Production of Milk Oligosaccharides in Plants

[001] This invention was made with government support under Dept of Energy award DEAC02-05CH1123 E The government has certain rights in the invention.

[002] Introduction

[003] Human breast milk is a complete and comprehensive food perfectly evolved to nourish and protect infants. A key component to the distinct bioactive properties of breast milk is the presence of a wide diversity of human milk oligosaccharides (HMOs) that are well documented in establishing the nascent gut microbiota of infants to prevent diseases and ensure healthy development. Although commercial infant formulas have tried to replicate the biochemical properties of breast milk, these synthetic cocktails are unable to recapitulate the unique health benefits of breast milk due to an inability to produce the diversity of HMOs found in human milk at scale.

[004] Currently, HMOs for infant formula have been largely produced via fermentation using engineered microbes; however, this approach is expensive and has been limited to the production of a small number of simple HMOs. Thus, there is great interest in developing new production hosts for HMOs. As such, plants serve as an attractive production platform, as they have an innate ability to generate a vast array of sugars and use them to produce complex oligosaccharide and polysaccharides. All HMOs can be grouped into three broad classes: neutral, fucosylated, and acidic. Here, we engineer plants to produce diverse sets of all three classes of HMOs (Figure 1 A). This includes the production of HMOs via transient expression and expression in stably transformed plants. Additionally, we increased accumulation of HMOs in plants through modulating nucleotide sugar pathways (Figure IB) to optimize the pool of substrate needed for HMO production. Finally, we demonstrate how plants can be used to make complex HMOs that have never before been produced through heterologous production, highlighting the advantages of engineering plants as an ideal production platform of HMOs.

[005] Summary of the Invention

[006] For this invention we chose plants serve as a production platform, as they have an innate ability to generate a vast array of sugars and use them to produce complex oligosaccharide and polysaccharides. Mammalian milk oligosaccharide (MMOs) can be grouped into three broad classes: neutral, fucosylated, and acidic. We engineered plants to produce diverse sets of all three classes of MMOs (Fig. 1 A). Additionally, we increased accumulation of MMOs in plants through modulating nucleotide sugar pathways to optimize the pool of substrate needed for MMO production (Fig. IB). Finally, we demonstrated how plants can be used to make complex MMOs that have never before been produced through heterologous production. Our approach allows the production of complex human milk oligosaccharides at an agricultural scale, permitting high production. This enables the supplementation of infant formula with complex MMOs, provide researchers with complex MMOs to conduct research on their bioactivity, and treat diseases in adults.

[007] The invention provides methods and compositions for plant production of mammalian (e.g. human) milk oligosaccharides.

[008] In an aspect the invention provides a method of producing a mammalian milk oligosaccharide (MMO) in a plant, comprising growing a plant comprising and expressing a recombinant MMO biosynthetic pathway sufficient to produce the MMO.

[009] In an aspect the invention provides a composition comprising a plant or plant part comprising and expressing a recombinant MMO biosynthetic pathway sufficient to produce the MMO.

[010] In an aspect the invention provides a composition comprising an MMO isolated from a plant comprising and expressing a recombinant MMO biosynthetic pathway sufficient to produce the MMO.

[Oil] In an aspect the invention provides a method of isolating an MMO comprising isolating the MMO from a plant comprising and expressing a recombinant MMO biosynthetic pathway sufficient to produce the MMO.

[012] In embodiments the invention provides a plant, method or composition herein, wherein the plant is further comprising and expressing a eukaryotic (e.g. human) or prokaryotic (e.g. bacterial, such as E. coli) nucleotide sugar biosynthetic pathway sufficient to increase the production of the MMO.

[013] In embodiments the invention provides a plant, method or composition herein, wherein the plant is further comprising and expressing a prokaryotic (e.g. bacterial, such as E. coli) nucleotide sugar biosynthetic pathway sufficient to increase the production of the MMO, wherein the use of a prokaryotic (as opposed to a eukaryotic) pathway substantially avoids pathway feedback inhibition in the plant.

[014] In embodiments the invention provides a plant, method or composition herein, wherein the plant is further comprising and expressing a prokaryotic (e.g. bacterial, such as E. coli) nucleotide sugar biosynthetic pathway sufficient to increase the production of the MMO, wherein the pathway comprises one, two or three pathways (and corresponding enzymes): [015] a) GDP-fucose (Man A, ManB, ManC, Gmd, WcaG), [016] b) UDP-galactose (pgi, pgm, GalU, GalE), and [017] c) UDP-GlcNAc (glmS, glmM, glmU).

[018] In embodiments the invention provides a plant, method or composition herein, wherein the MMO is of a mammal selected from human, cow and goat.

[019] In embodiments the invention provides a plant, method or composition herein, wherein the MMO is a human milk oligosaccharide (HMO).

[020] In embodiments the invention provides a plant, method or composition herein, the plant comprising and expressing a plurality of recombinant MMO biosynthetic pathways sufficient to produce a plurality (e.g. at least 2, 3, 4, 5, 6, 7, 8, 9 or 10) of different MMOs.

[021] In embodiments the invention provides a plant, method or composition herein, wherein the plant is a dicot or monocot plant, such as tobacco, potato, tomato, sorghum, soybean, sugarcane, Arabidopsis, rice, sugar beet, wheat, cassava, and oat.

[022] In embodiments the invention provides a plant, method or composition herein, further comprising isolating the resultant MMO from the plant.

[023] In aspects and embodiments the invention provides:

[024] a composition, plant or plant part herein, producing a mammalian milk oligosaccharide (MMO), comprising and expressing a recombinant MMO biosynthetic pathway sufficient to produce the MMO, wherein the pathway comprises recombinant enzymes:

[025] a β1 -4 galactosyltransferase (β1-4-GalT; e.g. GalTPMl 141 ), which glycosylates glucose using UDP-galactose to make lactose; and

[026] a β1-3 N-acetylglucosaminyltransferase ( β1-3-GnT; e.g. NmLgtA) generates lacto-N- triose (LNTII) through the transfer of GlcNAC from UDP-GlcNAc to the galactose in the lactose; and at least one of:

[027] a β1 -3 -galactosyltransferase (β1-3-GalT; e.g. Cvβ3GalT) and a β1-4-GalT (e.g. HP0826 or NmLgtB), convert the LNTII to lacto-N-tetraose (LNT) and laclo-N-neolelraose (LNnT), respectively.

[028] In aspects and embodiments the invention provides:

[029] a composition, plant or plant part herein, comprising and expressing of a β1-4-GalT (e.g. GalTPMl 141 ), a β1-3-GlcNAcT (e.g. NmLgtA), a β1-4-GalT (e.g. HP0826 or NmLgtB), and a β1-3-GalT (e.g. Cvβ3GalT) (Fig 1) to produce LNnT and LNT;

[030] a composition, plant or plant part herein producing non-fucosylated neutral MMOs (nMMOs, preferably nHMOs) comprising a lactose core with decorations of galactose and N- acetylglucos amine (GlcNAc);

[031] a composition, plant or plant part herein producing fucosylated MMOs (preferably HMOs), particularly comprising a lactose core decorated with one or more fucose moieties; [032] a composition, plant or plant part herein comprising and expressing a β1-4-GalT (e.g. GalTPM1141 ), a β1-3-GlcNAcT (e.g. NmLgtA), a β1-4-GalT (e.g. HP0826 or NmLgtB), a β1-3- GalT (e.g. Cvβ3GalT) and a al-2-fucosyltransferase (al-2-FucT) (e.g. Te2FT) (Fig 1) to produce lacto-N-fucopentaose I (LNFPI), a fucosylated pentasaccharide generated by the addition of anα-1, 2-linked fucose to the terminal galactose in laclo-A'-lelraose (LNT);

[033] a composition, plant or plant part herein, producing fucosyllactose, laclo-N-lelraose or lacto-N-neotetraose, and lacto-N-fucopentaose I (and particularly, 2’ FL, LNT, LNnT and LNFPI) respectively (Figure 3, Table 1).;

[034] a composition, plant or plant part herein, wherein to improve LNFPI yields, nucleotide sugar biosynthetic pathways (Figure IB) are expressed to increase the availability of GDP- fucose (Man A, ManB, ManC, Gmd, WcaG), UDP-galactose (pgi, pgm, GalU, GalE), and UDP- GlcNAc (glmS, glmM, glmU) combinatorially (Figure 4);

[035] a composition, plant or plant part herein, wherein when expressing the LNFPI biosynthetic pathway (Figure 1A) and the GDP-fucose biosynthetic pathway (Figure IB), there the plant metabolism harbors the innate ability to further glycosylate simple MMOs to create MMOs of higher complexity (such as lactodifucotetraose (LDFT) and lacto-N-difucohexaose I (LNDFHI); Figure 3, Table 1). that have not been previously produced through any other heterologous hosts;

[036] a composition, plant or plant part herein, producing acidic MMOs, particularly comprising a lactose or nMMO core with one or more N-acetylneuraminic acid (Neu5Ac) moieties, comprising in the plant

[037] a mammalian CMP-Neu5Ac pathway, a UDP-GlcNAc pathway and aMMO biosynthetic genes resulting in production of aMMOs, particularly production of CMP-Neu5Ac;

[038] a plant herein, comprising and expressing a three gene bacterial pathway (neuA, neuB, neuC) or a four gene mammalian pathway (GNE, NANS, NANP, CMAS) which utilize UDP- GlcNAc as a precursor for the production of CMP-Neu5Ac;

[039] a composition, plant or plant part herein, comprising and expressing a mammalian CMP- Neu5Ac pathway, GalTPM1141, NmLgtA, Cvβ3GalT, Hp0826 (or NmLgtB) and either PmSt3 or St6 (Figure 1A) to produce 3’SL, 6’SL and isomers of LST;

[040] a composition, plant or plant part herein, that is a stable, transgenic plant , comprising constructs for the constitutive production of 2’ FL and LNFPI (e.g. in stably transformed N. benthamiana); in embodiments, MMO10 (Figure 6) contains genes for the four enzymes required to produce lactose, 2’ FL, LNTII, LNT, and LNFPI connected via 2A peptides to allow multiple coding sequences to be driven by a single promoter, wherein each transcriptional unit is driven by a strong constitutive promoter to enable MMO production in all tissues, or MMO11 (Figure 6), which contains a GDP-D-mannose-4,6-dehydratase (Gmd) from the GDP-fucose pathway, wherein both constructs provide the production of multiple MMOs, specifically 2’FL (Figure 7) and LNFPI (Figure 8);

[041] a method of producing a mammalian milk oligosaccharide (MMO) in a plant herein, comprising growing the plant comprising and expressing a recombinant MMO biosynthetic pathway sufficient to produce the MMO;

[042] a composition comprising an MMO isolated from a plant herein comprising and expressing a recombinant MMO biosynthetic pathway sufficient to produce the MMO;

[043] a food product composition comprising composition, plant or plant part (such as leaves, stems, roots, seeds, fruit or flowers) herein comprising and expressing a recombinant MMO biosynthetic pathway sufficient to produce the MMO.

[044] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.

[045] Brief Description of the Drawings

[046] Figs 1A-B. A) MMO biosynthetic pathways used in this study. B) Nucleotide sugar biosynthetic pathways.

[047] Fig. 2. Production of neutral MMOs in planta. Extracted mass chromatogram identifying the in planta production of Lacto-N-tetraose/Lacto-N-neotetraose (LNT/LNnT), 4_1_0_0 , 5_2_0_0 and 6_1_0_0. Data collected using liquid chromatography-MS with a HILIC column. Numbered codes refer to the number of Hexose_HexNAc_Deoxyhexose_ N-acetylneuraminic acid saccharides in the oligosaccharide as determined by MS/MS fragmentation.

[048] Fig. 3. Production of fucosylated MMOs in planta. Extracted ion chromatogram identifying the in planta production of 2’-fucosyllactose (2’FL), lacto-N-fucopentaose I (LNFPI), lactodifucotetraose (LDFT), lacto-N-difucohexaose I (LNDFHI), and 4_1_1_0. Data collected using liquid chromatography-mass spectrometry with a HILIC column. Numbered codes refer to the number of Hexose_HexNAc_Deoxyhexose_ N-acetylneuraminic acid saccharides in the oligosaccharide as determined by MS/MS fragmentation.

[049] Figs 4A-B. Optimization of fucosylated MMO production. A) Quantification of LNFPI produced when expressing the LNFPI and nucleotide sugar biosynthetic pathways. B) Oligosaccharide profiles of leaf tissue expressing various nucleotide sugar biosynthetic pathways. Negative control produces negligible amounts of oligosaccharides compared to experimental samples. Hexose, HexNAc and deoxyhexose refer to the presence of at least one hexose sugar, N-acetylhexosamine sugar, and deoxyhexose sugar, respectively. [050] Fig. 5. Production of neutral MMOs in planta. Extracted ion chromatogram identifying the in planta production of 3’-sialyllactose (3’SL), 6’-sialyllactose (6’SL), sialyllacto-N- neotetraose d (LSTd), sialyllacto-N-neotetraose c (LSTc), and 4_1_0_1. Data was collected using liquid chromatography-mass spectrometry with a HILIC column. Numbered codes refer to the number of Hexose_HexNAc_Deoxyhexose_N-acetylneuraminic acid saccharides in the oligosaccharide as determined by MS/MS fragmentation.

[051] Fig. 6. Constructs used in the generation of stably transformed N. benthamiana. [052] Fig. 7. Identification of 2’-fucosyllactose (2’FL) produced in stably transform N. benthamiana. Data was collected using liquid chromatography-mass spectrometry with a porous graphitic carbon column.

[053] Fig. 8. Identification of lacto-N-fucopentaose I (LNFPI) produced in stably transform N. benthamiana. Data was collected using liquid chromatography-mass spectrometry with a porous graphitic carbon column.

[054] Description of Particular Embodiments of the Invention

[055] Plant material

[056] Nicotiana benthamiana was grown in 3.5 inch square pots in a controlled environment facility under a 12/12 day/night cycle (12 hours light, 12 hours dark) at -100 pmol photons m- ² sec ^-1 . Daytime temperatures were 26°C, and night temperatures were 25°C. Relative humidity was between 60 - 75%. Plants used in this study were 4 weeks old.

[057] Cloning

[058] Putative and known human milk oligosaccharide biosynthetic genes were either synthesized or acquired from other labs. Genes were PCR amplified . Amplified candidate genes were then cloned into the binary vector PMS057 using Golden Gate assembly, Gibson assembly, or standard digestion and ligation assembly. 2-4 μL of the assembly reactions were transformed into XL1B chemically competent E. coli cells via heat shock as previously described. Colonies were selected on LB agar plates containing 50 μg/mL kanamycin and sequence- verified using Sanger sequencing (McLab). Sequence verified plasmids were used to transform Agrobacterium tumefaciens str. GV3101 by electroporation as described by. Competent cells were then plated on LB agar plates containing 50 μg/mL rifampicin, 10 μg/mL gentamicin, 50 μg/mL kanamycin. [059] Transient expression in N. benthamiana

[060] Overnight cultures of A. tumefaciens str. GV3101 were grown in LB to an OD600 between 0.8 and 1.2. Cultures were centrifuged at 4000xG for 10 min and the supernatant was removed. Bacterial pellets were resuspended in infiltration media (10 mM MgCl ₂ , 10 rnM MES, 500 pM acetosyringone, pH: 5.6). Following an hour incubation, Agrobaclerium strains containing HMO biosynthetic genes were mixed in various combinations to a final OD600 of 0.5. A. tumefaciens strains were normalized to the level of the highest number of strains used in an experiment. For experiments that had less than the highest number of strains, an additional A. tumefaciens strain harboring the unrelated gene (dsRed) was added to reach a final OD600 of 0.5. An A. tumefaciens strain harboring the pl9 silencing suppressor was used in all experiments at the same concentration as other strains. A. tumefaciens suspensions were syringe infiltrated into the abaxial side of the seventh leaf of 4- week old N. benthamiana.

[061] Extractions

[062] N. benthamiana leaves were harvested 5 days post- infiltration. Major veins of N. benthamiana were removed from the leaf tissue, and the tissue was frozen in liquid nitrogen before lyophilization. Lyophilized leaf tissue was bead beaten using a single steel bead at 20 Hz for 10 min.

[063] Oligosaccharides were extracted from lyophilized leaf tissue by ethanol precipitation. To each sample, 1 mL of 80% ethanol was added before homogenization on a bead mill at 4 m/s for 1 min. Samples were then precipitated overnight at -20 °C and centrifuged at 10,000 rpm for 15 min. The supernatant was transferred to a 2 mL screw-cap tube. The pellet was washed twice by adding 500 μL of 80% ethanol, homogenizing via bead mill for 1 min, and centrifuging at 10,000 rpm for 15 min. The supernatant and washes were combined and dried in a vacuum centrifuge. Dried supernatants were reconstituted in 200 μL of water and subjected to both C18 and porous graphitized carbon (PGC) solid phase extractions (SPE) in 96- well plate format. Cl 8 cartridges containing 25 mg of stationary phase were first conditioned by two additions of 250 μL of acetonitrile (ACN) followed by four additions of 250 μL water. Samples were then loaded and eluted with two volumes of 200 μL water. PGC cartridges containing 40 mg of stationary phase were conditioned by addition of 400 μL water, 400 μL 80% (v/v) ACN and water, followed by two volumes of 400 μL water. The sample eluate from C18 SPE was then loaded, washed thrice with 500 μL water, and eluted using two volumes of 200 μL 40% (v/v) ACN and water. The purified extracts were dried in a vacuum centrifuge and reconstituted in 100 μL of water before injecting 5 μL for liquid chromatography-mass spectrometry (LC-MS) analysis.

[064] Initial chromatographic separation was carried out using a Thermo Scientific Vanquish UHPLC system equipped with a Waters BEH C18 Amide column (HILIC) (1.7 pm, 100 mm x 2.1 mm). A 10 min binary gradient was employed based on Xu et al. (2017): 0.0 - 4.0 min: 25- 35% A; 4.0 - 8.50 min, 35-65% A; 8.50 - 8.70 min: 25% A. Mobile phase A consisted of 3% ACN (v/v) in water with 0.1% formic acid, and mobile phase B consisted of 95% ACN (v/v) in water with 0.1% formic acid. [065] Chromatographic separation of oligosaccharides was carried out on an Agilent 1260 Infinity II LC equipped with a Hypercarb PGC column from Thermo Scientific (5 pm, 150 mm x 1 mm). A 45 min binary gradient was employed: 0.00-2.50 min: 0.0-0.0%A; 2.50-15.00 min: 0.0-16.0%B; 15.00-20.00 min: 16.0-58.0%B; 20.00-22.00 min: 58.0-100.0%B; 22.00-28.00 min: 100.0-100.0%B; 28.00-30.00 min: 100.0-0.0%B; 30.00-40.00 min: 0.0-0.0%B. Solvent A consisted of 3% (v/v) ACN in water + 0.1% formic acid and solvent B consisted of 90% (v/v) ACN in water + 0.1% formic acid.

[066] For identification of HMOs, a Thermo Scientific qExactive mass spectrometer equipped with an electrospray ionization source was operated in positive ionization mode with the following parameters: scan range = 133.4 - 2000 m/z, spray voltage = 2.5 IkVI, capillary temperature = 320°C, aux gas heater temperature = 325 °C, sheath gas flow rate = 25, aux gas flow rate = 8, sweep gas flow rate = 3. MS/MS analysis was performed using stepped collision energies of 20, 30, 40 [eV].

[067] For quantification of LNFPI and HMO profiling, mass spectral analysis was carried out on an Agilent 6530 Accurate-Mass Q-TOF MS operated in positive mode using data dependent acquisition. The gas temperatures were held at 150 °C. The fragmentor, skimmer, octopole, and capillary were operated at 70 V, 55 V, 750 V, and 1800 V, respectively. The collision energy was based on the empirically derived linear formula (1.8 x m/z - 3.6). The reference mass used for calibration was 922.009798 m/z. The Agilent MassHunter Qualitative software was used for data analysis. Oligosaccharides were identified using an in-house library, their MS/MS spectra, and comparison to either authenticated standards or a pool of human milk oligosaccharides of known composition. The reference mass used for calibration was 922.009798 m/z.

[068] Results

[069] Production of neutral HMOs

[070] Non-fucosylated neutral HMOs (nHMOs) are composed of a lactose core with various decorations of galactose and N-acetylglucosamine (GlcNAc). nHMOs are the most abundant class of HMOs in breast milk, comprising 42-55% of HMOs (Totten et al., 2012). While nHMOs provide various health benefits, they also serve as the core scaffold for the production of various complex HMOs.

[071] To produce nHMOs, lactose is first made by a β-1,4-galactosyltransferase, GalTPM1141 , which glycosylates glucose using UDP-galactose (Parschat et al., 2020). Following lactose production, a β-1,3-N-acetylglucosaminyltransferase, NmLgtA. generates lacto-V-lriose (LNTII) through the transfer of GlcNAC from UDP-GlcNAc to the galactose in lactose (McArthur et al., 2019). LNTII serves as a branch point for the production of various complex HMOs that can be further modified to generate large, complex HMOs. LNTII can then be used to generate lacto-N- tetraose (LNT) and lacto-N-neotetraose (LNnT), using β-1,3-galactosyltranserase, Cvβ3GalT, and a β-1,4-galactosyltransferase (e.g. HP0826 or NmLgtB}, respectively (Fang et al., 2018; McArthur et al., 2019). Expression of GalTPM1141, NmLgtA, HP0826 (or NmLgtB). and Cvβ3GalT (Figure 1) results in the production of ions with a m/z of 708.2565 and 708.2568 corresponding to LNnT and LNT respectively (Figure 2, Table 1). Analysis using HILIC and PGC chromatography with analytical standards, retention time and MS/MS fragmentation further validated the identification of these structures, demonstrating the ability of plants to make nHMOs. Additionally, neutral HMOs with a higher degree of polymerization composed of 4 hexose sugars/1 hexNAc sugar, 5 hexose sugars/2 hexNAc sugars, and 6 hexose sugars/1 hexNAc were identified (Figure 2, Table 1). These ions had values for m/z. of 870.3091, 1235.4430, and 1194.4120, respectively. This displays the ability of plants to create HMOs with a high degree of polymerization.

[072] Production and optimization of fucosylated HMOs

[073] Fucosylated HMOs (fHMOs) are a key component of breast milk and are characterized by the presence of a lactose or nHMO core decorated with one or more fucose moieties. While their abundance is dependent on the genotype of the mother, they are estimated to account for 35-50% of HMOs found in breast milk (Totten et al., 2012). fHMOs are of particular interest as they have distinct bioactivities from other HMOs. 2’-fucosyllactose (2’FL) is one of the most abundant HMOs found in human breast milk. Microbial production is currently able to produce 2’FL at a commercial scale; however, production of larger, abundant fHMOs such as lactodifucotetraose (LDFT), lacto-N-difucohexaose (LNDFH) and lacto-N-l'ucopcntaose (LNFP) has not been possible at a commercial scale.

[074] Lacto-N-f ucopenlaose 1 (LNFPl) is a fucosylated pentasaccharide generated by the addition of an α-1,2-linked fucose to the terminal galactose in lacto-N-tetraose (LNT). To produce LNFPl, we transiently expressed GalTPM1141, NmLgtA, Cvβ3GalT, HP0826 and Te2FT in N. benthamiana (Figure 1). The expression of this pathway alone resulted in the production of an ions with m/z of 489.1824, 708.2568, 708.2565 and 854.3150, corresponding to fucosyllactose, lacto-N-tetraose or lacto-N-neotetraose, and lacto-N-fucopentaose I, respectively (Figure 3, Table 1). Analysis using HILIC and PGC chromatography with analytical standards, retention time and MS/MS fragmentation identified these structures as 2’FL, LNT, LNnT and LNFPl (Table 1). Leaves expressing the LNFPl pathway produced LNFPl at a concentration of 809 μg/g DW (Figure 4A). To improve LNFPl yields, nucleotide sugar biosynthetic pathways (Figure IB) were expressed to increase the availability of GDP- fucose {Man A, ManB, ManC, Gmd, WcaG), UDP-galactose (pgi, pgm, GalU, GalE), and UDP- GlcNAc (glmS, glmM, glmU) combinatorially (Figure 4). The highest LNFPl yields were obtained by the expression of the GDP-fucose pathway with the LNFPI pathway, reaching a concentration of 1075 μg/g DW (1.33-fold improvement) (Figure 4A). The increased yield following the expression of the GDP-fucose pathway suggests that native GDP-fucose levels are limiting (Figure 4A); thus, our pathway engineering efforts demonstrate a metabolic strategy to address this bottleneck and improve overall yields of fHMOs like LNFPI.

[075] HMO composition was also affected by changes in nucleotide sugar levels. Expression of the GDP-fucose pathway dramatically increased the relative amounts of oligosaccharides containing a deoxyhexose moiety, which likely corresponds to an increased abundance of fucosy lated oligosaccharides (Figure 4B). Expression of the UDP-GlcNAc pathway increased the relative amounts of oligosaccharides containing at least one hexose and one N- acetylhexosamine (hexNAc) (Figure 4B). Together, these results display our ability to generate neutral and fucosylated HMOs in a single leaf and alter HMO composition by overexpression of various nucleotide sugar biosynthetic pathways.

[076] In addition to producing the expected HMOs when expressing the LNFPI biosynthetic pathway (Figure 1 A) and the GDP-fucose biosynthetic pathway (Figure IB), we identified the unanticipated production of two additional ions of m/z 635.2411 and 1000.3700. Analysis using HILIC and PGC chromatography with analytical standards, retention time and MS/MS fragmentation, the ion with m/z 635.2411 was identified as the HMO, lactodifucotetraose (LDFT), and the ion with m/z 1000.3700 was identified as the HMO, lacto-N-difucohexaose I (LNDFHI) (Figure 3, Table 1). Since none of the enzymes used in this pathway are reported to have either α-1,3- or α-1, 4-fucosyltransferase activity, we suspect that these additional glycosylations are products of endogenous plant enzymes; thus demonstrating some of the natural unanticipated advantages of using plants as a chassis for the production of HMOs. The discovery that plants have enzymes to fill in the last steps involved in making HMOs such as LDFT and LNDFHI highlight how plants may serve as a more attractive host for production of HMOs than current microbial platforms. Specifically, plant metabolism harbors the innate ability to further glycosylate simple HMOs to create HMOs of higher complexity that have not been previously produced through any other heterologous hosts.

[077] Production of acidic HMOs

[078] Acidic HMOs (aHMOs) are a class of HMO characterized by the presence of a lactose or nHMO core with one or more N -acetylneuraminic acid (Neu5Ac) moieties. aHMOs represent a structurally diverse class of HMOs, accounting for 12-14% of HMOs found in human breast milk (Totten et al., 2012). While aHMOs are not currently commercially available, simple aHMOs, such as 3’-sialyl1actose (3’SL) and 6’-sialyllactose (6’SL), have been produced in E. coll (Ruffing & Chen, 2006); however, microbial systems have been unable to produce larger aHMOs, such as isomers of sialyllacto-N-tetraose (LST). To overcome this limitation, we sought to produce both simple and complex aHMOs using transient expression in N. benthamiana. [079] Since plants do not natively produce CMP-Neu5Ac, we tested a three gene bacterial pathway (neuA, neuB, neuC) and a four gene mammalian pathway (GNE, NANS, NANP, CMAS) which utilize UDP-GlcNAc as a precursor for the production of CMP-Neu5Ac (Castilho et al., 2008; Vimr et al., 2004). Expression of the bacterial CMP-Neu5Ac pathway and aHMO biosynthetic genes did not produce any aHMOs. Expression of the mammalian CMP-Neu5Ac pathway, UDP-GlcNAc pathway and aHMO biosynthetic genes resulted in the successful production of aHMOs, displaying the successful production of CMP-Neu5Ac.

[080] Simple aHMOs, such as 3’SL and 6’SL, are produced through the α2-3 or α2-6 sialylation of lactose. LST is a complex pentasaccharide aHMO with multiple isomers. Production of LST requires the production of Lacto-N-neotetraose (LNnT) and its subsequent addition of an a-2,6-linked or a-2,3-linked Neu5Ac. To produce 3’SL, 6’SL and isomers of LST, we expressed a mammalian CMP-Neu5Ac pathway, GalTPMl 141 , NmLgtA, Cvβ3GalT, Hp0826 (or NmLgtB) and either PmSt3 or S16 (Figure 1 A). Following HILIC LC-MS analysis a peak was identified for m/z 634.2189, indicating sialyllactose was present (Figure 5). Further analysis using PGC chromatography with analytical standards, retention time and MS/MS fragmentation indicates the presence of two sialyllactose isomers, corresponding to 3’SL and 6’SL (Figure 5, Table 1). Multiple peaks are also present for m/z 999.3483, indicating the production of LST. Further analysis using PGC chromatography with analytical standards, retention time, and MS/MS fragmentation indicate the production of LSTc and LSTd in planta (Figure 5, Table 1 ). This is especially notable, as this is the first successful production of LST in a heterologous host.

[081] The production of 3’SL, 6’SL, LNnT, LNT, and multiple isomers of LST in a heterologous host has never been described before. Additionally, this is the first demonstration of producing LST isomers in a heterologous host, displaying the utility of plants as a platform. The production of aHMOs will serve as a starling point for the production of HMOs decorated with both fucose and Neu5Ac moieties.

[082] Production of HMOs in stably transformed plants

[083] To test the ability of stable transgenic plants to produce HMOs, we generated two constructs for the constitutive production of 2’ FL and LNFPI in stably transformed N. benthamiana. HMO10 (Figure 6) contains genes for the four enzymes required to produce lactose, 2 ’FL, LNTII, LNT, and LNFPI connected via 2A peptides to allow multiple coding sequences to be driven by a single promoter. Each transcriptional unit is driven by a strong constitutive promoter to enable HMO production in all tissues. To explore the effects of overexpressing portions of the GDP-fucose pathway, we also generated stable lines expressing HM011 (Figure 6), which contains a GDP-D-mannose-4,6-dehydratase (Gmd) from the GDP- fucose pathway. Both constructs enabled the production of multiple HMOs, specifically 2’ FL (Figure 7) and LNFPI (Figure 8).

[084] References

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[093] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

[094] Table 1. Chromatographic retention time and mass spectrum peaks of HMOs identified using liquid chromatography-mass spectrometry with a porous graphitic carbon column. * denotes identification with an authenticated standard. [095] HMO Gene Sequences: Gene Name, Accession, Nucleotide and Amino acid sequence