by elution with water. The eluate was concentrated, passed through a Bio-Gel P-2 gel filtration column, and eluted with water. The fractions containing the pure LDFT product were collected and lyophilized.

Strains and Plasmids

[0090] All strains used in this study are listed in Tables 1 and 3. All plasmids and primers are listed on Tables 4 and 5. Gene deletions and integrations were constructed using CRISPR-Cas9-mediated homologous recombination (Jiang et al., 2015). Linear DNA repair fragments for gene deletions were constructed by PCR assembly or amplification from genomic DNA using primers listed in Tables 4 and 6. The linear DNA repair fragment for ss9::P.sub.lacUV5:T7rnap was PCR amplified from repair plasmid pAL1856 constructed from pSS9 template (Addgene plasmid #71655) (Bassalo et al., 2016) listed in Tables 3 & 6. All genomic modifications were PCR and sequence verified.

TABLE-US-00001 TABLE 1 Strain list Strain no. E. coli strain Plasmid Key Genotype 1 AL3535 As BL21 Star (DE3), but lacZ 2 AL3535 pAL1779/pAL1817 lacZ, P.sub.T7:fkp-wbgL, P.sub.T7:Hp3/4ft 3 BL21 Star pAL1834 P.sub.T7:sfgfp (DE3) 4 AL3535 pAL1834 lacZ, P.sub.T7: sfgfp 5 AL3600 As AL62, but ss9::PlacUV5:T7rnap 6 AL3601 As AL1050, but ss9::PlacUV5:T7rnap 7 AL3600 pAL1834 P.sub.T7:sfgfp 8 AL3601 pAL1834 P.sub.T7:sfgfp 9 AL3606 As Strain 6, but fucU 10 AL3659 As Strain 9, but lacZ 11 AL3659 pAL1779/pAL1817 fucU, lacZ, P.sub.T7:fkp-wbgL 12 AL3659 pAL1834 fucU, lacZ, P.sub.T7:sfgfp 13 AL3585 As AL1050, but fucU 14 AL3664 As Strain 13, but lacZ 15 AL3732 As Strain 14, but ss9::P.sub.lacUV5:T7rnap 16 AL3732 pAL1834 fucU, lacZ, ss9::P.sub.lacUV5:T7rnap, P.sub.T7:sfgfp 17 AL1050 pAL421 P.sub.LlacO1:sfgfp 18 AL3664 pAL421 fucU, lacZ, P.sub.LlacO1:sfgfp 19 AL3732 pAL421 fucU, lacZ, ss9::P.sub.lacUV5:T7rnap, P.sub.LlacO1:sfgfp 20 AL1050 pAL2054 P.sub.lacUV5:sfgfp 21 AL3585 pAL2054 fucU, P.sub.lacUV5:sfgfp 22 AL3664 pAL2054 fucU, lacZ, P.sub.lacUV5:sfgfp 23 AL3664 pAL1759/pAL1760 fucU, lacZ, P.sub.LlacO1:fkp-wbgL, P.sub.LtetO1:Hp3/4ft 24 AL3664 pAL2027/pAL1760 fucU, lacZ, P.sub.LlacO1:fkp-wbgL, BBa_K1824896:lacY, P.sub.LtetO1:Hp3/4ft 25 AL3664 pAL2028/pAL1760 fucU, lacZ, P.sub.LlacO1:fkp-wbgL, BBa_K1824896:fucP, P.sub.LtetO1:Hp3/4ft 26 AL3664 pAL2029/pAL1760 fucU, lacZ, P.sub.LlacO1:fkp-wbgL, BBa_K1824896:lacY-fucP, P.sub.LtetO1:Hp3/4ft 27 AL3664 pAL2059/pAL1760 fucU, lacZ, P.sub.LlacO1:fkp, BBa_K1824896:lacY-fucP, P.sub.LtetO1:Hp3/4ft

TABLE-US-00002 TABLE 2 Plasmid list Plasmid Genotype Reference pAL421 P.sub.LlacO1:sfgfp; ColE1; amp.sup.r This study pAL1759 P.sub.LlacO1:fkp-wbgL; ColE1; amp.sup.r This study pAL1760 P.sub.LtetO1:Hp3/4ft; ColA; kan.sup.r This study pAL1779 P.sub.T7: fkp-wbgL; pBR322; amp.sup.r This study pAL1817 P.sub.T7:Hp3/4ft; ColA; kan.sup.r This study pAL1834 P.sub.T7:sfgfp; pBR322; amp.sup.r This study pAL2027 BBa_K1824896:lacY; P.sub.LlacO1:fkp-wbgL; This study pBR322; amp.sup.r pAL2028 BBa_K1824896:fucP; P.sub.LlacO1:fkp-wbgL This study pBR322; amp.sup.r pAL2029 BBa_K1824896:lacY-fucP; P.sub.LlacO1:fkp-wbgL; This study pBR322; amp.sup.r pAL2054 P.sub.lacUV5:sfgfp; ColE1; amp.sup.r This study pAL2059 BBa_K1824896:lacY-fucP; P.sub.LlacO1:fkp; pBR322; This study amp.sup.r
Other plasmids information is in Table 4.

TABLE-US-00003 TABLE 3 Strains used in this study Strain Genotype Source XL-1 Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac Agilent [F proAB lacIq ZM15 Tn10 (tet.sup.r)] BL21 Star F.sup. ompT hsdS.sub.B (r.sub.B.sup., m.sub.B.sup.) gal dcm rne131 (DE3) ThermoFisher (DE3) (AL15) BW25113 Z1 lacI.sup.+rrnB.sub.T14 lacZ.sub.WJ16 hsdR514 This study (AL62) araBAD.sub.AH33 rhaBAD.sub.LD78 rph-1 (araB-D)567 (rhaD-B)568 lacZ4787(::rrnB- 3) hsdR514 rph-1 attB::lacI.sup.q tetR spec.sup.r MG1655 Z1 F lambda- ilvG- rfb-50 rph-1 attB::lacI.sup.q tetR spec.sup.r (Yoneda et al. (AL1050) 2014) AL3271 As BW25113, but F [proAB lacIq ZM15 Tn10 (tet.sup.r)] This study fucU AL3535 As BL21 Star (DE3), but lacZ This study AL3585 As AL1050, but fucU This study AL3600 As AL62, but ss9::P.sub.lacUV5:T7rnap This study AL3601 As AL1050, but ss9::P.sub.lacUV5:T7rnap This study AL3606 As AL3601, but fucU This study AL3659 As AL3606, but lacZ This study AL3664 As AL3585, but lacZ This study AL3732 As AL3664, but ss9::P.sub.lacUV5:T7rnap This study

TABLE-US-00004 TABLE 4 Plasmids used in this study Plasmid Genotype Source pCas P.sub.cas:cas9 P.sub.araB:Red lacI.sup.q P.sub.trc:sgRNA Addgene #62225 pMB1 repA101(Ts) kan.sup.r (Jiang et al., 2015) pTargetF sgRNA-pmB1 pMB1 spec.sup.r Addgene #62226 (Jiang et al., 2015) pss9 integration HR1.sup.#-P.sub.T7A1:gfpUV-HR2{circumflex over ()} pBR322 tet.sup.r Addgene #71655 template (Bassalo et al., 2016) pAL421 P.sub.LlacO1:sfgfp ColE1 amp.sup.r This study pAL631 P.sub.LlacO1:sfgfp ColE1 kan.sup.r This study pAL1023 P.sub.LtetO1 ColA kan.sup.r This study pAL1354 P.sub.LlacO1 ColE1 amp.sup.r This study pAL1687 P.sub.T7:fkp pBR322 amp.sup.r This study pAL1688 P.sub.T7:wbgL pBR322 amp.sup.r This study pAL1689 P.sub.T7:Hp3/4ft pBR322 amp.sup.r This study pAL1759 P.sub.LlacO1:fkp-wbgL ColE1 amp.sup.r This study pAL1760 P.sub.LtetO1:Hp3/4ft ColA kan.sup.r This study pAL1762 sgRNA-ss9 pMB1 spec.sup.r This study pAL1779 P.sub.T7:fkp-wbgL pBR322 amp.sup.r This study pAL1817 P.sub.T7:Hp3/4ft ColA kan.sup.r This study pAL1783 HR1.sup.#-P.sub.T7A1:gfpUV-HR2{circumflex over ()} pBR322 amp.sup.r This study pAL1834 P.sub.T7:sfgfp pBR322 amp.sup.r This study pAL1845 lacZ HR1.sup.#-HR2{circumflex over ()} ColE1 amp.sup.r This study pAL1846 sgRNA-lacZ pMB1 spec.sup.r This study pAL1851 sgRNA-lacZ pMB1 amp.sup.r This study pAL1853 sgRNA-ss9 pMB1 amp.sup.r This study pAL1854 P.sub.lacUV5:lacZ-T7rnap ColE1 amp.sup.r This study pAL1855 P.sub.lacUV5:T7rnap ColE1 amp.sup.r This study pAL1856 HR1-P.sub.lacUV5:T7rnap-HR2 pBR322 amp.sup.r This study pAL1864 sgRNA-fucU pMB1 amp.sup.r This study pAL2026 BBa_K1824896*, P.sub.LlacO1:fkp-wbgL This study colE1 amp.sup.r pAL2027 BBa_K1824896*:lacY, P.sub.LlacO1;fkp-wbgL This study ColE1 amp.sup.r pAL2028 BBa_K1824896*:fucP, P.sub.LlacO1:fkp-wbgL This study ColE1 amp.sup.r pAL2029 BBa_K1824896*:lacY-fucP, P.sub.LlacO1:fkp- This study wbgL ColE1 amp.sup.r pAL2054 P.sub.lacUV5:sfgfp ColE1 amp.sup.r This study pAL2059 BBa_K1824896*:lacY, P.sub.LlacO1:fkp ColE1 This study amp.sup.r .sup.#upstream homologous region, {circumflex over ()}downstream homologous region, *iGEM part #: BBa_K1824896

TABLE-US-00005 TABLE5 Oligonucleotidesusedinthisstudy SEQID Plasmid(s) UsedforPCR NO Name Sequence5.fwdarw.3 produced and/orsequencing 27 AZ52 GTCTTGTCGATCAGGATGATC pAL1817 28 AZ55 CGAGCCCGTATAAACTGAAAGC pAL1760 29 AZ56 CTAGGTCTAGGGCGGCGGATTTG pAL1759, pAL1845, pAL1854,pAL2054 30 AZ57 CGTAAGATACTGACAGAAAACGC pAL1759, pAL1779,pAL2059 31 AZ60 GGAGGAAGGAAAGAATATCTGG pAL1759 32 AZ61 GTGACTTTATTGGCTGCTATTCC pAL1759 33 AZ64 CAAATAGGGGTTCCGCGCACAT pAL1759, pAL1779, pAL1854,pAL1855 34 AZ65 GATATGACTGTTCTCGATCCA pAL1759 35 AZ82 CCCTGGCAAATGTTGATTGA fucUupstream 36 AZ83 CAGGCTGTTACCAAAGAAGT fucUdownstream 37 AZ105 CGGCCTTATTGTCTCTCTGC pAL1817 38 AZ154 CCTAGGTCTAGGGCGGCGGATTTG pAL2059 39 AZ155 CATTATAACATTCTTCAAGCAGCC pAL2026,pAL2059 40 AZ224 AATTCATTAAAGAGGAGAAAAGATATA pAL1759 CCATGGGCAGCAG 41 AZ225 CATATGTATATCTCCTTCTTTTATGATCG pAL1759 TGATACTTGGAATC 42 AZ226 AAGAAGGAGATATACATATGAGCATTA pAL1759 TTCG 43 AZ227 TTAGCAGCCGGATCTCAGTG pAL1759 44 AZ228 CACTGAGATCCGGCTGCTAAGGTACCTA pAL1759 ATCTAGAGGCATC 45 AZ229 TTTCTCCTCTTTAATGAATTCGGTCAGTG pAL1759 CGTCC 46 AZ230 AATTCATTAAAGAGGAGAAACATATGTT pAL1760 CCAACCGCTGCTG 47 AZ231 CTCTAGAGTCATTAGGTACCGCTTTGTT pAL1760 AGCAGCCGGATC 48 AZ233 TTTCTCCTCTTTAATGAATTCGG pAL1760 49 AZ259 GAATTCGGTCAGTGCGTCCTGCTG pAL2027,pAL2028 50 AZ274 AAGGATCCGGCTGCTAACAAAAGGAGA pAL1779 TATACATATGAGC 51 AZ275 ACTCAGCTTCCTTTCGGGCTAGCAGCCG pAL1779 GATCTCAGTG 52 AZ276 AGCCCGAAAGGAAGCTGAGTTGGCTGC pAL1779 TG 53 AZ277 TTGTTAGCAGCCGGATCCTTATGATCGT pAL1779 GATACTTG 54 AZ293 ATGATTGAACAAGATGGATTGCACGC pAL1817 pAL1817 55 AZ294 AGGAGAGCGTTCACCGACAAAACGCCA pAL1817 GCAACGCGG 56 AZ295 AATCCATCTTGTTCAATCATACTCTTCCT pAL1817 TTTTCAATATTATTGAAGCATTTATCAG GG 57 AZ307 CACTTTACTACCCACGCCGC BL21Star(DE3) attBlocus 58 AZ308 GACTGGCAGCAACAGGTGGC BL21Star(DE3) attBlocus 59 AZ309 GTTGAGCTACAGGCGGTCAG ss9::P.sub.lacUV5:T7rnap 60 AZ310 ATTTACTAACTGGAAGAGGC pAL1854, pAL1856, ss9::P.sub.lacuV5:T7rnap 61 AZ311 CATTGAGTCAACCGGAATGG pAL1854, pAL1856, ss9::.sub.PlacuV5:T7rnap 62 AZ312 AAACCAATCGGTAAGGAAGG pAL1854, pAL1856, ss9::P.sub.lacUV5:T7rnap 63 AZ313 TTTTACCGTTCACGCGCTGG ss9::P.sub.lacUV5:T7rnap 64 AZ336 TGGTGCCGCGCGGCAGCCATATGGGTCA pAL1834 TCACCACCATCATC 65 AZ337 TTCGGGCTAGCAGCCGGATCTTATTTGT pAL1834 ACAGTTCGTCCATGCCG 66 AZ338 GATCCGGCTGCTAGCCCGAAAGGAAGC pAL1834 TGAGTTGGCTG 67 AZ339 ATGGCTGCCGCGCGGCACCAG pAL1834 68 AZ340 AATGCGCGCCATTACCGAGTCCG pAL1845 lacZupstream 69 AZ341 AGCTGTTTCCTGTGTGAAATTGTTATCC pAL1845 GC 70 AZ342 ATTTCACACAGGAAACAGCTTAATAACC pAL1845 GGGCAGGCCATGTCTG 71 AZ343 ACTTTCTCAATAAATGCCTCTACTGCTG pAL1845 lacZdownstream GCGCACC 72 AZ344 GAGGCATTTATTGAGAAAGTTAATCTAG pAL1845 AGGCATCAAATAAAACGAAAGGCTCAG TCG 73 AZ345 ACTCGGTAATGGCGCGCATTGGTCAGTG pAL1845 CGTCCTGCTGATG 74 AZ347 GCCGACACCAGTTTTAGAGCTAGAAATA pAL1846 G 75 AZ348 TCCGCCGCCTACTAGTATTATACCTAGG pAL1846 ACTGAG 76 AZ359 CAGCGGTGGAGTGCAATGTCATGAGTAT pAL1851 TCAACATTTCCG 77 AZ360 ATCGACTGGCGAGCGGCATCTTACCAAT pAL1851 GCTTAATCAGTG 78 AZ361 GATGCCGCTCGCCAGTCGATTGGC pAL1851 79 AZ362 GACATTGCACTCCACCGCTGATGAC pAL1851 80 AZ364 TCCGGATTTACTAACTGGAAGAGGCACT pAL1855 AAATG 81 AZ365 AGCTGTTTCCTGTGTGAAATTGTTATCC pAL1855 GCTC 82 AZ366 CCTTTCGTCTTCACCTCGAGTCACTCATT pAL1854 AGGCACCCCAGGC 83 AZ367 GGTACCTTAGCAGCCGGATCTTACGCGA pAL1854 ACGCGAAGTCCGAC 84 AZ368 GATCCGGCTGCTAAGGTACCTAATCTAG pAL1854 AGGC 85 AZ369 CTCGAGGTGAAGACGAAAGGGCCT pAL1854 86 AZ370 AGTTGATATGTCAAACAGGTTCACTCAT pAL1856 TAGGCACCCCAGGC 87 AZ371 CGGCGCTCAGTTGGAATTCAACAACAGA pAL1856 TAAAACGAAAGGCC 88 AZ372 TGAATTCCAACTGAGCGCCGGTC pAL1856 89 AZ373 ACCTGTTTGACATATCAACTGCGCC pAL1856 90 AZ384 GTGATGATGGGTTTTAGAGCTAGAAATA pAL1864 GC 91 AZ385 CAGCGGCGGTACTAGTATTATACCTAGG pAL1864 AC 92 AZ403 ACTCTTCCTTTTTCAATATTATTGAAGCA pAL2027, TTTATCAGGG pAL2028, pAL2029, pAL2054,pAL2059 93 AZ411 CGCGCGGCACACTAGTATTATACCTAGG pAL2029 AC 94 AZ710 GTGCCACCTGACGTCTAAGACTAGTACT pAL2026 pAL2026 CTAGTATTTCTCCTCTTTA 95 AZ711 GCTACTAGAGTACTAGAGTACTAGAGAT pAL2026 TAAAGAGGAGAAATACTAGAGTACTAG TCTTA 96 AZ712 TCTCTAGTACTCTAGTACTCTAGTAGCT pAL2026 AGCACTGTACCTAGGACTGAGCTAGCCG T 97 AZ713 ACGCCTATTTTTATAGGTTAATGTCATG pAL2026 ATAATAATGGTTTTGACGGCTAGCTCAG TCC 98 AZ714 TGACATTAACCTATAAAAATAGGCGTAT pAL2026 CACGAGGCCCTTTCGTCTTCACCTCGAG AAT 99 AZ715 TTGTTATCCGCTCACAATGTCAATTGTTA pAL2026 pAL2026 TCCGCTCACAATTCTCGAGGTGAAGACG AA 100 AZ716 CAATTGACATTGTGAGCGGATAACAAG pAL2026 101 AZ717 TCTTAGACGTCAGGTGGCACTTTTCG pAL2026, pAL2027, pAL2028. pAL2029 102 AZ718 GTGCCACCTGACGTCTAAGATTAAGCGA pAL2027 CTTCATTCACCTG 103 AZ719 GAGAAATACTAGAGTACTAGATGTACTA pAL2027 TTTAAAAAACACAAACTTTTGGATG 104 AZ720 CTAGTACTCTAGTATTTCTCCTCTTTAAT pAL2027, CTCTAGTAC pAL2028 105 AZ721 GTGCCACCTGACGTCTAAGATCAGTTAG pAL2028, TTGCCGTTTGAGAAC pAL2029 106 AZ722 GAGAAATACTAGAGTACTAGATGGGAA pAL2028 ACACATCAATACAAACGCAGAG 107 AZ723 GAAAGAGGGGACAAACTAGTATGGGAA pAL2029 ACACATCAATACAAACG 108 AZ724 TTGTCCCCTCTTTCTCTAGATTAAGCGAC pAL2029 TTCATTCACCTGACG 109 AZ819 CTAACTGGAAGAGGCACTAAATGGGTC pAL2054 ATCACCACCATCATCACG 110 AZ820 GGTACCTTAGCAGCCGGATCTTATTTGT pAL2054 ACAGTTCGTCCATGCCG 111 AZ821 GATCCGGCTGCTAAGGTACCTAATC pAL2054 112 AZ822 TTAGTGCCTCTTCCAGTTAGTAAATCCG pAL2054 G 113 AZ851 CTGCTAAGGTACCTAATCTAGAGGCATC pAL2059 114 AZ852 CCGGATCTTATGATCGTGATACTTGGAA pAL2059 TC 115 JO232 GGTTCCGCGCACATTTCCC pAL1845 116 MMM40 GAGTCAGTGAGCGAGGAAGC pAL1846, pAL1851,pAL1864 117 MMM131 GCTTGGTTGAGAATACGCCG pAL1856,ss9 upstream 118 MMM132 GCCTACGATTACGCATGGCTTG pAL1856,ss9 downstream 119 SD62 GGCCCTTTCGTCTTCACCTCGAG pAL1760 120 SL005 AACGCAGTCAGGCACCGTGTATGAGTAT pAL1783 pAL1783 TCAACATTTCCG 121 SL006 GAGGTGCCGCCGGCTTCCATTTACCAAT pAL1783 pAL1783 GCTTAATCAGTG 122 SL007 ATGGAAGCCGGCGGCACCTC pAL1783 123 SL008 ACACGGTGCCTGACTGCGTTAGC pAL1783 124 YT167 TAATGACTCTAGAGGCATCAAATAA pAL1760 125 YT054 TTGTCGGTGAACGCTCTCCTG pAL1817 pAL1817 126 YT400 ATGGGTCATCACCACCATCATCA pAL1834 127 YT430 CCAGTAGTAGGTTGAGGCCGTTGAG pAL1834 128 YT092 CTACTCAGGAGAGCGTTCAC pAL1760 129 YT101 GCTTCCCAACCTTACCAGAG pAL1760 130 YTC427 CAAGCAGCAGATTACGCGCAG pAL1851

TABLE-US-00006 TABLE6 GuideforCRISPR-Cas9-mediategenedeletionsandinsertions pTargetF PCRLinearRepairFragment Modification Plasmid 20bpsgRNAsequence5.fwdarw.3 Primers Template fucU pAL1864 ACCGCCGCTGGTGATGATGG AZ82(F),AZ83 AL3271 (SEQIDNO:131) (R) gDNA lacZ pAL1851 AGGCGGCGGAGCCGACACCA AZ340(F), pAL1845 (SEQIDNO:132) AZ343(R) ss9::P.sub.lacUV5:T7rnap pAL1853 TCTGGCGCAGTTGATATGTA MMM131(F), pAL1856 (SEQIDNO:133) MMM132(R)

[0091] Plasmids for sfGFP fluorescence assays, LDFT production, and 3-FL production were constructed using sequence and ligation independent cloning (SLIC) (Li and Elledge, 2007). Plasmids encoding sgRNAs for CRISPR-Cas9-mediated homologous recombination were constructed with Q5 site-directed mutagenesis using a modified template pTargetF (Addgene plasmid #62226). Templates used for DNA amplification and cloning are listed in Table 7. All plasmids were verified by PCR and Sanger sequencing. Culture conditions

[0092] Overnight cultures were grown at 37 C., 250 rpm, in 3 mL of Luria-Bertani (LB) media with appropriate antibiotics. Antibiotic concentrations were as follows: spectinomycin (50 g/mL), ampicillin (200 g/mL), and kanamycin (50 g/mL). Growth assays were carried out in M9 minimal medium (33.7 mM Na.sub.2HPO4, 22 mM KH.sub.2PO4, 8.6 mM NaCl, 9.4 mM NH4Cl, 1 mM MgSO.sub.4, 0.1 mM CaCl.sub.2)) including 1000A5 trace metal mix (2.86 g H.sub.3BO.sub.3, 1.81 g MnCl.sub.2.Math.4H2O, 0.079 g CuSO.sub.4.Math.5H2O, 49.4 mg Co(NO.sub.3).sub.2.Math.6H2O per liter water). LDFT production was carried out in M9 minimal medium supplemented with 5 g/L yeast extract (M9P). Optical densities were measured at 600 nm (OD.sub.600) with a Synergy H1 hybrid plate reader (BioTek Instruments, Inc.).

Growth Assays

[0093] Overnight cultures were inoculated at 1% in 3 mL of M9 minimal medium supplemented with 1 g/L D-lactose or 1 g/L L-fucose. Cultures were grown at 37 C., 250 rpm, for 24 h and OD.sub.600 was measured.

Fluorescence Assays

[0094] Overnight cultures were inoculated at 1% in 3 mL of LB media and grown at 37 C., 250 rpm, until OD.sub.600 reached 0.4-0.6. Cultures were respectively induced with isopropyl -D-1-thiogalactopyranoside (IPTG, 1.0 mM) and grown at 37 C., 250 rpm, for 24 h.

TABLE-US-00007 TABLE 7 Plasmid construction guide PCR for Vector PCR for Insert(s) Primer Primer Sequence of Plasmid (F) (R) Template Primer (F) Primer (R) Template Interest pAL1759 AZ228 AZ229 pAL1354 AZ224 AZ225 pAL1687 fkp AZ226 AZ227 pAL1688 wbgL pAL1760 YT167 AZ233 pAL1023 AZ230 AZ231 pAL1689 Hp3/4ft pAL1779 AZ276 AZ277 pAL1687 AZ274 AZ275 pAL1688 wbgL pAL1817 AZ294 AZ295 pAL1689 AZ293 YT054 pAL1023 ColA-kan.sup.r pAL1762* MMM139 MMM140 pTargetF pAL1783 SL007 SL008 pss9 SL005 SL006 pAL1354 amp.sup.r pAL1845 AZ344 AZ345 pAL1354 AZ340 AZ341 AL1050 gDNA 400 bp upstream HR1 lacZ AZ342 AZ343 AL1050 gDNA 400 bp downstream HR2 lacZ pAL1854 AZ368 AZ369 pAL1759 AZ366 AZ367 BL21 Star (DE3) P.sub.lacUV5:lacZ gDNA T7rnap pAL1855* AZ364 AZ365 pAL1854 pAL1856 AZ372 AZ373 pAL1783 AZ370 AZ371 pAL1855 P.sub.lacUV5:T7rnap pAL1846* AZ347 AZ348 pTargetT pAL1851 AZ361 AZ362 pAL1846 AZ359 AZ360 pAL1687 amp.sup.r pAL1853 AZ361 AZ362 pAL1762 AZ359 AZ360 pAL1687 amp.sup.r pAL1864* AZ384 AZ385 pAL1851 pAL1834 AZ338 AZ339 pAL1687 AZ336 AZ337 pAL421 sfgfp pAL2026 AZ716 AZ717 pAL1759 AZ710, AZ712, AZ711, AZ713, N/A BBa_K1824896 AZ714 AZ715 pAL2027 AZ720 AZ717 pAL2026 AZ718 AZ719 AL1050 gDNA lacY pAL2028 AZ721 AZ722 pAL2026 AZ718 AZ719 AL1050 gDNA fucP pAL2029 AZ724 AZ717 pAL2027 AZ721 AZ723 AL1050 gDNA fucP pAL2054 AZ821 AZ822 pAL1855 AZ819 AZ820 pAL631 sfgfp pAL2059* AZ851 AZ852 pAL2029 *Q5-site directed mutagenesis (NEB).
Fluorescence emission was measured at 510 nm with a Synergy H1 hybrid plate reader (BioTek Instruments, Inc.).

LDFT Production

[0095] Overnight cultures were inoculated at 1% in 3 mL of M9P supplemented with 5 g/L glucose, 10 g/L glycerol, or 20 g/L glycerol. Cultures were grown at 37 C., 250 rpm, until OD.sub.600 reached 0.4-0.6. Appropriate concentrations of lactose, L-fucose, IPTG, and anhydrotetracycline (aTc) were added and the cultures were grown at 30 C., 250 rpm, for 24 h. The produced LDFT was confirmed by high resolution electrospray ionization mass spectrometry using a Thermo Electron LTQ-Orbitrap Hybrid MS at the Mass Spectrometry Facility in the University of California, Davis.

HPLC Analysis

[0096] To measure glycerol, L-fucose, lactose, 2-FL, 3-FL, and LDFT, cell culture supernatant was analyzed using HPLC (Shimadzu) equipped with a refractive index detector (RID) 10 A and a Luna Omega HILIC Sugar column (Phenomenex). The mobile phase consisted of 100% 70:30 HPLC-grade acetonitrile:MilliQ water was run at a flow rate of 1.0 mL/min for 12 min, with the column oven at 35 C. and RID cell temperature at 40 C.

[0097] To prepare samples for HPLC analysis, 125 L of culture was collected and spun down at 17,000 g for 5 min. 15 L of culture supernatant or compound standard in water was diluted with 45 L of MilliQ water and 180 L of acetonitrile. The mixture was vortexed and spun down at 17,000 g for 5 min. 40 L of each sample was injected into the column for analysis.

Results

[0098] Pathway Design for LDFT Production in E. coli

[0099] HMO production does not naturally occur in E. coli, therefore the following three enzymes were employed for the production of LDFT: a bifunctional L-fucokinase/GDP-L-fucose pyrophosphorylase (Fkp) from Bacteroides fragilis (Yi et al., 2009), an 1-2-fucosyltransferase (WbgL) from E. coli 0126 (Engels and Elling, 2014; McArthur et al., 2019), and 1-3/4-fucosyltransferase (Hp3/4FT) from Helicobacter pylori UA948 (Rasko et al., 2000; Yu et al., 2017). Acceptor substrate specificity studies of both WbgL and Hp3/4FT have been reported (Engels and Elling, 2014; Ma et al., 2006; McArthur et al., 2019; Yu et al., 2017). WbgL exhibits high activity towards non-fucosylated acceptor substrates, such as lactose, N-acetyllactosamine (LacNAc), and lactulose, and no activity towards 3-FL. Hp3/4FT has been shown to be highly active towards LacNAc and 2-fucosyl-LacNAc with low activity towards lactose. The acceptor preferences of the fucosyltransferases allow sequential fucosylation of lactose for the formation of LDFT in the presence of both fucosyltransferases. Fkp uses one ATP and GTP to convert L-fucose to GDP-fucose, which is taken as a donor substrate by WbgL to fucosylate lactose at the C2 position, forming the intermediate 2-FL (FIG. 1). Due to its structural similarity to 2-fucosyl-LacNAc, 2-FL was hypothesized to be a suitable acceptor substrate for fucosylation by Hp3/4FT to produce LDFT, which is expected to be secreted to the supernatant by native membrane exporter SetA (Liu et al., 1999).

LDFT Production in E. coli B Strains

[0100] The relatively low soluble expression level of recombinant fucosyltransferases was of initial concern as a potential cause of bottlenecks for synthesizing fucosylated HMOs in microbial hosts (Nidetzky et al., 2018). In this study, the C-terminal 34-amino acid hydrophobic sequence of Hp3/4FT was truncated to increase its solubility (Yu et al., 2017). To increase the expression of fucosyltransferases, E. coli B strain BL21 Star (DE3) was selected as an LDFT production host. BL21 Star (DE3) is widely used for recombinant protein expression and is capable of high expression via the two-step IPTG-inducible T7 bacteriophage promoter (Rosano and Ceccarelli, 2014). The fkp and wbgL genes were cloned together into an expression vector under a T7-promoter (P.sub.T7, pAL1779, Table 2) and the truncated Hp3/4 ft gene was cloned into a second expression vector under P.sub.T7 (pAL1817, Table 2).

[0101] Lactose and L-fucose were used as starting substrates for LDFT production, but E. coli is known to catabolize these two sugars for growth. It was hypothesized that minimizing assimilation of L-fucose and lactose for cellular growth would contribute to maximization of LDFT production. Therefore, the strain's ability to grow on these two carbon sources was evaluated to determine which carbon assimilating pathways to remove. Although the BL21 Star (DE3) encodes all genes involved in L-fucose degradation, the strain was not able to grow on L-fucose as the sole carbon source (FIG. 2A). The strain was able to grow on lactose as the sole carbon source (FIG. 2A). When the lacZ gene encoding for a -galactosidase was deleted in the strain (Table 1: Strain 1), lactose did not enable growth anymore (FIG. 2).

[0102] The two plasmids containing the LDFT production pathway (pAL1779 and pAL1817, Tables 2 & 4) were introduced into Strain 1 to form Strain 2 (Table 1). To determine the best carbon source for growth and production, Strain 2 was grown in parallel with glucose, a common feedstock known for its catabolite repression towards lactose importation (Bruckner and Titgemeyer, 2002), and glycerol, an inexpensive feedstock that does not cause catabolite repression. Under both of these culturing conditions, Strain 2 did not produce LDFT nor its precursor, 2-FL. To examine the expression from P.sub.T7, the plasmid containing sfgfp under P.sub.T7 (pAL1843, Table 2) was introduced into BL21 Star (DE3) and Strain 1 to form Strains 3 and 4, respectively (Table 1). Strain 3 produced a strong fluorescent signal after IPTG induction while Strain 4 did not produce fluorescence signal in either induction conditions, suggesting that T7 RNA polymerase expression was lacking (FIG. 2B). Sequencing of the attB integration locus in Strain 1 revealed an excision of the DE3 lysogen containing P.sub.lacUV5:lacZ-T7rnap. Several attempts were made to remove lacZ from BL21 Star (DE3) without off-target modifications to the DE3 lysogen but resulted in failure.

Introduction of the T7 RNAP Gene into K-12 Derivative Strains

[0103] Due to difficulties in genetically modifying BL21 Star (DE3), P.sub.lacUV5:T7rnap was integrated into the E. coli K-12 derivative strains, BW25113 Z1 and MG1655 Z1 (Table 3). The Z1 fragment containing laci.sup.q, tetR, and spec.sup.r was integrated into the attB site of these strains. It has been shown that many regions in the E. coli genome are stable and high-efficiency integration sites for heterologous genes (Bassalo et al., 2016), therefore intergenic locus ss9 was chosen as the insertion site for P.sub.lacUV5:T7rnap. The P.sub.lacUV5:T7rnap cassette was integrated into ss9 of BW25113 Z1 and MG1655 Z1 to form Strains 5 and 6, respectively (Table 1).

[0104] pAL1834 containing P.sub.T7:sfgfp was introduced into Strains 5 and 6 to form Strains 7 and 8, respectively (Table 1) to assess the repression and induction efficiencies of P.sub.T7 through a fluorescence assay. Tight repression of GFP expression without IPTG was observed in Strains 7 and 8 (FIG. 3A). IPTG induction in Strains 7 and 8 increased GFP fluorescence 95-fold and 440-fold, respectively (FIG. 3A). Strain 6 was chosen as the base strain for further genetic modification due to its tighter repression and stronger inducibility of P.sub.T7. The growth of Strain 6 on L-fucose and lactose was tested. Strain 6 was able to grow on L-fucose or lactose as a sole carbon source (FIG. 3B). To remove L-fucose and lactose assimilation, fucU encoding an L-fucose mutarotase and lacZ were deleted to form Strain 10 (Table 1). Strain 10 was not able to grow on L-fucose or lactose as a sole carbon source (FIG. 3B).

[0105] The LDFT production plasmids (pAL1779 and pAL1817, Table 2) were introduced into Strain 10 to form Strain 11 (Table 1). Strain 11 was grown to test LDFT production from lactose and L-fucose. Glucose or glycerol was used to maintain cellular growth. Under both conditions, LDFT was not produced in Strain 11. This prompted the examination of the T7 RNA polymerase expression system in Strain 10. pAL1834 containing P.sub.T7:sfgfp was introduced into Strain 10 to form Strain 12 (Table 1). Strain 12 produced strong GFP fluorescence without IPTG induction, indicating the expression from P.sub.T7 was leaky in Strain 12 (FIG. 3C). In Strain 10, a mutation in the promoter region of the P.sub.lacUV5:T7rnap cassette was found. The deletion of lacZ in Strain 9 without incurring P.sub.lacUV5 mutations was attempted several times, but the attempts were unsuccessful. Without wishing to be bound by any particular theory, it is believed that that the mutations in P.sub.lacUV5 are correlated with the CRISPR-Cas9-mediated gene removal of lacZ due to the similarity of the lacZ promoter to P.sub.lacUV5.

[0106] To avoid the potential sequence similarity issues observed for P.sub.lacUV5 and the native lacZ promoter, the three modifications into MG1655 Z1 were introduced in a different order. First, fucU and lacZ in MG1655 Z1 were deleted to form Strain 13 (fucU) and Strain 14 (fucU lacZ)). Then, P.sub.lacUV5: T7rnap was integrated into the ss9 locus to form Strain 15 (Table 1). Strain 15 was unable to grow on L-fucose or lactose as a sole carbon source (FIG. 3D). Although the P.sub.lacUV5:T7rnap cassette in Strain 15 had no mutations, Strain 15 with pAL1834 harboring P.sub.T7:sfgfp (Table 1: Strain 16) showed leaky GFP expression without IPTG. To determine if other lac-based promoters are deregulated by the strain modifications, pAL421 containing P.sub.LlacO1:sfgfp was introduced into MG1655 z1, Strains 14 and 15 to form Strains 17, 18, and 19, respectively (Table 1) to assess the regulation of the lac-based promoter in these strains. The expressions from P.sub.LlacO1 without IPTG were well repressed in Strains 17, 18 and 19 (FIG. 4A). Next, pAL2045 containing P.sub.lacUV5:sfgfp was introduced into MG1655 z1, Strains 13 and 14 to form Strains 20, 21, and 22, respectively (Table 1). The expression of sfgfp in Strains 21 and 22 was leakier than that in Strain 20 (FIG. 4B), suggesting that the deletion of fucU caused the leaky expression of P.sub.lacUV5.

Production of LDFT in K-12 Derivative Strains

[0107] Rather than pursuing alternative promoters for T7rnap, other induction systems for the LDFT biosynthetic pathway genes were used. The fkp and wbgL genes were cloned under P.sub.LlacO1 (pAL1759, Tables 2 & 4) and the Hp3/4 ft gene was cloned under an aTc-inducible promoter P.sub.LetO1 (pAL1760, Tables 2 & 4) (Lutz and Bujard, 1997). The LDFT production plasmids (pAL1759 and pAL1760) were introduced to Strain 14 to form Strain 23 (Table 1). Strain 23 was grown in M9P containing L-fucose and lactose with glucose or glycerol. After 24 h, Strain 23 produced 0.08 g/L 2-FL and 0.16 g/L LDFT under the glycerol conditions, but neither were produced under the glucose conditions (FIG. 5).

Enhancing Substrate Levels by Overexpressing Transporter Genes

[0108] Intracellular availability of L-fucose and lactose is important for efficient LDFT production. It was hypothesized that additional expression of the substrate transporter genes would increase the substrate supply and improve LDFT production. The lactose and L-fucose membrane symporter genes, lacY and fucP, were expressed under a constitutive promoter (iGEM part No. BBa_K1824896, Tables 2 & 4). The lacY gene was expressed from the fkp-wbgL plasmid pAL2027 (Tables 2 & 4). The LDFT production plasmids with lacY (pAL2027 and pAL1760) were introduced into Strain 14 to form Strain 24 (Table 1) but the overexpression of lacY did not improve LDFT production (FIG. 6). The fucP gene was expressed from the fkp-wbgL plasmid pAL2028 (Table 2). The LDFT production plasmids with fucP (pAL2028 and pAL1760) were introduced into Strain 14 to form Strain 25 (Table 1). After 24 h, Strain 25 produced 0.9 g/L LDFT, a 6.9-fold improvement compared to Strain 23.

[0109] Next, both lacY and fucU were expressed from the fkp-wbgL plasmid pAL2029 (Table 2). The LDFT-production plasmids with lacY and fucU (pAL2029 and pAL1760) were introduced into Strain 14 to form Strain 26 (Table 1). Strain 26 produced 1.1 g/L LDFT after 24 h, representing 59% of the theoretical maximum yield (TMY) from lactose and accumulated 0.17 g/L 2-FL and/or 3-FL (FIG. 6). As the HPLC and the MS methods used were unable to discriminate between the two mono-fucosylated lactose, the combined concentrations of 2-FL and 3-FL are reported here.

Tuning of the Expression Levels of the LDFT Biosynthetic Pathway Genes

[0110] To fine-tune the nucleotide activation of L-fucose and the fucosylation reactions, a range of IPTG concentrations (0, 25, 50, 100, and 1,000 M) were screened for the expression of P.sub.LlacO1:fkp-wbgL in the presence of 100 ng/mL aTc for induction of P.sub.LtetO1:Hp3/4 ft. The best growth, greatest lactose and L-fucose consumption, and the highest level LDFT production (1.6 g/L, 89% of TMY) was observed with 50 M IPTG (FIG. 7A). A range of aTc concentrations (0, 25, 50 and 100 ng/mL) were tested for the LDFT production in the presence of 50 M IPTG to determine if adjusting Hp3/4FT expression levels could improve LDFT production. Strain 26 produced more LDFT with higher concentrations of aTc (FIG. 7B). Thus, the induction condition with 50 M IPTG and 100 ng/mL aTc was used for further studies.

Characterization of LDFT Production

[0111] The LDFT production profile in Strain 26 was characterized for 12 h post-induction by monitoring substrate, intermediate, side product, and LDFT levels using HPLC (FIG. 8). LDFT was first detected at 5 h, and between 5 to 10 h the production rate was 0.24 g/L/h (FIG. 8). Monofucosides (2-FL/3-FL) were accumulated up to 0.3 g/L until lactose was depleted at 8 h and remained constant at 0.3 g/L between 8 to 12 h. The lack of monofucoside consumption after 8 h indicated that most of the remaining monofucoside was 3-FL, which was the side product produced by Hp3/4FT from lactose that cannot be fucosylated further by WbgL to produce LDFT.

[0112] When WbgL and Hp3/4FT are expressed at the same time, both enzymes can compete to fucosylate lactose into 2-FL and 3-FL, respectively. In the presence of lactose and 2-FL, Hp3/4FT can also convert the respective acceptor substrates into 3-FL and LDFT. It was hypothesized that the delayed induction of Hp3/4 ft would decrease the competition between WbgL and Hp3/4FT for lactose and decrease the production of the side product, 3-FL. Therefore, delaying of the Hp3/4FT expression was tested by adding 100 ng/mL aTc at 2, 4, and 6 h. However, the delayed expressions of Hp3/4 ft resulted in increased monofucoside accumulation and decreased LDFT production (FIG. 9). This increase in monofucoside in the supernatant suggests that 2-FL formed by WbgL may be secreted to media and its reimport may be limited, which decreases the substrate availability of Hp3/4FT for LDFT production.

[0113] To examine the import efficiency of 2-FL, 2-FL was fed to the production cultures. The wbgL gene was removed from pAL2029 to form pAL2059 (Table 2). pAL2059 and pAL1760 were introduced into Strain 14 to form Strain 27 (Table 1). Strain 27 was grown in M9P with 10 g/L glycerol. Cultures were induced with 50 M IPTG and 100 ng/mL aTc and supplemented with 1.42 g/L of 2-FL (mole equivalent to 1 g/L lactose) and 0.5 g/L L-fucose. Lactose was not fed to the cultures and wbgL was not present in system, making it unlikely for Strain 27 to produce 2-FL and 3-FL. Under these conditions, LDFT should be produced only from the fed 2-FL. Strain 27 produced only 0.4 g/L LDFT in 24 h, further supporting that the import of 2-FL is not efficient in E. coli (FIG. 10).

LDFT Production with Higher Substrate Concentrations

[0114] Strain 26 consumed 1 g/L lactose within 8 h and LDFT production reached completion at 12 h post-induction (FIG. 8). To evaluate LDFT production with higher substrate concentrations, Strain 26 was grown in M9P with 20 g/L glycerol and various amounts of lactose and L-fucose (1, 2, or 3 g/L) for 24 h. In conditions with only lactose or L-fucose as the added substrate, Strain 26 did not produce any detectable amounts of fucosides. In the presence of both substrates, the increase in LDFT yield was nearly proportional to the increase of substrate concentrations (FIG. 11). Strain 26 consumed 3.0 g/L lactose and 2.6 g/L L-fucose and produced 5.1 g/L LDFT in 24 h. LDFT was produced at 910% of TMY.

Discussion

[0115] LDFT has been identified as an effective gastrointestinal and immunological modulator and has the potential to be developed to treat human diseases. Its high cost and limited commercial access make LDFT a desirable target for production in microbial hosts. Systems developed in E. coli, B. subtilis, and S. cerevisiae have successfully produced HMOs such as 2-FL, 3-FL, LNT, and LNnT, which represent only a small fraction of over 200 naturally occurring HMOs. Developing microbial production systems dedicated to synthesizing HMOs with a higher structural complexity is still challenging. In this study, a microbial system that specifically and efficiently produces LDFT was established.

[0116] The greatest challenge of this study was pairing an 1-2-fucosyltransferase with an 1-3-fucosyltransferase that can efficiently produce LDFT with minimal accumulation of monofucoside intermediates. WbgL was chosen to drive lactose fucosylation into 2-FL because it expresses well in E. coli and has been characterized to prefer 1-4-linked galactose substrates, such as lactose and LacNAc (Engels and Elling, 2014). From acceptor substrate screenings of 1-3-fucosylatransferases, Hp3/4FT was annotated with high activity towards 2-fucosyl LacNAc, which suggested 2-FL may also be a suitable acceptor for Hp3/4FT (Ma et al., 2006; Yu et al., 2017). Characterization of LDFT production as described herein demonstrated that Hp3/4FT had preferential activity towards 2-FL over lactose and LDFT was formed as the dominant product (FIG. 8). The presence of residual monofucosides indicates possible formation of the side product 3-FL, which is an unsuitable acceptor for WbgL (Engels and Elling, 2014). Monofucoside titers were relatively low and can be separated from LDFT in downstream purification processes. Other fucosyltransferases that may be employed include, but are not limited to, 1-2-fucosyltransferases such as Hm2FT (GenBank: CBG40460), E. coli 0128:B12 1-2-fucosyltransferase WbsJ (GenBank: AA037698.1), H. pylori UA1234 1-2-fucosyltransferase (Hp2FTa) (GenBank: AAD29863.1), H. pylori UA802 1-2-fucosyltransferase (Hp2FTb) (GenBank: AAC99764.1), and related eukaryotic 1-2-fucosyltransferases (see, e.g., cazy.org/GT11_characterized.html); as well as 1-3-fucosyltransferases such as H. pylori ATCC43504 1-3-fucosyltransferase (Hp3FT) (GenBank: AAB93985), H. pylori J99 1-3-fucosyltransferase (Hp3FT) (GenBank: AAD06169.1, AAD06573.1), H. pylori NCTC11639 1-3-fucosyltransferase (Hp3FT) (GenBank: AAB93985), and related eukaryotic 1-3-fucosyltransferases and 1-3/4-fucosyltransferases (see, e.g., cazy.org/GT10_characterized.html). It is possible to screen 1-3-fucosyltransferases for lower activity towards lactose and higher activity towards 2-FL, and also pursue protein engineering strategies to expand 1-2-fucosyltransferase's acceptor substrate range to 3-FL so that this side product can be fucosylated into LDFT.

[0117] The rate of LDFT formation was dictated by carbon catabolite repression (CCR) and the activity of sugar transporters, which firmly control the import of carbohydrates across the inner membrane (Grke and Stlke, 2008). It has been shown that import of glucose through the phosphotransferase system inhibits transcription of lac operon genes, including lacY. From the experiments described above, glucose conditions led to suppressed LDFT production while glycerol conditions resulted in improved LDFT production. This suggests glucose inhibits lactose import whereas glycerol allows for lactose import through sufficient lacY expression. Although glucose is a traditional carbon feedstock for microbial fermentation, it is unsuitable for HMO production systems that use lactose as a substrate. In the absence of CCR, LDFT production was still limited by the native expression levels of lacY and fucP (FIG. 6). Additional expression of fucP increased LDFT production by 6.9-fold to 0.9 g/L (FIG. 6), indicating that L-fucose import was one of the bottlenecks for LDFT production. While native expression levels of lacY without CCR were adequate for supplying lactose, overexpression of lacY and fucU further balanced the donor-acceptor substrate ratio and improved LDFT titers to 1.1 g/L in 24 h (FIG. 6).

[0118] Lastly, balancing expression levels of the LDFT biosynthetic pathway genes (fkp, wbgL, and Hp3/4 ft) increased efficiency of LDFT production. Decreasing expression of fkp reduces excessive ATP and GTP consumption in GDP-fucose production, potentially relieving the metabolic burden of regenerating nucleotide cofactors (FIG. 7A). Decreasing expression of wbgL helps synchronize 2-FL production with Hp3/4FT's slower turnover rate, streamlining 2-FL towards LDFT production (FIG. 7A). Decreasing or delaying Hp3/4 ft expression causes build-up of 2-FL, which is rapidly exported from the cell (FIG. 7B). It has been hypothesized that LacY is an importer for 2-FL (Shin et al., 2020), but enhanced lacY expression was still insufficient for LDFT production from 2-FL feeding (FIG. 10). Expression of additional heterologous importers may improve 2-FL transport. Fucosyllactose transporters have been identified in gut prebiotic Bifidobacterium species and are ideal candidates for screening in further studies to improve LDFT production (Sakanaka et al., 2019). Examples of such transporters include ABC transporters FL transporter-1 and FL transporter 2-from Bifidobacterium longum subsp. infantis ATCC 15697. FL transporter-1 is made up of the domains set forth in SEQ ID NOS:19-21 and transports 2-fucosyllactose and 3-fucosyllactose, while FL transporter-2 is made up of the domains set forth in SEQ ID NOS:22-24 and transports 2-FL, 3-FL, LDFT, and LNFP I.

[0119] Due to concerns about strain virulence for the production of bioactive compounds, the HMO production technologies can be translated to nonpathogenic generally-recognized-as-safe (GRAS) strains such as Bacillus subtilis, Corynebacterium glutamicum, and Saccharomyces cerevisiae (Becker et al., 2018; Kaspar et al., 2019; Lian et al., 2018). For example, lactose transporters can also be introduced into hosts such as C. glutamicum as described by Shen et al. (Microb Cell Fact (2019) 18:51). Expression of known FucU and LacZ homologes (e.g., B. subtilis homologs set forth in SEQ ID NO:25 and SEQ ID NO:26), can be reduced or eliminated as described above for E. coli. Alternatively, host cells such as S. cerevisiae which are not known to express FucU homologs would not require such modifications. Advancements in GRAS strains' synthetic biology toolbox such as genome editing, vector expression systems, and tuning of gene expression has improved their industrial application in producing nutraceuticals, food additives and biofuels. Some of these GRAS hosts also enable post-translational modification of enzymes and localization of proteins into organelles or on membranes. Development of GRAS HMO fucosylation systems would also forge production routes for other fucosylated compounds for pharmaceutical research.

REFERENCES

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IV. Exemplary Embodiments

[0172] Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the claims and the following embodiments: [0173] 1. A recombinant cell for production of an oligosaccharide product, the recombinant cell comprising: [0174] a polynucleotide encoding a first glycosyltransferase polypeptide having a first substrate selectivity, and [0175] a polynucleotide encoding a second glycosyltransferase polypeptide having a second substrate selectivity. [0176] 2. The recombinant cell of embodiment 1, further comprising one or more polynucleotides selected from the group consisting of: [0177] a polynucleotide encoding a nucleotide sugar pyrophosphorylase polypeptide, [0178] a monosaccharide transporter polypeptide, and [0179] an oligosaccharide transporter polypeptide. [0180] 3. The recombinant cell of embodiment 1 or 2, for production of an oligosaccharide comprising two or more fucose moieties, comprising: [0181] a polynucleotide encoding a first fucosyltransferase polypeptide having a first substrate selectivity, and [0182] a polynucleotide encoding a second fucosyltransferase polypeptide having a second substrate selectivity; [0183] and optionally comprising one or more of: [0184] a polynucleotide encoding a nucleotide sugar pyrophosphorylase polypeptide, [0185] a polynucleotide encoding a lactose transporter polypeptide, and [0186] a polynucleotide encoding an L-fucose transporter polypeptide. [0187] 4. The recombinant cell of embodiment 2 or embodiment 3, wherein the nucleotide sugar pyrophosphorylase polypeptide is a bifunctional glycokinase and nucleotide sugar pyrophosphorylase polypeptide. [0188] 5. The recombinant cell of embodiment 3 or embodiment 4, wherein the first fucosyltransferase polypeptide is an 1-2-fucosyltransferase polypeptide. [0189] 6. The recombinant cell of embodiment 5, wherein the 1-2-fucosyltransferase polypeptide is an E. coli 0126 1-2-fucosyltransferase WbgL polypeptide. [0190] 7. The recombinant cell of embodiment 5 or embodiment 6, wherein the 1-2-fucosyltransferase polypeptide is an E. coli O126 1-2-fucosyltransferase (WbgL) polypeptide (GenBank: ABE98421.1), an H. mustelae 12198 1-2-fucosyltransferase (Hm2FT) polypeptide (GenBank: CBG40460), an E. coli 0128:B12 1-2-fucosyltransferase (WbsJ) polypeptide (GenBank: AA037698.1), an H. pylori UA1234 1-2-fucosyltransferase (Hp2FTa) polypeptide (GenBank: AAD29863.1), or an H. pylori UA802 1-2-fucosyltransferase (Hp2FTb) polypeptide (GenBank: AAC99764.1). [0191] 8. The recombinant cell of any one of embodiments 3-7, wherein the second fucosyltransferase polypeptide is an 1-3-fucosyltransferase polypeptide. [0192] 9. The recombinant cell of embodiment 8, wherein the 1-3-fucosyltransferase polypeptide is a truncated 1-3-fucosyltransferase polypeptide. [0193] 10. The recombinant cell of embodiment 8 or embodiment 9, wherein the 1-3-fucosyltransferase polypeptide is an H. pylori UA948 1-3/4-fucosyltransferase (Hp3/4FT) polypeptide (GenBank: AAF35291.2), an H. pylori ATCC43504 1-3-fucosyltransferase (Hp3FT) polypeptide (GenBank: AAB93985), an H. pylori J99 1-3-fucosyltransferase (Hp3FT) polypeptide (GenBank: AAD06169.1, AAD06573.1), an H. pylori NCTC11637 1-3-fucosyltransferase (Hp3FT) polypeptide (GenBank: AAB93985), a B. fragilis NCTC 9343 1-3/4-fucosyltransferase polypeptide (GenBank: CAH09495.1), or an H. hepaticus ATCC 51449 Hh0072 polypeptide (GenBank: AAP76669.1). [0194] 11. The recombinant cell of any one of embodiments 2-10, wherein the nucleotide sugar pyrophosphorylase polypeptide is a B. fragilis bifunctional L-fucokinase/GDP-L-fucose pyrophosphorylase (Fkp) polypeptide. [0195] 12. The recombinant cell of any one of embodiments 3-11, which is transformed with a first expression vector comprising: [0196] the polynucleotide encoding the first fucosyltransferase polypeptide, [0197] the polynucleotide encoding the nucleotide sugar pyrophosphorylase polypeptide, [0198] the polynucleotide encoding the lactose transporter polypeptide, and [0199] the polynucleotide encoding the L-fucose transporter polypeptide. [0200] 13. The recombinant cell of any one of embodiments 3-12, wherein the polynucleotide encoding the first fucosyltransferase polypeptide and the polynucleotide encoding the nucleotide sugar pyrophosphorylase polypeptide are operably linked to a first inducible promoter. [0201] 14. The recombinant cell of embodiment 13, wherein the first inducible promoter is a P.sub.LLacO1 promoter. [0202] 15. The recombinant cell of any one of embodiments 3-14, wherein the polynucleotide encoding the second fucosyltransferase polypeptide is operably linked to a second inducible promoter. [0203] 16. The recombinant cell of embodiment 15, wherein the second inducible promoter is a P.sub.LtetO1 promoter. [0204] 17. The recombinant cell of any one of embodiments 3-16, wherein the L-fucose transporter polypeptide is an E. coli FucP polypeptide. [0205] 18. The recombinant cell of any one of embodiments 3-17, wherein the lactose transporter polypeptide is an E. coli LacY polypeptide. [0206] 19. The recombinant cell of any one of embodiments 3-18, wherein the polynucleotide encoding the lactose transporter polypeptide and the polynucleotide encoding the L-fucose transporter polypeptide are operably linked to a constitutive promoter. [0207] 20. The recombinant cell of any one of embodiments 1-19, which is modified to eliminate or reduce expression of an L-fucose mutarotase. [0208] 21. The recombinant cell of embodiment 20, wherein the L-fucose mutarotase is E. coli fucU. [0209] 22. The recombinant cell of any one of embodiments 1-21, which is modified to reduce or eliminate expression of a -galactosidase. [0210] 23. The recombinant cell of embodiment 22, wherein the -galactosidase is E. coli LacZ. [0211] 24. The recombinant cell of any one of embodiments 1-23, further comprising an polynucleotide encoding an additional transporter polypeptide. [0212] 25. The recombinant cell of embodiment 24, wherein the additional transporter polypeptide is a Bifidobacterium fucosyllactose transporter polypeptide. [0213] 26. The recombinant cell of any one of embodiments 1-25, which is an E. coli cell, a B. subtilis cell, a C. glutamicum cell, or an S. cerevisiae cell. [0214] 27. The recombinant cell of embodiment 26, which is an E. coli BW25113 Z1 cell or an E. coli MG1655 Z1 cell. [0215] 28. A method for producing an oligosaccharide product comprising two or more fucose moieties, the method comprising culturing a recombinant cell according to any one of embodiments 1-27 in a cell culture medium comprising L-fucose, an oligosaccharide acceptor, and a carbon source; [0216] wherein first glycosyltransferase is a first fucosyltransferase; the second glycosyltransferase is a second fucosyltransferase; and [0217] wherein the cell is cultured under conditions in which the first fucosyltransferase polypeptide, the second fucosyltransferase polypeptide, and the oligosaccharide acceptor is converted to the difucosylated oligosaccharide. [0218] 29. The method of embodiment 28, wherein the oligosaccharide transporter polypeptide is a lactose transporter polypeptide; the monosaccharide transporter polypeptide is an L-fucose transporter polypeptide; and the nucleotide sugar pyrophosphorylase polypeptide, the lactose transporter polypeptide, and the L-fucose transporter polypeptide are expressed under the culture conditions. [0219] 30. The method of embodiment 28 or embodiment 29, wherein the oligosaccharide acceptor is lactose and the oligosaccharide product is lactodifucotetraose (LDFT). [0220] 31. The method of any one of embodiments 28-29, wherein the carbon source comprises glucose, glycerol, or a combination thereof. [0221] 32. The method of any one of embodiments 28-31, wherein expression of the nucleotide sugar pyrophosphorylase polypeptide and the first fucosyltransferase polypeptide is induced at a level corresponding to 30-40% of maximum level (e.g., with isopropyl -D-1-thiogalactopyranoside in an amount around 50 M). [0222] 33. The method of embodiment 32, wherein expression of the second fucosyltransferase polypeptide is induced at a maximum level (e.g., with anhydrotetracycline at around 100 ng/mL).

[0223] Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, one of skill in the art will appreciate that certain changes and modifications can be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

TABLE-US-00008 V.InformalSequenceListing SEQIDNO:1.E.coli01261-2-fucosyltransferase(WbgL)(GenBank:ABE98421.1) 1 MSIIRLQGGLGNQLFQFSFGYALSKINGTPLYFDISHYAENDDHGGYRLNNLQIPEEYLQ 61 YYTPKINNIYKLLVRGSRLYPDIFLFLGFCNEFHAYGYDFEYIAQKWKSKKYIGYWQSEH 121 FFHKHILDLKEFFIPKNVSEQANLLAAKILESQSSLSIHIRRGDYIKNKTATLTHGVCSL 181 EYYKKALNKIRDLAMIRDVFIFSDDIFWCKENIETLLSKKYNIYYSEDLSQEEDLWLMSL 241 ANHHIIANSSFSWWGAYLGSSASQIVIYPTPWYDITPKNTYIPIVNHWINVDKHSSC SEQIDNO:2.H.pyloriUA9481-3/4-fucosyltransferase(Hp3/4FT)(GenBank: AAF35291.2), 1 MFQPLLDAFIDSTHLDETTHKPPLNVALANWWPLKNSEKKGERDFILHFILKQRYKIILH 61 SNPNEPSDLVFGNPLEQARKILSYQNTKRVFYTGENEVPNFNLEDYAIGFDELDENDRYL 121 RMPLYYAYLHYKAMLVNDTTSPYKLKALYTLKKPSHKFKENHPNLCALIHNESDPWKRGE 181 ASFVASNPNAPIRNAFYDALNAIEPVASGGSVKNTLGYKVKNKNEFLSQYKENLCFENSQ 241 GYGYVTEKILDAYFSHTIPIYWGSPSVAKDFNPKSFVNVHDENNEDEAIDYIRYLHAHQN 301 AYLDMLYENPLNTIDGKAGFYQDLSFEKILDFFKNILENDTIYHCNDAHYSALHRDLNEP 361 LVSVDDLRRDHDDLRVNYDDLRVNYDDLRVNYDDLRVNYDDLRVNYDDLRRDHDDLRRDH 421 ERLLSKATPLLELSQNTSFKIYRKAYQKSLPLLRAIRRWVRK SEQIDNO:3.BacteroidesfragilisbifunctionalLfucokinase/GDP-L-fucose pyrophosphorylase(BfFKP)(GenBank:CAH08307.1) 1 MQKLLSLPSNLVQSFHELERVNRTDWFCTSDPVGKKLGSGGGTSWLLEECYNEYSDGATE 61 GEWLEKEKRILLHAGGQSRRLPGYAPSGKILTPVPVERWERGQHLGQNLLSLQLPLYEKI 121 MSLAPDKLHTLIASGDVYIRSEKPLQSIPEADVVCYGLWVDPSLATHHGVFASDRKHPEQ 181 LDEMLQKPSLAELESLSKTHLFLMDIGIWLLSDRAVEILMKRSHKESSEELKYYDLYSDF 241 GLALGTHPRIEDEEVNTLSVAILPLPGGEFYHYGTSKELISSTLSVQNKVYDQRRIMHRK 301 VKPNPAMFVQNAVVRIPLCAENADLWIENSHIGPKWKIASRHIITGVPENDWSLAVPAGV 361 CVDVVPMGDKGFVARPYGLDDVFKGDLRDSKTTLTGIPFGEWMSKRGLSYTDLKGRTDDL 421 QAVSVFPMVNSVEELGLVLRWMLSEPELEEGKNIWLRSEHFSADEISAGANLKRLYAQRE 481 EFRKGNWKALAVNHEKSVFYQLDLADAAEDFVRLGLDMPELLPEDALQMSRIHNRMLRAR 541 ILKLDGKDYRPEEQAAFDLLRDGLLDGISNRKSTPKLDVYSDQIVWGRSPVRIDMAGGWT 601 DTPPYSLYSGGNVVNLAIELNGQPPLQVYVKPCKDFHIVLRSIDMGAMEIVSTFDELQDY 661 KKIGSPFSIPKAALSLAGFAPAFSAVSYASLEEQLKDEGAGIEVTLLAAIPAGSGLGTSS 721 ILASTVLGAINDFCGLAWDKNEICQRTLVLEQLLTTGGGWQDQYGGVLQGVKLLQTEAGF 781 AQSPLVRWLPDHLFTHPEYKDCHLLYYTGITRTAKGILAEIVSSMELNSSLHLNLLSEMK 841 AHALDMNEAIQRGSFVEFGRLVGKTWEQNKALDSGTNPPAVEAIIDLIKDYTLGYKLPGA 901 GGGGYLYMVAKDPQAAVRIRKILTENAPNPRARFVEMTLSDKGFQVSRS SEQIDNO:4.E.colistr.K-12substr.MG1655LacY(GenBank:AAC73446.1) 1 MYYLKNTNFWMFGLFFFFYFFIMGAYFPFFPIWLHDINHISKSDTGIIFAAISLESLLFQ 61 PLFGLLSDKLGLRKYLLWIITGMLVMFAPFFIFIFGPLLQYNILVGSIVGGIYLGFCENA 121 GAPAVEAFIEKVSRRSNFEFGRARMFGCVGWALCASIVGIMFTINNQFVFWLGSGCALIL 181 AVLLFFAKTDAPSSATVANAVGANHSAFSLKLALELFRQPKLWFLSLYVIGVSCTYDVED 241 QQFANFFTSFFATGEQGTRVFGYVTTMGELLNASIMFFAPLIINRIGGKNALLLAGTIMS 301 VRIIGSSFATSALEVVILKTLHMFEVPFLLVGCFKYITSQFEVRESATIYLVCFCFFKQL 361 AMIFMSVLAGNMYESIGFQGAYLVLGLVALGFTLISVFTLSGPGPLSLLRRQVNEVA SEQIDNO:5.E.coliK-12substr.MG1655FucP(AAC75843.1) 1 MGNTSIQTQSYRAVDKDAGQSRSYIIPFALLCSLFFLWAVANNLNDILLPQFQQAFTLTN 61 FQAGLIQSAFYFGYFIIPIPAGILMKKLSYKAGIITGLFLYALGAALFWPAAEIMNYTLE 121 LVGLFIIAAGLGCLETAANPFVTVLGPESSGHERLNLAQTFNSFGAIIAVVFGQSLILSN 181 VPHQSQDVLDKMSPEQLSAYKHSLVLSVQTPYMIIVAIVLLVALLIMLTKFPALQSDNHS 241 DAKQGSFSASLSRLARIRHWRWAVLAQFCYVGAQTACWSYLIRYAVEEIPGMTAGFAANY 301 LTGTMVCFFIGRFTGTWLISRFAPHKVLAAYALIAMALCLISAFAGGHVGLIALTLCSAF 361 MSIQYPTIFSLGIKNLGQDTKYGSSFIVMTIIGGGIVTPVMGFVSDAAGNIPTAELIPAL 421 CFAVIFIFARFRSQTATN SEQIDNO:6.E.coliK-12substr.MG1655FucU(AAC75846.1) 1 MLKTISPLISPELLKVLAEMGHGDEIIFSDAHFPAHSMGPQVIRADGLLVSDLLQAIIPL 61 FELDSYAPPLVMMAAVEGDTLDPEVERRYRNALSLQAPCPDIIRINRFAFYERAQKAFAI 121 VITGERAKYGNILLKKGVTP SEQIDNO:7.E.coliLacZK-12substr.MG1655(AAC73447.1) 1 MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWR 61 FAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNVTYPITVNPPFVPTENP 121 TGCYSLTFNVDESWLQEGQTRIIFDGVNSAFHLWCNGRWVGYGQDSRLPSEFDLSAFLRA 181 GENRLAVMVLRWSDGSYLEDQDMWRMSGIFRDVSLLHKPTTQISDEHVATRENDDESRAV 241 LEAEVQMCGELRDYLRVTVSLWQGETQVASGTAPFGGEIIDERGGYADRVTLRLNVENPK 301 LWSAEIPNLYRAVVELHTADGTLIEAEACDVGFREVRIENGLLLLNGKPLLIRGVNRHEH 361 HPLHGQVMDEQTMVQDILLMKQNNENAVRCSHYPNHPLWYTLCDRYGLYVVDEANIETHG 421 MVPMNRLTDDPRWLPAMSERVTRMVQRDRNHPSVIIWSLGNESGHGANHDALYRWIKSVD 481 PSRPVQYEGGGADTTATDIICPMYARVDEDQPFPAVPKWSIKKWLSLPGETRPLILCEYA 541 HAMGNSLGGFAKYWQAFRQYPRLQGGFVWDWVDQSLIKYDENGNPWSAYGGDFGDTPNDR 601 QFCMNGLVFADRTPHPALTEAKHQQQFFQFRLSGQTIEVTSEYLFRHSDNELLHWMVALD 661 GKPLASGEVPLDVAPQGKQLIELPELPQPESAGQLWLTVRVVQPNATAWSEAGHISAWQQ 721 WRLAENLSVTLPAASHAIPHLTTSEMDFCIELGNKRWQFNRQSGFLSQMWIGDKKQLLTP 781 LRDQFTRAPLDNDIGVSEATRIDPNAWVERWKAAGHYQAEAALLQCTADTLADAVLITTA 841 HAWQHQGKTLFISRKTYRIDGSGQMAITVDVEVASDTPHPARIGLNCQLAQVAERVNWLG 901 LGPQENYPDRLTAACFDRWDLPLSDMYTPYVFPSENGLRCGTRELNYGPHQWRGDFQFNI 961 SRYSQQQLMETSHRHLLHAEEGTWLNIDGFHMGIGGDDSWSPSVSAEFQLSAGRYHYQLV 1021 WCQK SEQIDNO:8.H.mustelae121981-2-fucosyltransferase(Hm2FT)(GenBank: CBG40460), 1 MDFKIVQVHGGLGNQMFQYAFAKSLQTHLNIPVLLDTTWFDYGNRELGLHLEPIDLQCAS 61 AQQIAAAHMQNLPRLVRGALRRMGLGRVSKEIVFEYMPELFEPSRIAYFHGYFQDPRYFE 121 DISPLIKQTFTLPHPTEHAEQYSRKLSQILAAKNSVFVHIRRGDYMRLGWQLDISYQLRA 181 IAYMAKRVQNLELFLFCEDLEFVQNLDLGYPFVDMTTRDGAAHWDMMLMQSCKHGIITNS 241 TYSWWAAYLIKNPEKIIIGPSHWIYGNENILCKDWVKIESQFETKS SEQIDNO:9.E.coli0128:B121-2-fucosyltransferase(WbsJ)(GenBank:AAO37698.1), 1 MEVKIIGGLGNQMFQYATAFAIAKRTHQNLTVDISDAVKYKTHPLRLVELSCSSEFVKKA 61 WPFEKYLESEKIPHEMKKGMFRKHYVEKSLEYDPDIDTKSINKKIVGYFQTEKYFKEFRH 121 ELIKEFQPKTKENSYQNELLNLIKENDTCSLHIRRGDYVSSKIANETHGTCSEKYFERAI 181 DYLMNKGVINKKTLLFIFSDDIKWCRENIFFNNQICFVQGDAYHVELDMLLMSKCKNNII 241 SNSSFSWWAAWLNENKNKTVIAPSKWFKKDIKHDIIPESWVKL SEQIDNO:10.H.pyloriUA12341-2-fucosyltransferase(Hp2FTa)(GenBank: AAD29863.1), 1 MAFKVVQICGGLGNQMFQYAFAKSLQKHSNTPVLLDITSFDWSNRKMQLELFPIDLPYAS 61 EKEIAIAKMQHLPKLVRNVLKCMGEDRVSQEIVFEYEPKLLKTSRLTYFYGYFQDPRYED 121 AISPLIKQTFTLPPPPENGNNKKKEEEYHRKLALILAAKNSVEVHIRRGDYVGIGCQLGI 181 DYQKKALEYMAKRVPNMELFVFCEDLEFTQNLDLGYPEMDMTTRDKEEEAYWDMLLMQSC 241 KHGIIANSTYSWWAAYLINNPEKIIIGPKHWLFGHENILCKEWVKIESHFEVKSQKYNA SEQIDNO:11.H.pyloriUA8021-2-fucosyltransferase(Hp2FTb)(GenBank: AAC99764.1). 1 MAFKVVQICGGLGNQMFQYAFAKSLQKHLNTPVLLDTTSFDWSNRKMQLELFPIDLPYAN 61 AKEIAIAKMQHLPKLVRDALKYIGFDRVSQEIVFEYEPKLLKPSRLTYFFGYFQDPRYED 121 AISSLIKQTFTLPPPPENNKNNNKKEEEYQRKLSLILAAKNSVFVHIRRGDYVGIGCQLG 181 IDYQKKALEYMAKRVPNMELFVFCEDLKFTQNLDLGYPFTDMTTRDKEEEAYWDMLLMQS 241 CKHGIIANSTYSWWAAYLMENPEKIIIGPKHWLFGHENILCKEWVKIESHFEVKSQKYNA SEQIDNO:12.H.pyloriATCC435041-3-fucosyltransferase(Hp435043FT)(GenBank: AAB93985), 1 MPLYYDRLHHKAESVNDTTAPYKIKGNSLYTLKKPSHCFKENHPNLCALINNESDPLKRG 61 FASEVASNANAPMRNAFYDALNSIEPVTGGGAVKNTLGYKVGNKSEFLSQYKENLCFENS 121 QGYGYVTEKIIDAYFSHTIPIYWGSPSVAKDENPKSFVNVHDENNEDEAIDYVRYLHTHP 181 NAYLDMLYENPLNTLDGKAYFYQNLSFKKILDFFKTILENDTIYHNNPFIFYRDLNEPLV 241 SIDNLRINYDNLRVNYDDLRVNYDDLRVNYDDLRINYDDLRINYDDLRINYERLLQNASP 301 LLELSQNTSFKIYRKIYQKSLPLLRVIRRWVKK SEQIDNO:13.H.pyloriJ991-3-fucosyltransferase(HpJ993FT)(GenBank: AAD06169.1), 1 MFQPLLDAYTDSTRLDETDYKPPLNIALANWWPLDKRESKGFRRFILYFILSQRYTITLH 61 QNPNEPSDLVFGSPIGSARKILSYQNTKRVFYTGENEVPNFNLFDYAIGFDELDERDRYL 121 RMPLYYASLHYKAESVNDTTAPYKLKDNSLYALKKPSHHFKENHPNLCAVVNDESDPLKR 181 GFASFVASNPNAPIRNAFYDALNSIEPVTGGGSVKNTLGYNVKNKSEFLSQYKENLCFEN 241 TQGYGYVTEKIIDAYFSHTIPIYWGSPSVAKDENPKSFVNVCDFKNFDEAIDYVRYLHTH 301 PNAYLDMLYENPLNTLDGKAYFYQNLSFKKILDFFKTILENDTIYHDNPFIFYRDLNEPL 361 VAIDDLRVNYDDLRVNYDDLRVNYDDLRVNYDDLRVNYDDLRVNYDDLRVNYDRLLQNAS 420 PLLELSQNTTFKIYRKAYQKSLPLLRTIRRWVKK SEQIDNO:14.H.pyloriJ991-3-fucosyltransferase(HpJ993FT)(GenBank: AAD06573.1), 1 MFQPLLDAFIESTPIKKKITFKSPPPPLKIAVANWWGGAEEFKKSTLYFILSQRYTITLH 61 QNPNEPSDLVLGSPIGSARKILSYQNTKRVFYTGENEVPNFNLFDYAIGFDELDERDRYL 121 RMPLYYASLHYKAESVNDTTAPYKLKDNSLYALKKPSHHFKENHPNLCAVVNDESDPLKR 181 GFASEVASNPNAPIRNAFYDALNSIEPVTGGGSVKNTLGYNVKNKSEFLSQYKENLCFEN 241 TQGYGYVTEKIIDAYFSHTIPIYWGSPSVAKDENPKSFVNVCDFKNFDEAIDYVRYLHTH 301 PNAYLDMLYENPLNTLDGKAYFYQNLSFKKILDFFKTILENDTIYHDNPFIFYRDLNEPL 361 VAIDDLRVNYDDLRVNYDDLRVNYDDLRVNYDRLLQNASPLLELSQNTTFKIYRKAYQKS 421 LPLLRAIRRWVKKLGL SEQIDNO:15.H.pyloriNCTC116371-3-fucosyltransferase(Hp116373FT)(GenBank: AAB93985). 1 MPLYYDRLHHKAESVNDTTAPYKIKGNSLYTLKKPSHCFKENHPNLCALINNESDPLKRG 61 FASFVASNANAPMRNAFYDALNSIEPVTGGGAVKNTLGYKVGNKSEFLSQYKENLCFENS 121 QGYGYVTEKIIDAYFSHTIPIYWGSPSVAKDENPKSFVNVHDENNEDEAIDYVRYLHTHP 181 NAYLDMLYENPLNTLDGKAYFYQNLSFKKILDFFKTILENDTIYHNNPFIFYRDLNEPLV 241 SIDNLRINYDNLRVNYDDLRVNYDDLRVNYDDLRINYDDLRINYDDLRINYERLLQNASP 301 LLELSQNTSFKIYRKIYQKSLPLLRVIRRWVKK SEQIDNO:16.B.fragilisNCTC93431-3/1-4-fucosyltransferase(Bf3/4ft)(GenBank: CAH09495.1) 1 MDILILFYNTMWGFPLEFRKEDLPGGCVITTDRNLIAKADAVVFHLPDLPSVMEDEIDKR 61 EGQLWVGWSLECEENYSWTKDPEFRESEDLWMGYHQEDDIVYPYYGPDYGKMLVTARREK 121 PYKKKACMFISSDMNRSHRQEYLKELMQYTDIDSYGKLYRNCELPVEDRGRDTLLSVIGD 181 YQFVISFENAIGKDYVTEKFFNPLLAGTVPVYLGAPNIREFAPGENCFLDICTFDSPEGV 241 AAFMNQCYDDEALYERFYAWRKRPLLLSFTNKLEQVRSNPLIRLCQKIHELKLGGI SEQIDNO:17.H.hepaticusATCC51449Hh0072(GenBank:AAP76669.1) 1 MKDDLVILHPDGGIASQIAFVALGLAFEQKGAKVKYDLSWFAEGAKGEWNPSNGYDKVYD 61 ITWDISKAFPALHIEIANEEEIERYKSKYLIDNDRVIDYAPPLYCYGYKGRIFHYLYAPF 121 FAQSFAPKEAQDSHTPFAALLQEIESSPSPCGVHIRRGDLSQPHIVYGNPTSNEYFAKSI 181 ELMCLLHPQSSFYLESDDLAFVKEQIVPLLKGKTYRICDVNNPSQGYLDLYLLSRCRNII 241 GSQGSMGEFAKVLSPHNPLLITPRYRNIFKEVENVMCVNWGESVQHPPLVCSAPPPLVSQ 301 LKRNAPLNSRLYKEKDNASA SEQIDNO:18.A.thalianaFKGP(UniProt:Q9LNJ9) 1 MSKQRKKADLATVLRKSWYHLRLSVRHPTRVPTWDAIVLTAASPEQAELYDWQLRRAKRM 61 GRIASSTVTLAVPDPDGKRIGSGAATLNAIYALARHYEKLGFDLGPEMEVANGACKWVRF 121 ISAKHVLMLHAGGDSKRVPWANPMGKVFLPLPYLAADDPDGPVPLLEDHILAIASCARQA 181 FQDQGGLFIMTGDVLPCFDAFKMTLPEDAASIVTVPITLDIASNHGVIVTSKSESLAESY 241 TVSLVNDLLQKPTVEDLVKKDAILHDGRTLLDTGIISARGRAWSDLVALGCSCQPMILEL 301 IGSKKEMSLYEDLVAAWVPSRHDWLRTRPLGELLVNSLGRQKMYSYCTYDLQFLHFGTSS 361 EVLDHLSGDASGIVGRRHLCSIPATTVSDIAASSVILSSEIAPGVSIGEDSLIYDSTVSG 421 AVQIGSQSIVVGIHIPSEDLGTPESFREMLPDRHCLWEVPLVGHKGRVIVYCGLHDNPKN 481 SIHKDGTFCGKPLEKVLFDLGIEESDLWSSYVAQDRCLWNAKLFPILTYSEMLKLASWLM 541 GLDDSRNKEKIKLWRSSQRVSLEELHGSINFPEMCNGSSNHQADLAGGIAKACMNYGMLG 601 RNLSQLCHEILQKESLGLEICKNFLDQCPKFQEQNSKILPKSRAYQVEVDLLRACGDEAK 661 AIELEHKVWGAVAEETASAVRYGFREHLLESSGKSHSENHISHPDRVFQPRRTKVELPVR 721 VDFVGGWSDTPPWSLERAGYVLNMAITLEGSLPIGTIIETTNQMGISIQDDAGNELHIED 781 PISIKTPFEVNDPFRLVKSALLVTGIVQENFVDSTGLAIKTWANVPRGSGLGTSSILAAA 841 VVKGLLQISNGDESNENIARLVLVLEQLMGTGGGWQDQIGGLYPGIKFTSSFPGIPMRLQ 901 VVPLLASPQLISELEQRLLVVFTGQVRLAHQVLHKVVTRYLQRDNLLISSIKRLTELAKS SEQIDNO:19.B.longumsubsp.infantisATCC15697FLtransporter-1domainBlon-0341 (ACJ51465.1) 1 MTNATAQPDTSVMRKPKRQYIGILYCLPYVVVFLFGMIVPMFYALYLSFFKQSLLGGTTE 61 AGFDNFIRAFKDEALWGGFRNVLIYAAIQIPMNLILSLVAALVLDSQRIRHIAVPRILLF 121 LPYAVPGVIAALMWGYIYGDKYGLFGQIAGMFGVAAPNMLSKQLMLFAIANICTWCFLGY 181 NMLIYYSALIGIPNDLYESARIDGASELRIAWSVKIPQIKSTIVMTVLESVIGTLQLENE 241 PNILRTSAPDVINSSYTPNIYTYNLAFNGQNVNYAAAVSLVIGIIVMALVAVVKIIGNKW 301 ENK SEQIDNO:20.B.longumsubsp.infantisATCC15697FLtransporter-1domainBlon-0342 (ACJ51466.1) 1 MSEAIARPRSKSLQRRDAKLALKASKHYKRMQQREPAPKLTGKQRVLNWLLHIIMAVMVI 61 YCLVPLLWVVFSSTKTSEGIFSSFGLWEDDKNVEWQNVQDTFAYQHGVYTRWLENTIMYA 121 VVAGVGATIIATFAGYAIATMRFPGRNALLAVTLAFMSIPSTVITVPLFLMYSKIGLVGT 181 PWAVIIPQLATPFGLYLMIIYAQTSIPVSLIEAAKLDGANTWTIFWKVGFPLLSPGFVTV 241 LLFTLVGVWNNYFLPLIMLTNTNDYPLTVGLNMWLKMGAQGTSDGQVPNNLIITGSLIAV 301 VPLIIAFMFLQKYWQSGLAAGSVKQ SEQIDNO:21.B.longumsubsp.infantisATCC15697FLtransporter-1domainBlon-0343 (ACJ51467.1) 1 MTHKGVIMKKSIRLIAAVAALAMTAGAAACGSGTSQKNNKADVSLNDINSALTDTSKTTD 61 LTVWAYSAKQIEGPVKAFQERYPHIKINFVNTGAASDHFTKFQNVVSANKGVPDVVQMSI 121 SEYEQYAVSGALLNFESDEIEKAWGTQYAQAAWKNVHFGGGLYGTPQDAAPLALYVRKDI 181 LDEHGLKVPTTWQEFYDEGVKLHKQDPSKYMGFISSSDTSLFGVLRTVGAKPWTVKDTTN 241 IDFSLTTGRVAEFIKFIQKCLDDGVLRAAATGTDEFNREVNDGVYATRLEGCWQGNIYKD 301 QNPSLKGKMVVAHPLAWGNDGESYQSESTGSMFSVSSATPKDKQAAALAFIQWVNGSKDG 361 VSEFLTANKGNYFMASNYYQKDKSKRDQQETDGYFANTNVNEIYFESMDKVNMDWDYIPF 421 PAQLTVAFGDTVAPALTGKGDLLTAFTKLQDNLKSYAEDNGFKVTTDAD SEQIDNO:22.B.longumsubsp.infantisATCC15697FLtransporter-2domainBlon-2202 (ACJ51465.1) 1 MKKSIRLVAAIAALAMTAGISACGSSTNGNQAKSDVTAQDVENALTDTSKNVELTVWAYS 61 AKQMEPTVKAFEKKYPHIKINFVNTGAAEDHFTKFQNVVQAQKDIPDVVQMSANKFQQFA 121 VSGALLNFANDSIEKAWSKLYTKTAWAQVHYAGGLYGAPQDATPLANYVRKDILDEHNLQ 181 VPESWEDIYNEGIKLHKEDSNKYMGILGSDISFFTNLYRSVGARLWKVNSVDDVELTMNS 241 GKAKEFTEFLQKCLKDGVLEGGTVFTDEFNRSINDGRYATFINENWMGNTYKEQNPSLKG 301 KMVVAAPPSWKGQPYQSSSVGSMMSVSAACPKEKQAAALAFINWLDSDKDAIQSWQDTNN 361 GNFFMAASVYQDDENQRNKKETDGYYANDDVNAVYFDSMDKVNTDWEYLPFMSQVEVVEN 421 DVIVPEMNENGDLVGAMAKAQQKLKAYAEDNGFKVTTDAD SEQIDNO:23.B.longumsubsp.infantisATCC15697FLtransporter-2domainBlon-2203 (ACJ53263.1) 1 MSEAIARPRSKSLQRRDAKLALKASKHYKRMQQREPAPKLTGKQRVLNWLLHIIMAVMVI 61 YCLVPLLWVVFSSTKTSEGIFSSFGLWEDDKNVFWQNVQDTFAYQHGVYTRWLENTIMYA 121 VVAGVGATIIATFAGYAIATMRFPGRNALLAVTLAFMSIPSTVITVPLELMYSKIGLVGT 181 PWAVIIPQLATPFGLYLMIIYAQTSIPVSLIEAAKLDGANTWTIFWKVGFPLLSPGFVTV 241 LLFTLVGVWNNYFLPLIMLTNTNDYPLTVGLNMWLKMGAQGTSDGQVPNNLIITGSLIAV 301 VPLIIAFMFLQKYWQSGLAAGSVKQ SEQIDNO:24.B.longumsubsp.infantisATCC15697FLtransporter-2domainBlon-2204 (ACJ53264.1) 1 MTNATAQPDTSVMRKPKRQYIGILYCLPYVVVFLFGMIVPMFYALYLSFFKQSLLGGTTF 61 AGFDNFIRAFKDEALWGGFRNVLIYAAIQIPMNLILSLVAALVLDSQRIRHIAVPRILLE 121 LPYAVPGVIAALMWGYIYGDKYGLFGQIAGMFGVAAPNMLSKQLMLFAIANICTWCFLGY 181 NMLIYYSALIGIPNDLYESARIDGASELRIAWSVKIPQIKSTIVMTVLESVIGTLQLENE 241 PNILRTSAPDVINSSYTPNIYTYNLAFNGQNVNYAAAVSLVIGIIVMALVAVVKIIGNKW 301 ENK SEQIDNO:25.B.subtilusFucUhomolog(WP_158321581.1) 1 MLKGIPAILSPDLMKVLMEMGHGDEIVLADGNFPSASHAQNLLRCDGHGIPALLEAILKE 61 FPLDTYVEHPVTLMDVVEGEQFQPTIWQDFEKVIQKEHGPALQMEYLDRFTFYERAKKAY 121 AIVATGEAAQYANIILKKGVVK SEQIDNO:26.B.subtilusLacZhomolog(MBA5241670.1) 1 MEVTDVRLRVDRENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRFAWFP 61 APEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNVTYPITVNPPFVPTENPTGCYS 121 LTFNVDESWLQEGQTRIIFDGVNSAFHLWCNGRWVGYGQDSRLPSEFDLSAFLRAGENRL 181 AVMVLRWSDGSYLEDQDMWRMSGIFRDVSLLHKPTTQISDFHVATRENDDFSRAVLEAEV 241 QMCGELRDYLRVTVSLWQGETQVASGTAPFGGEIIDERGGYADRVTLRLNVENPKLWSAE 301 IPNLYRAVVELHTADGTLIEAEACDVGFREVRIENGLLLLNGKPLLIRGVNRHEHHPLHG 361 QVMDEQTMVQDILLMKQNNFNAVRCSHYPNHPLWYTLCDRYGLYVVDEANIETHGMVPMN 421 RLTDDPRWLPAMSERVTRMVQRDRNHPSVIIWSLGNESGHGANHDALYRWIKSVDPSRPV 481 QYEGGGADTTATDIICPMYARVDEDQPFPAVPKWSIKKWLSLPGETRPLILCEYAHAMGN 541 SLGGFAKYWQAFRQYPRLQGGFVWDWVDQSLIKYDENGNPWSAYGGDFGDTPNDRQFCMN 601 GLVFADRTPHPALTEAKHQQQFFQFRLSGQTIEVTSEYLFRHSDNELLHWMVALDGKPLA 661 SGEVPLDVAPQGKQLIELPELPQPESAGQLWLTVRVVQPNATAWSEAGHISAWQQWRLAE 721 NLSVTLPAASHAIPHLTTSEMDFCIELGNKRWQFNRQSGFLSQMWIGDKKQLLTPLRDQF 781 TRAPLDNDIGVSEATRIDPNAWVERWKAAGHYQAEAALLQCTADTLADAVLITTAHAWQH 841 QGKTLFISRKTYRIDGSGQMAITVDVEVASDTPHPARIGLNCQLAQVAERVNWLGLGPQE 901 NYPDRLTAACFDRWDLPLSDMYTPYVEPSENGLRCGTRELNYGPHQWRGDFQFNISRYSQ 961 QQLMETSHRHLLHAEEGTWLNIDGFHMGIGGDDSWSPSVSAELQLSAGRYHYQLVWCQK