ZHANG ANGELA (US)
CHEN XI (US)
BAI YUANYUAN (US)
YU HAI (US)
MCARTHUR JOHN B (US)
US6461835B1 | 2002-10-08 | |||
US20170081353A1 | 2017-03-23 | |||
US20200181665A1 | 2020-06-11 | |||
US20110111458A1 | 2011-05-12 |
EMINE SEYDAMETOVA, JIWON YU, JONGHYEOK SHIN, YOONJUNG PARK, CHAKHEE KIM, HOOYEON KIM, SEOK HYEON YU, YONGCHEOL PARK, DAE-HYUK KWEO: "Search for bacterial α1,2-fucosyltransferases for whole-cell biosynthesis of 2′-fucosyllactose in recombinant Escherichia coli", MICROBIOLOGICAL RESEARCH, vol. 222, 1 May 2019 (2019-05-01), DE , pages 35 - 42, XP055698081, ISSN: 0944-5013, DOI: 10.1016/j.micres.2019.02.009
ENGELS ET AL: "WbgL: a novel bacterial alpha1,2-fucosyltransferase for the synthesis of 2'-fucosyllactose", GLYCOBIOLOGY, vol. 24, 1 January 2014 (2014-01-01), pages 170 - 178, XP002765456
YU HAI, LI YANHONG, WU ZHIGANG, LI LEI, ZENG JIE, ZHAO CHAO, WU YIJING, TASNIMA NOVA, WANG JING, LIU HUAIDE, GADI MADHUSUDHAN REDD: "H. pylori α1–3/4-fucosyltransferase (Hp3/4FT)-catalyzed one-pot multienzyme (OPME) synthesis of Lewis antigens and human milk fucosides", CHEMICAL COMMUNICATIONS, vol. 53, no. 80, 5 October 2017 (2017-10-05), UK , pages 11012 - 11015, XP093087394, ISSN: 1359-7345, DOI: 10.1039/C7CC05403C
MIKIYASU SAKANAKA, HANSEN MORTEN EJBY, GOTOH AINA, KATOH TOSHIHIKO, YOSHIDA KEISUKE, ODAMAKI TOSHITAKA, YACHI HIROYUKI, SUGIYAMA Y: "Evolutionary adaptation in fucosyllactose uptake systems supports bifidobacteria-infant symbiosis", SCIENCE ADVANCES, vol. 5, no. eaaw7696, 28 August 2019 (2019-08-28), US , pages 1 - 15, XP055709822, ISSN: 2375-2548, DOI: 10.1126/sciadv.aaw7696
PARK DONGKYU, RYU KYOUNG-SEOK, CHOI DONGWOOK, KWAK JAECHAN, PARK CHANKYU: "Characterization and role of fucose mutarotase in mammalian cells", GLYCOBIOLOGY, vol. 17, no. 9, 29 June 2007 (2007-06-29), pages 955 - 962, XP093096480, DOI: 10.1093/glyco/cwm066
WHAT IS CLAIMED IS: 1 1. A recombinant cell for production of an difucosylated oligosaccharide 2 product, the recombinant cell comprising: 3 a polynucleotide encoding an α1-2-fucosyltransferase polypeptide, and 4 a polynucleotide encoding an α1-3-fucosyltransferase polypeptide. 1 2. The recombinant cell of claim 1, further comprising one or more 2 polynucleotides selected from the group consisting of: 3 a polynucleotide encoding a nucleotide sugar pyrophosphorylase polypeptide, 4 a polynucleotide encoding a lactose transporter polypeptide, and 5 a polynucleotide encoding an L-fucose transporter polypeptide. 1 3. The recombinant cell of claim 1, further comprising: 2 a polynucleotide encoding a nucleotide sugar pyrophosphorylase polypeptide, 3 a polynucleotide encoding a lactose transporter polypeptide, and 4 a polynucleotide encoding an L-fucose transporter polypeptide. 1 4. The recombinant cell of claim 1, wherein the α1-2-fucosyltransferase 2 polypeptide is an E. coli 0126 α1-2-fucosyltransferase WbgL polypeptide. 1 5. The recombinant cell of claim 1, wherein the α1-3-fucosyltransferase 2 polypeptide is a truncated α1-3-fucosyltransferase polypeptide. 1 6. The recombinant cell of claim 1, wherein the α1-3-fucosyltransferase 2 polypeptide is an H. pylori UA948 α1-3/4-fucosyltransferase (Hp3/4FT) polypeptide. 1 7. The recombinant cell of claim 2, wherein the nucleotide sugar 2 pyrophosphorylase polypeptide is a bifunctional glycokinase and nucleotide sugar 3 pyrophosphorylase polypeptide. 1 8. The recombinant cell of claim 2, wherein the nucleotide sugar 2 pyrophosphorylase polypeptide is a.B.fragilis bifunctional L-fucokinase/GDP-L-fucose 3 pyrophosphorylase (Fkp) polypeptide. 1 9. The recombinant cell of claim 2, wherein the lactose transporter 2 polypeptide is an E. coll LacY polypeptide. 1 10. The recombinant cell of claim 2, wherein the L-fucose transporter 2 polypeptide is an E. coll FucP polypeptide. 1 11. The recombinant cell of claim 1, which is modified to eliminate or 2 reduce expression of an L-fucose mutarotase. 1 12. The recombinant cell of claim 11, wherein the L-fucose mutarotase is 2 E. coll fucU. 1 13. The recombinant cell of claim 1, which is modified to eliminate or 2 reduce expression of a β-galactosidase. 1 14. The recombinant cell of claim 13, wherein the P-galactosidase is E. 2 coll LacZ. 1 15. The recombinant cell of claim 1, further comprising an polynucleotide 2 encoding an additional transporter polypeptide. 1 16. The recombinant cell of claim 15, wherein the additional transporter 2 polypeptide is a Bifidobacterium fucosy llactose transporter polypeptide. 1 17. The recombinant cell claim 1, which is an E. coli cell, a B. subtilis cell, 2 a C. glutamicum cell, or an S. cerevisiae cell. 1 18. A method for producing an oligosaccharide product comprising two or 2 more fucose moieties, the method comprising culturing a recombinant cell according to any 3 one of claim 1 in a cell culture medium comprising L-fucose, an oligosaccharide acceptor, 4 and a carbon source; 5 wherein the cell is cultured under conditions in which the α1-2- 6 fucosyltransferase polypeptide and the α1-3 -fucosy Itransferase polypeptide are expressed, 7 and 8 wherein the oligosaccharide acceptor is converted to the difucosylated 9 oligosaccharide. 1 19. The method of claim 18, wherein the oligosaccharide acceptor is 2 lactose and the oligosaccharide product is lactodifucotetraose (LDFT). |
#upstream homologous region, ^downstream homologous region, *iGEM part #: BBa_K1824896 Table 5. Oligonucleotides used in this study
Table 6. Guide for CRISPR-Cas9-mediate gene deletions and insertions [0073] 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 [0074] 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 µg/mL), ampicillin (200 µg/mL), and kanamycin (50 µg/mL). Growth assays were carried out in M9 minimal medium (33.7 mM Na 2 HPO 4 , 22 mM KH 2 PO 4 , 8.6 mM NaCl, 9.4 mM NH4Cl, 1 mM MgSO4, 0.1 mM CaCl 2 ) including 1000 × A5 trace metal mix (2.86 g H3BO3, 1.81 g MnCl 2 ·4H 2 O, 0.079 g CuSO 4 ·5H 2 O, 49.4 mg Co(NO 3 ) 2 ·6H 2 O 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 600 ) with a Synergy H1 hybrid plate reader (BioTek Instruments, Inc.). Growth Assays [0075] 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 OD600 was measured. Fluorescence Assays [0076] Overnight cultures were inoculated at 1% in 3 mL of LB media and grown at 37 °C, 250 rpm, until OD600 reached 0.4–0.6. Cultures were respectively induced with isopropyl Δ- D-1-thiogalactopyranoside (IPTG, 1.0 mM) and grown at 37 °C, 250 rpm, for 24 h.
Fluorescence emission was measured at 510 nm with a Synergy H1 hybrid plate reader (BioTek Instruments, Inc.). LDFT production [0077] 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 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 [0078] 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. [0079] To prepare samples for HPLC analysis, 125 μL of culture was collected and spun down at 17,000 g for 5 min.15 μL of culture supernatant or compound standard in water was diluted with 45 μL of MilliQ water and 180 μL of acetonitrile. The mixture was vortexed and spun down at 17,000 g for 5 min.40 μL of each sample was injected into the column for analysis. RESULTS Pathway design for LDFT production in E. coli [0080] 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 α1–2- fucosyltransferase (WbgL) from E. coli O126 (Engels and Elling, 2014; McArthur et al., 2019), and α1–3/4-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 [0081] 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 T7 , pAL1779, Table 2) and the truncated Hp3/4ft gene was cloned into a second expression vector under PT7 (pAL1817, Table 2). [0082] 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 β-galactosidase was deleted in the strain (Table 1: Strain 1), lactose did not enable growth anymore (FIG.2). [0083] 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 (Brückner 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 PT7, the plasmid containing sfgfp under P 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 λDE3 lysogen containing P lacUV5 :lacZ Δ ΔT7rnap. Several attempts were made to remove lacZ from BL21 Star (DE3) without off-target modifications to the λDE3 lysogen but resulted in failure. Introduction of the T7 RNAP gene into K-12 derivative strains [0084] Due to difficulties in genetically modifying BL21 Star (DE3), P lacUV5 :T7rnap was integrated into the E. coli K-12 derivative strains, BW25113 Z1 and MG1655 Z1 (Table 3). The Z1 fragment containing lacI q , tetR, and spec 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 PlacUV5:T7rnap. The PlacUV5:T7rnap cassette was integrated into ss9 of BW25113 Z1 and MG1655 Z1 to form Strains 5 and 6, respectively (Table 1). [0085] pAL1834 containing PT7: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 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 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). [0086] 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 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 T7 was leaky in Strain 12 (FIG.3C). In Strain 10, a mutation in the promoter region of the PlacUV5:T7rnap cassette was found. The deletion of lacZ in Strain 9 without incurring P 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 lacUV5 are correlated with the CRISPR-Cas9-mediated gene removal of lacZ due to the similarity of the lacZ promoter to PlacUV5. [0087] To avoid the potential sequence similarity issues observed for PlacUV5 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 ( ΔfucU) and Strain 14 ( ΔfucU ΔlacZ)). Then, PlacUV5: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 PlacUV5:T7rnap cassette in Strain 15 had no mutations, Strain 15 with pAL1834 harboring P 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 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 LlacO1 without IPTG were well repressed in Strains 17, 18 and 19 (FIG.4A). Next, pAL2045 containing PlacUV5: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 lacUV5 . Production of LDFT in K-12 derivative strains [0088] 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 PLlacO1 (pAL1759, Tables 2 & 4) and the Hp3/4ft gene was cloned under an aTc-inducible promoter P LtetO1 (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 [0089] 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. [0090] 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 [0091] 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 μM) were screened for the expression of P LlacO1 :fkp-wbgL in the presence of 100 ng/mL aTc for induction of PLtetO1:Hp3/4ft . 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 μM 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 μM 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 μM IPTG and 100 ng/mL aTc was used for further studies. Characterization of LDFT production [0092] 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. [0093] 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 Hp3/4ft 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 Hp3/4ft 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. [0094] 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 μM 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 [0095] 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 91% of TMY. DISCUSSION [0096] 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. [0097] The greatest challenge of this study was pairing an α1–2-fucosyltransferase with an α1–3-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 α1–4-linked galactose substrates, such as lactose and LacNAc (Engels and Elling, 2014). From acceptor substrate screenings of α1–3-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, α1–2- fucosyltransferases such as Hm2FT (GenBank: CBG40460), E. coli O128:B12 α1–2- fucosyltransferase WbsJ (GenBank: AAO37698.1), H. pylori UA1234 α1–2- fucosyltransferase (Hp2FTa) (GenBank: AAD29863.1), H. pylori UA802 α1–2- fucosyltransferase (Hp2FTb) (GenBank: AAC99764.1), and related eukaryotic α1–2- fucosyltransferases (see, e.g., cazy.org/GT11_characterized.html); as well as α1–3- fucosyltransferases such as H. pylori ATCC43504 α1–3-fucosyltransferase (Hp3FT) (GenBank: AAB93985), H. pylori J99 α1–3-fucosyltransferase (Hp3FT) (GenBank: AAD06169.1, AAD06573.1), H. pylori NCTC11639 α1–3-fucosyltransferase (Hp3FT) (GenBank: AAB93985), and related eukaryotic α1–3-fucosyltransferases and α1–3/4- fucosyltransferases (see, e.g., cazy.org/GT10_characterized.html). It is possible to screen α1– 3-fucosyltransferases for lower activity towards lactose and higher activity towards 2’-FL, and also pursue protein engineering strategies to expand α1–2-fucosyltransferase’s acceptor substrate range to 3-FL so that this side product can be fucosylated into LDFT. [0098] 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 (Görke and Stülke, 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). [0099] Lastly, balancing expression levels of the LDFT biosynthetic pathway genes (fkp, wbgL, and Hp3/4ft) 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 Hp3/4ft 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. [0100] 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. 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The one-pot multienzyme (OPME) synthesis of human blood group H antigens and a human milk oligosaccharide (HMOS) with highly active Thermosynechococcus elongatus α1–2-fucosyltransferase. Chem. Commun.52, 3899–3902. IV. Exemplary Embodiments [0101] Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the claims and the following embodiments: 1. A recombinant cell for production of an oligosaccharide product, the recombinant cell comprising: a polynucleotide encoding a first glycosyltransferase polypeptide having a first substrate selectivity, and a polynucleotide encoding a second glycosyltransferase polypeptide having a second substrate selectivity. 2. The recombinant cell of embodiment 1, further comprising one or more polynucleotides selected from the group consisting of: a polynucleotide encoding a nucleotide sugar pyrophosphorylase polypeptide, a monosaccharide transporter polypeptide, and an oligosaccharide transporter polypeptide. 3. The recombinant cell of embodiment 1 or 2, for production of an oligosaccharide comprising two or more fucose moieties, comprising: a polynucleotide encoding a first fucosyltransferase polypeptide having a first substrate selectivity, and a polynucleotide encoding a second fucosyltransferase polypeptide having a second substrate selectivity; and optionally comprising one or more 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. 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. 5. The recombinant cell of embodiment 3 or embodiment 4, wherein the first fucosyltransferase polypeptide is an α1–2-fucosyltransferase polypeptide. 6. The recombinant cell of embodiment 5, wherein the α1–2- fucosyltransferase polypeptide is an E. coli O126 α1–2-fucosyltransferase WbgL polypeptide. 7. The recombinant cell of embodiment 5 or embodiment 6, wherein the α1–2-fucosyltransferase polypeptide is an E. coli O126 α1–2-fucosyltransferase (WbgL) polypeptide (GenBank: ABE98421.1), an H. mustelae 12198 α1–2-fucosyltransferase (Hm2FT) polypeptide (GenBank: CBG40460), an E. coli O128:B12 α1–2-fucosyltransferase (WbsJ) polypeptide (GenBank: AAO37698.1), an H. pylori UA1234 α1–2-fucosyltransferase (Hp2FTa) polypeptide (GenBank: AAD29863.1), or an H. pylori UA802 α1–2- fucosyltransferase (Hp2FTb) polypeptide (GenBank: AAC99764.1). 8. The recombinant cell of any one of embodiments 3-7, wherein the second fucosyltransferase polypeptide is an α1–3-fucosyltransferase polypeptide. 9. The recombinant cell of embodiment 8, wherein the α1–3- fucosyltransferase polypeptide is a truncated α1–3-fucosyltransferase polypeptide. 10. The recombinant cell of embodiment 8 or embodiment 9, wherein the α1–3-fucosyltransferase polypeptide is an H. pylori UA948 α1–3/4-fucosyltransferase (Hp3/4FT) polypeptide (GenBank: AAF35291.2), an H. pylori ATCC43504 α1–3- fucosyltransferase (Hp3FT) polypeptide (GenBank: AAB93985), an H. pylori J99 α1–3- fucosyltransferase (Hp3FT) polypeptide (GenBank: AAD06169.1, AAD06573.1), an H. pylori NCTC11637 α1–3-fucosyltransferase (Hp3FT) polypeptide (GenBank: AAB93985), a B. fragilis NCTC 9343 α1–3/4-fucosyltransferase polypeptide (GenBank: CAH09495.1), or an H. hepaticus ATCC 51449 Hh0072 polypeptide (GenBank: AAP76669.1). 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. 12. The recombinant cell of any one of embodiments 3-11, which is transformed with a first expression vector comprising: the polynucleotide encoding the first fucosyltransferase polypeptide, the polynucleotide encoding the nucleotide sugar pyrophosphorylase polypeptide, the polynucleotide encoding the lactose transporter polypeptide, and the polynucleotide encoding the L-fucose transporter polypeptide. 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. 14. The recombinant cell of embodiment 13, wherein the first inducible promoter is a PLlacO1 promoter. 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. 16. The recombinant cell of embodiment 15, wherein the second inducible promoter is a PLtetO1 promoter. 17. The recombinant cell of any one of embodiments 3-16, wherein the L- fucose transporter polypeptide is an E. coli FucP polypeptide. 18. The recombinant cell of any one of embodiments 3-17, wherein the lactose transporter polypeptide is an E. coli LacY polypeptide. 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. 20. The recombinant cell of any one of embodiments 1-19, which is modified to eliminate or reduce expression of an L-fucose mutarotase. 21. The recombinant cell of embodiment 20, wherein the L-fucose mutarotase is E. coli fucU. 22. The recombinant cell of any one of embodiments 1-21, which is modified to reduce or eliminate expression of a β-galactosidase. 23. The recombinant cell of embodiment 22, wherein the β-galactosidase is E. coli LacZ. 24. The recombinant cell of any one of embodiments 1-23, further comprising an polynucleotide encoding an additional transporter polypeptide. 25. The recombinant cell of embodiment 24, wherein the additional transporter polypeptide is a Bifidobacterium fucosyllactose transporter polypeptide. 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. 27. The recombinant cell of embodiment 26, which is an E. coli BW25113 Z1 cell or an E. coli MG1655 Z1 cell. 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; wherein first glycosyltransferase is a first fucosyltransferase; the second glycosyltransferase is a second fucosyltransferase; and 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. 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. 30. The method of embodiment 28 or embodiment 29, wherein the oligosaccharide acceptor is lactose and the oligosaccharide product is lactodifucotetraose (LDFT). 31. The method of any one of embodiments 28-29, wherein the carbon source comprises glucose, glycerol, or a combination thereof. 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 β-D-1-thiogalactopyranoside in an amount around 50 µM). 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). [0102] 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.