Alpha (1,3) fucosyltransferases for use in the production of fucosylated oligosaccharides

Abstract

The invention relates to methods and compositions for the production of fucosylated oligosaccharides.

Claims

1. A method for producing a fucosylated oligosaccharide in a bacterium comprising expressing a lactose-accepting α(1,3) fucosyltransferase enzyme in a host bacterium, wherein the amino acid sequence of said enzyme comprises at least 90% identity to full length CafC (SEQ ID NO: 2), thereby producing an α(1,3) fucosylated oligosaccharide.

2. The method of claim 1, wherein the amino acid sequence of said enzyme comprises at least 95% sequence identity to full length CafC (SEQ ID NO: 2).

3. The method of claim 1, wherein said amino acid sequence comprises at least 50% or at least 80% identity to the CafC active site region 2 (residues 116-202 of SEQ ID NO:2).

4. The method of claim 1, wherein (a) said α(1,3) fucosyltransferase enzyme comprises CafC, or a functional variant or fragment thereof; or (b) said α(1,3) fucosyltransferase enzyme comprises the amino acid sequence of SEQ ID NO: 2 (CafC), or a functional fragment thereof.

5. The method of claim 1, further comprising providing the bacterium a nucleic acid construct comprising an isolated nucleic acid encoding the α(1,3) fucosyltransferase enzyme.

6. The method of claim 5, wherein said nucleic acid is operably linked to one or more heterologous control sequences that direct the production of the enzyme in the bacterium.

7. The method of claim 6, wherein said heterologous control sequence comprises a bacterial promoter and operator, a bacterial ribosome binding site, a bacterial transcriptional terminator, or a plasmid selectable marker.

8. The method of claim 1, further comprising retrieving the fucosylated oligosaccharide from said bacterium or from a culture supernatant of said bacterium.

9. The method of claim 1, wherein said fucosylated oligosaccharide comprises 3-fucosyllactose (3-FL) or lactodifucotetraose (LDFT).

10. The method of claim 1, wherein said bacterium is E. coli.

11. The method of claim 10, further comprising culturing said bacterium in the presence of tryptophan and in the absence of thymidine.

12. The method of claim 10, wherein an endogenous lacZ gene and an endogenous lacI gene of said bacterium are deleted.

13. The method of claim 12, wherein said bacterium comprises a lacIq gene promoter immediately upstream of a lacY gene.

14. The method of claim 10, wherein an endogenous wcaJ gene of said bacterium is deleted.

15. The method of claim 10, wherein said bacterium comprises a null mutation in a lon gene.

16. The method of claim 10, wherein said bacterium accumulates intracellular lactose in the presence of exogenous lactose.

17. The method of claim 10, wherein said bacterium accumulates intracellular guanosine diphosphate (GDP)-fucose.

18. The method of claim 10, wherein said bacterium further expresses an α(1,2) fucosyltransferase enzyme, an α(1,4) fucosyltransferase enzyme, and/or an additional α(1,3) fucosyltransferase enzyme.

19. The method of claim 5, wherein the nucleic acid construct further comprises an isolated nucleic acid encoding an α(1,2) fucosyltransferase enzyme and/or an additional α(1,3) fucosyltransferase enzyme.

20. The method of claim 10, further comprising culturing said bacterium in the presence of a nitrogen-rich additive.

21. The method of claim 20, wherein the nitrogen-rich additive comprises casamino acids (CAA), yeast extract (YE), or a protein hydrolysate.

22. The method of claim 1, wherein said α(1,3) fucosyltransferase enzyme comprises the amino acid sequence of SEQ ID NO: 2 (CafC).

23. The method of claim 1, wherein the method further comprises culturing the bacterium in the presence of lactose to produce the fucosylated oligosaccharide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic illustration showing the synthetic pathway of the major neutral fucosyl-oligosaccharides found in human milk.

(2) FIG. 2 is a schematic demonstrating metabolic pathways and the changes introduced into them to engineer 3-fucosyllactose (3-FL) synthesis in Escherichia coli (E. coli). Specifically, the lactose synthesis pathway and the GDP-fucose synthesis pathway are illustrated. In the GDP-fucose synthesis pathway: manA=phosphomannose isomerase (PMI), manB=phosphomannomutase (PMM), manC=mannose-1-phosphate guanylyltransferase (GMP), gmd=GDP-mannose-4,6-dehydratase, fcl=GDP-fucose synthase (GFS), and AwcaJ=mutated UDP-glucose lipid carrier transferase.

(3) FIG. 3 is a scheme outlining the two sequential database screens that led to the discovery of the several novel α(1,3) fucosyltransferases of this invention.

(4) FIG. 4 is a series of photographs showing thin layer chromatography analysis of 3-FL produced in E. coli strains by candidate α(1,3) fucosyltransferases that were identified in an initial database screen utilizing the FutA sequence as the query. FIG. 4A shows significant production of 3-FL by FutA, CafA, and CafB. FIG. 4B shows significant production of 3-FL by FutA and CafC. FIG. 4C shows significant production of 3-FL by CafF.

(5) FIG. 5 is a series of photographs showing protein expression of Caf genes in an E. coli production strain.

(6) FIG. 6 is a photograph showing thin layer chromatography analysis of 3-FL produced in E. coli strains by 12 candidate α(1,3) fucosyltransferases identified in a second database screen that used a sequence alignment of CafC and CafF as the query. The figure shows significant production of 3-FL by FutA, CafC, CafF and also by the new candidate enzymes CafL, CafN, CafO, CafQ, CafU and CafV.

(7) FIG. 7 is a schematic demonstrating metabolic pathways and the changes introduced into them to engineer lactodifucotetraose (LDFT) synthesis in Escherichia coli (E. coli).

(8) FIG. 8 shows the expression of LDFT in host bacteria expressing an α(1,3) fucosyltransferase (CafA, CafC, CafF) in combination with an α(1,2) fucosyltransferase (wbgL).

(9) FIG. 9 is a schematic demonstrating metabolic pathways and the changes introduced into them to engineer lacto-N-fucopentaose (LNF III, Le.sup.x) synthesis in Escherichia coli (E. coli).

(10) FIG. 10 shows synthesis of LNF III by attachment of fucose to LNnT.

(11) FIG. 11 is a diagram of plasmid pG364 (pEC2-cafF-rcsA-thyA).

(12) FIG. 12 is a diagram of plasmid pG365 (pEC2-cafA-rcsA-thyA).

(13) FIG. 13 is a diagram of plasmid pG366 (pEC2-cafC-rcsA-thyA).

(14) FIG. 14 is a diagram of plasmid pG369 (pEC2-wbgL-cafA-rcsA-thyA).

(15) FIG. 15 is a diagram of plasmid pG370 (pEC2-wbgL-cafF-rcsA-thyA).

(16) FIG. 16 is a diagram of plasmid pG371 (pEC2-wbgL-cafC-rcsA-thyA).

(17) FIG. 17 is a diagram of plasmid pG367 (pEC2′-LgtA-4GalT-cafD-ThyA).

(18) FIG. 18 is a sequence alignment of FutA (SEQ ID NO: 54) with 8 lactose-utilizing “Caf” α(1,3) fucosyltransferases (i.e. CafF (SEQ ID NO: 1), CafC (SEQ ID NO: 2), CafV (SEQ ID NO: 17), CafN (SEQ ID NO: 9), CafL (SEQ ID NO: 7), CafO (SEQ ID NO: 10), CafQ (SEQ ID NO: 12), and CafU (SEQ ID NO: 16)) discovered in the computational screens of this invention. Conserved regions important for substrate binding and catalysis are delineated by thick bars above the sequences. Within those bars the white dots indicate the four completely conserved residues at the catalytic active site. Consensus sequences is SEQ ID NO: 62.

(19) FIG. 19 is a sequence alignment across “active site region 2” (corresponding to FutA residues 180-266) of CafC with 8 other lactose-utilizing “Caf” α(1,3) fucosyltransferases (i.e. CafV (SEQ ID NO: 17), CafN (SEQ ID NO: 9), CafL (SEQ ID NO: 7), CafO (SEQ ID NO: 10), CafQ (SEQ ID NO: 12), CafU (SEQ ID NO: 16), CafF (SEQ ID NO: 1) and FutA (SEQ ID NO: 54)). Conserved regions important for substrate binding and catalysis are delineated by thick bars above the sequences. Within those bars the white dots indicate three completely conserved residues at this region of the catalytic active site. Consensus sequences is SEQ ID NO: 63.

(20) FIG. 20 is a pairwise comparison table of the alignment of FIG. 19, presenting percent identities across “active site region 2” (corresponding to FutA residues 180-266) of CafC with 8 other lactose-utilizing “Caf” α(1,3) fucosyltransferases (i.e. CafV, CafN, CafL, CafO, CafQ, CafU, CafF and FutA).

(21) FIG. 21 is a diagram of plasmid pG420 (pEC2-cafC-cafN-rcsA-thyA).

(22) FIG. 22A-FIG. 22C are images of thin layer chromatography analysis of culture supernatants for various strains of the invention. FIG. 22A is an image of thin layer chromatography analysis of culture supernatants from a pEC2-P.sub.L—CafC-rcsA-thyA (pG366) strain. FIG. 22B is an image of thin layer chromatography analysis of culture supernatants from a pEC2-P.sub.L—CafC-CafN-rcsA-thyA (pG420) strain. FIG. 22C is an image of thin layer chromatography analysis of culture supernatants

DETAILED DESCRIPTION

(23) While some studies suggest that human milk glycans could be used as antimicrobial anti-adhesion agents, the difficulty and expense of producing adequate quantities of these agents of a quality suitable for human consumption has limited their full-scale testing and perceived utility. What has been needed is a suitable method for producing the appropriate glycans in sufficient quantities at reasonable cost. Prior to the invention described herein, there were attempts to use several distinct synthetic approaches for glycan synthesis. Some chemical approaches can synthesize oligosaccharides (Flowers, H. M. Methods Enzymol 50, 93-121 (1978); Seeberger, P. H. Chem Commun (Camb) 1115-1121 (2003)), but reactants for these methods are expensive and potentially toxic (Koeller, K. M. & Wong, C. H. Chem Rev 100, 4465-4494 (2000)).

(24) Enzymes expressed from engineered organisms (Albermann, C., Piepersberg, W. & Wehmeier, U. F. Carbohydr Res 334, 97-103 (2001); Bettler, E., Samain, E., Chazalet, V., Bosso, C., et al. Glycoconj J 16, 205-212 (1999); Johnson, K. F. Glycoconj J 16, 141-146 (1999); Palcic, M. M. Curr Opin Biotechnol 10, 616-624 (1999); Wymer, N. & Toone, E. J. Curr Opin Chem Biol 4, 110-119 (2000)) provide a precise and efficient synthesis (Palcic, M. M. Curr Opin Biotechnol 10, 616-624 (1999)); Crout, D. H. & Vic, G. Curr Opin Chem Biol 2, 98-111 (1998)), but the high cost of the reactants, especially the sugar nucleotides, limits their utility for low-cost, large-scale production. Microbes have been genetically engineered to express the glycosyltransferases needed to synthesize oligosaccharides from the bacteria's innate pool of nucleotide sugars (Endo, T., Koizumi, S., Tabata, K., Kakita, S. & Ozaki, A. Carbohydr Res 330, 439-443 (2001); Endo, T., Koizumi, S., Tabata, K. & Ozaki, A. Appl Microbiol Biotechnol 53, 257-261 (2000); Endo, T. & Koizumi, S. Curr Opin Struct Biol 10, 536-541 (2000); Endo, T., Koizumi, S., Tabata, K., Kakita, S. & Ozaki, A. Carbohydr Res 316, 179-183 (1999); Koizumi, S., Endo, T., Tabata, K. & Ozaki, A. Nat Biotechnol 16, 847-850 (1998)).

(25) One strategy for efficient, industrial-scale synthesis of HMOS is the metabolic engineering of bacteria. This approach involves the construction of microbial strains overexpressing heterologous glycosyltransferases, membrane transporters for the import of precursor sugars into the bacterial cytosol, and possessing enhanced pools of regenerating nucleotide sugars for use as biosynthetic precursors (Dumon, C., Samain, E., and Priem, B. (2004). Biotechnol Prog 20, 412-19; Ruffing, A., and Chen, R. R. (2006). Microb Cell Fact 5, 25). A key aspect of this approach is the heterologous glycosyltransferase selected for overexpression in the microbial host. The choice of glycosyltransferase can significantly affect the final yield of the desired synthesized oligosaccharide, given that enzymes can vary greatly in terms of kinetics, substrate specificity, affinity for donor and acceptor molecules, stability and solubility. A few glycosyltransferases derived from different bacterial species have been identified and characterized in terms of their ability to catalyze the biosynthesis of HMOS in E. coli host strains (Dumon, C., et al. (2006). Chembiochem 7, 359-365; Dumon, C., Samain, E., and Priem, B. (2004). Biotechnol Prog 20, 412-19; Li, M., Liu, X. W., Shao, J., Shen, J., Jia, Q., Yi, W., Song, J. K., Woodward, R., Chow, C. S., and Wang, P. G. (2008). Biochemistry 47, 378-387). The identification of additional glycosyltransferases with faster kinetics, greater affinity for nucleotide sugar donors and/or acceptor molecules, or greater stability within the bacterial host significantly improves the yields of therapeutically useful HMOS. Prior to the invention described herein, chemical syntheses of HMOS were possible, but were limited by stereo-specificity issues, precursor availability, product impurities, and high overall cost (Flowers, H. M. Methods Enzymol 50, 93-121 (1978); Seeberger, P. H. Chem Commun (Camb) 1115-1121 (2003); Koeller, K. M. & Wong, C. H. Chem Rev 100, 4465-4494 (2000)). The invention overcomes the shortcomings of these previous attempts by providing new strategies to inexpensively manufacture large quantities of human milk oligosaccharides (HMOS) for use as dietary supplements.

(26) Prior to the invention described herein, there was a growing need to identify and characterize additional glycosyltransferases that are useful for the synthesis of HMOS in metabolically engineered bacterial hosts.

(27) Advantages provided by the invention include efficient expression of the enzyme, improved stability and/or solubility of the fucosylated oligosaccharide product β-FL, LDFT and LNF III,) and reduced toxicity to the host organism. The invention features novel α(1,3) FTs suitable for expression in production strains for increased efficacy and yield of fucosylated HMOS compared to α(1,3) FTs currently utilized in the field.

(28) Human Milk Glycans

(29) Human milk contains a diverse and abundant set of neutral and acidic oligosaccharides (Kunz, C., Rudloff, S., Baier, W., Klein, N., and Strobel, S. (2000). Annu Rev Nutr 20, 699-722; Bode, L. (2006). J Nutr 136, 2127-130). More than 130 different complex oligosaccharides have been identified in human milk, and their structural diversity and abundance is unique to humans. Although these molecules may not be utilized directly by infants for nutrition, they nevertheless serve critical roles in the establishment of a healthy gut microbiome (Marcobal, A., Barboza, M., Froehlich, J. W., Block, D. E., et al. J Agric Food Chem 58, 5334-5340 (2010)), in the prevention of disease (Newburg, D. S., Ruiz-Palacios, G. M. & Morrow, A. L. Annu Rev Nutr 25, 37-58 (2005)), and in immune function (Newburg, D. S. & Walker, W. A. Pediatr Res 61, 2-8 (2007)). Despite millions of years of exposure to human milk oligosaccharides (HMOS), pathogens have yet to develop ways to circumvent the ability of HMOS to prevent adhesion to target cells and to inhibit infection. The ability to utilize HMOS as pathogen adherence inhibitors promises to address the current crisis of burgeoning antibiotic resistance. Human milk oligosaccharides produced by biosynthesis represent the lead compounds of a novel class of therapeutics against some of the most intractable scourges of society.

(30) Role of Human Milk Glycans in Infectious Disease

(31) Human milk glycans, which comprise both unbound oligosaccharides and their glycoconjugates, play a significant role in the protection and development of the infant gastrointestinal (GI) tract. Neutral fucosylated oligosaccharides, including α(1,3) fucosylated oligosaccharides, protect infants against several important pathogens. Milk oligosaccharides found in various mammals differ greatly, and the composition in humans is unique (Hamosh M., 2001 Pediatr Clin North Am, 48:69-86; Newburg D. S., 2001 Adv Exp Med Biol, 501:3-10). Moreover, glycan levels in human milk change throughout lactation and also vary widely among individuals (Morrow A. L. et al., 2004 J Pediatr, 145:297-303; Chaturvedi P et al., 2001 Glycobiology, 11:365-372). Approximately 200 distinct human milk oligosaccharides have been identified and combinations of simple epitopes are responsible for this diversity (Newburg D. S., 1999 Curr Med Chem, 6:117-127; Ninonuevo M. et al., 2006 J Agric Food Chem, 54:7471-74801).

(32) Human milk oligosaccharides are composed of 5 monosaccharides: D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc), and sialic acid (N-acetyl neuraminic acid, Neu5Ac, NANA). Human milk oligosaccharides are usually divided into two groups according to their chemical structures: neutral compounds containing Glc, Gal, GlcNAc, and Fuc, linked to a lactose (Galβ1-4Glc) core, and acidic compounds including the same sugars, and often the same core structures, plus NANA (Charlwood J. et al., 1999 Anal Biochem, 273:261-277; Martin-Sosa et al., 2003 J Dairy Sci, 86:52-59; Parkkinen J. and Finne J., 1987 Methods Enzymol, 138:289-300; Shen Z. et al., 2001 J Chromatogr A, 921:315-321).

(33) Approximately 70-80% of oligosaccharides in human milk are fucosylated, and their synthetic pathways are believed to proceed as shown in FIG. 1. A smaller proportion of the oligosaccharides are sialylated or both fucosylated and sialylated, but their synthetic pathways are not fully defined. Understanding of the acidic (sialylated) oligosaccharides is limited in part by the ability to measure these compounds. Sensitive and reproducible methods for the analysis of both neutral and acidic oligosaccharides have been designed. Human milk oligosaccharides as a class survive transit through the intestine of infants very efficiently, being essentially indigestible (Chaturvedi, P., Warren, C. D., Buescher, C. R., Pickering, L. K. & Newburg, D. S. Adv Exp Med Biol 501, 315-323 (2001)).

(34) Human Milk Glycans Inhibit Binding of Enteropathogens to their Receptors

(35) Human milk glycans have structural homology to cell receptors for enteropathogens and function as receptor decoys.

(36) For example, 3-fucosyllactose (3FL) is one of the most abundant fucosylated oligosaccharides present in human milk and is thought to function with other HMOS to promote the growth of beneficial commensal bacteria in the infant gut, such as Bifidobacterium spp (Marcobal, A., et al. (2010). Consumption of human milk oligosaccharides by gut-related microbes. J Agric Food Chem 58, 5334-340.; Asakuma, S., et al. (2011). Physiology of the consumption of human milk oligosaccharides by infant-gut associated bifidobacteria. J Biol Chem; Sela, D. A., et al. (2012). Bifidobacterium longum subsp. infantis ATCC 15697 α-fucosidases are active on fucosylated human milk oligosaccharides. Appl Environ Microbiol 78, 795-803.; Garrido, D., et al. (2012). A molecular basis for bifidobacterial enrichment in the infant gastrointestinal tract. Adv Nutr 3, 415S-421S.). Indeed, it has been shown that 3FL can be utilized for growth by several different Bifidobacterium spp. In vitro when provided as the sole sugar source (Yu, Z. T., et al. (2012). The Principal Fucosylated Oligosaccharides of Human Milk Exhibit Prebiotic Properties on Cultured Infant Microbiota. Glycobiology). Furthermore, it has been demonstrated that 3FL was consumed in the context of an in vitro infant fecal microbiota culture system, providing further evidence that 3FL is a substrate for beneficial commensal microbes in the infant gut (Yu, Z. T., et al. (2012). The Principal Fucosylated Oligosaccharides of Human Milk Exhibit Prebiotic Properties on Cultured Infant Microbiota. Glycobiology). In addition, several bacterial and viral pathogens target host cell molecules with structural similarity to 3FL for cell-surface binding in the process of initiating infection. Several studies have shown that 3FL can prevent the binding of pathogens to their target molecules or host cells via a competition mechanism, suggesting that 3FL will also be useful as an anti-infective molecule (Huang et al, 2003; Coppa et al 2006; Chessa et al, 2008). Structurally, 3FL consists of a fucose molecule α1,3 linked to the glucose portion of lactose (Galβ1-4(Fucα1-3)Glc) (FIG. 1). This structure is highly similar to that of the Lewis x (Le.sup.x) histo-blood group antigen (Galβ1,4(Fucα1,3)GlcNAcβ-R), a common epitope of glycoproteins and glycolipids that has a role in many different biological processes (Rudloff, S., and Kunz, C. (2012). Milk oligosaccharides and metabolism in infants. Adv Nutr 3, 398S-405S.).

(37) LDFT is a di-fucosylated HMOS and has the structure Fucα1,2Galβ1,4(Fucα1,3)Glc. LDFT is one of the most abundant HMOS found in human milk (Newburg et al., 2000; Warren et al., 2001). LDFT has been shown to be utilized as a sugar source for growth in vitro by beneficial, commensal bacteria of the infant gut (i.e. Bifidobacteria spp.) and will therefore have utility as an important prebiotic, or “Bifidogenic” factor (Asakuma, S., et al. (2011). Physiology of the consumption of human milk oligosaccharides by infant-gut associated bifidobacteria. J Biol Chem; Yu, Z. T., et al. (2012). The Principal Fucosylated Oligosaccharides of Human Milk Exhibit Prebiotic Properties on Cultured Infant Microbiota. Glycobiology; Blank, D., et al. (2012). Human milk oligosaccharides and Lewis blood group: individual high-throughput sample profiling to enhance conclusions from functional studies. Adv Nutr 3, 440S-49S.). Furthermore, LDFT is structurally highly similar to the histo-blood group antigen Lewis Y (Le.sup.y). Many bacterial and viral pathogens target molecules on the surface of host cells with structural similarity to the Lewis Y epitope for binding in the process of initiating infection, such as at the lining of the gut. Orally administered LDFT could serve as a structural mimic of host cell receptors and therefore prevent the binding of pathogens to the intestinal epithelium via a competition mechanism (Ruiz-Palacios, G. M., et al. (2003). Campylobacter jejuni binds intestinal H(O) antigen (Fuc alpha 1, 2Gal beta 1, 4GlcNAc), and fucosyloligosaccharides of human milk inhibit its binding and infection. J Biol Chem 278, 14112-120.; Morrow, A. L., et al. (2004). Human milk oligosaccharide blood group epitopes and innate immune protection against campylobacter and calicivirus diarrhea in breastfed infants. Adv Exp Med Biol 554, 443-46.; Sharon, N. (2006). Carbohydrates as future anti-adhesion drugs for infectious diseases. Biochim Biophys Acta 1760, 527-537.; Bode, L., and Jantscher-Krenn, E. (2012). Structure-function relationships of human milk oligosaccharides. Adv Nutr 3, 383S-391S.).

(38) LNF III has the structure Galβ1-4(Fucα1,3)GlcNacβ1-3Galβ1-4Glc, and contains the Le.sup.x antigen structure. LNF III is likely to serve as a prebiotic factor for the growth of commensal microbes in the infant gut, and also may prevent the binding of microbial pathogens to the intestinal epithelia via receptor mimicry.

(39) Several pathogens utilize sialylated glycans as their host receptors, such as influenza (Couceiro, J. N., Paulson, J. C. & Baum, L. G. Virus Res 29, 155-165 (1993)), parainfluenza (Amonsen, M., Smith, D. F., Cummings, R. D. & Air, G. M. J Virol 81, 8341-8345 (2007), and rotoviruses (Kuhlenschmidt, T. B., Hanafin, W. P., Gelberg, H. B. & Kuhlenschmidt, M. S. Adv Exp Med Biol 473, 309-317 (1999)). The sialyl-Lewis X epitope is used by Helicobacter pylori (Mandavi, J., Sondén, B., Hurtig, M., Olfat, F. O., et al. Science 297, 573-578 (2002)), Pseudomonas aeruginosa (Scharfman, A., Delmotte, P., Beau, J., Lamblin, G., et al. Glycoconj J 17, 735-740 (2000)), and some strains of noroviruses (Rydell, G. E., Nilsson, J., Rodriguez-Diaz, J., Ruvoën-Clouet, N., et al. Glycobiology 19, 309-320 (2009)).

(40) Identification of Novel α(1,3) Fucosyltransferases

(41) The present invention provides novel α(1,3) fucosyltransferase enzymes (α(1,3) FTs). The α(1,3) FTs of the invention provide advantages over known α(1,3) fucosyltransferase enzymes, such advantages including improved yield, improved specificity, and reduced toxicity to host cells.

(42) Not all α(1,3)fucosyltransferases can utilize lactose as an acceptor substrate. An acceptor substrate includes, for example, a carbohydrate, an oligosaccharide, a protein or glycoprotein, a lipid or glycolipid, e.g., N-acetylglucosamine, N-acetyllactosamine, galactose, fucose, sialic acid, glucose, lactose, or any combination thereof. A preferred alpha (1,3) fucosyltransferase utilizes GDP-fucose as a donor, and lactose is the acceptor for that donor.

(43) A method of identifying novel α(1,2)fucosyltransferase enzymes capable of utilizing lactose as an acceptor was previously carried out (as described in PCT/US2013/051777, hereby incorporated by reference in its entirety) using the following steps: 1) performing a computational search of sequence databases to define a broad group of simple sequence homologs of any known, lactose-utilizing α(1,2)fucosyltransferase (e.g. in this case Helicobacter pylori 26695 FutC); 2) using the list of homologs from step 1 to derive a search profile containing common sequence and/or structural motifs shared by the members of the broad group, e.g. by using computer programs such as MEME (Multiple Em for Motif Elicitation. http://meme.sdsc.edu/meme/cgi-bin/meme.cgi (accessed Aug. 5, 2014)) or PSI-BLAST (Position-Specific Iterated BLAST) (Blast. http://ncbi.nlm.nih.gov/blast (accessed Aug. 4, 2014); with additional information at openstax CNX. http://cnx.org/content/m11040/latest/(accessed Aug. 5, 2014)); 3) searching sequence databases (e.g., using computer programs such as PSI-BLAST, or MAST (Motif Alignment Search Tool. http://meme.sdsc.edu/meme/cgi-bin/mast.cgi (accessed Aug. 5, 2014)); using this derived search profile as query, and identifying “candidate sequences” whose simple sequence homology to the original lactose-accepting α(1,2)fucosyltransferase is 50% or less; 4) scanning the scientific literature and developing a list of “candidate organisms” known to express α(1,2)fucosyl-glycans, or whose natural habitat is known to include processes and interactions involving α(1,2)fucosyl-glycans; 5) selecting only those “candidate sequences” that are derived from “candidate organisms” to generate a list of “candidate lactose-utilizing enzymes”; and 6) expressing each “candidate lactose-utilizing enzyme” and testing for lactose-utilizing α(1,2)fucosyltransferase activity.

(44) The percentage sequence identity threshold in step (3) above is 50% or less, such as less than 50%. Preferably, the % sequence identity threshold is 45% or less, more preferably 42% or less. A preferred % sequence identity threshold is 6%-42%. The threshold was set to select candidate sequences which are more distantly-related to the query α(1,2)fucosyltransferase (e.g. in this case Helicobacter pylori 26695 FutC), and to exclude more closely related candidate sequences.

(45) Example α(1,2) fucosyltransferases include but are not limited to: Helicobacter pylori FutC (GenBank Accession AAD29869.1; Helicobacter mustelae 12198 FutL (GenBank Accession YP_003517185.1); Bacteroides vulgatus ATCC 8482 FutN (GenBank Accession YP_001300461.1); Escherichia coli UMEA 3065-1 WbgL (GenBank Accession WP_021554465.1); Escherichia coli WbsJ (GenBank Accession AA037698.1); Prevotella melaninogenica ATCC 25845 FutO (GenBank Accession YP_003814512.1); Clostridium bolteae 90A9 FutP (GenBank Accession WP_002570768.1); Lachnospiraceae bacterium 3_1_57FAA_CT1 FutQ (GenBank Accession WP_009251343.1); Methanosphaerula palustris E1-9c FutR (GenBank Accession YP_002467213.1); Tannerella sp. CAG:118 FutS (GenbBank WP_021929367.1); Bacteroides caccae ATCC 43185 FutU (GenBank Accession WP_005675707.1); Butyrivibrio sp. AE2015 FutV (GenBank Accesion WP_022772718.1); Prevotella sp. CAG:891 FutW (GenBank Accession WP_022481266.1); Parabacteroides johnsonii CL02T12C29 FutX (GenBank Accession WP_008155883.1); Salmonella enterica subsp. enterica serovar Poona str. ATCC BAA-1673 FutZ (GenBank Accession WP_023214330.1); and Bacteroides sp. CAG:633 (GenBank Accesion WP_022161880.1).

(46) The MEME suite of sequence analysis tools (MEME. http://meme.sdsc.edu/meme/cgi-bin/meme.cgi (accessed Aug. 5, 2014)) is optionally used as an alternative to PSI-BLAST. Sequence motifs are discovered using the program “MEME”. These motifs can then be used to search sequence databases using the program “MAST”. The BLAST and PSI-BLAST search algorithms are other well-known alternatives.

(47) An α(1,3) FT from H. pylori strain 26695 termed FutA has been utilized by others to catalyze the synthesis of 3FL in metabolically engineered E. coli (Dumon, C. et al. (2006). Production of Lewis x tetrasaccharides by metabolically engineered Escherichia coli. Chembiochem 7, 359-365.; Dumon, C. et al. (2004). Assessment of the two Helicobacter pylori alpha-1,3-fucosyltransferase ortholog genes for the large-scale synthesis of LewisX human milk oligosaccharides by metabolically engineered Escherichia coli. Biotechnol Prog 20, 412-19.), however the overall yield of 3FL obtained using this enzyme is low. Moreover FutA is promiscuous in its specificity, i.e. the enzyme will not only form an α-fucose linkage at the 3-position of glucose at the reducing end of sugar acceptors, but additionally will form α-fucose linkages at the 3-position of internal N-acetyl-glucosamine (GlcNAc) moieties. Thus FutA cannot be utilized effectively for the production of lacto-N-fucopentaose III (LNF-III, Lewis X) using lacto-N-neotetraose (LNnT) as the acceptor sugar. In addition FutA also catalyzes, at a low level, the promiscuous insertion of an α-fucose linkage at the 2-position of the galactose moiety of lactose. This latter activity, although it may sometime compromise the purity of a desired product in a particular biosynthesis, can also sometimes be advantageous, leading to the production of useful oligosaccharides as side products. The compositions and methods described herein overcomes these problems by providing novel α(1,3) fucosyltransferases, which generate higher 3-fucosyllactose yields, enable the production of LNF-III, and/or possess properties leading to either enhanced or reduced levels of oligosaccharide side products. The novel α(1,3) fucosyltransferases of the present invention therefore provide advantages over known α(1,3) fucosyltransferases, including FutA.

(48) FutA: SEQ ID NO: 54

(49) TABLE-US-00002 (SEQ ID NO: 54) MFQPLLDAFIESASIEKMASKSPPPPLKIAVANWWGDEEIKEFKKSVLY FILSQRYAITLHQNPNEFSDLVFSNPLGAARKILSYQNTKRVFYTGENE SPNFNLFDYAIGFDELDFNDRYLRMPLYYAHLHYKAELVNDTTAPYKLK DNSLYALKKPSHHFKENHPNLCAVVNDESDLLKRGFASFVASNANAPMR NAFYDALNSIEPVTGGGSVRNTLGYKVGNKSEFLSQYKFNLCFENSQGY GYVTEKILDAYFSHTIPIYWGSPSVAKDFNPKSFVNVHDFNNFDEAIDY IKYLHTHPNAYLDMLYENPLNTLDGKAYFYQDLSFKKILDFFKTILEND TIYHKFSTSFMWEYDLHKPLVSIDDLRVNYDDLRVNYDRLLQNASPLLE LSQNTTFKIYRKAYQKSLPLLRAVRKLKKLGL
Identification of Alternative α(1,3) Fucosyltransferases

(50) To identify novel α(1,3)fucosyltransferases, two sequential database screens were performed. An outline of these two sequential screens is shown in FIG. 3.

(51) First, the sequence of a single known lactose-accepting α(1,3)fucosyltransferase (i.e. H. pylori strain 26695 FutA) was used to search public databases to find simple homologs that might represent additional lactose-accepting α(1,3)fucosyltransferases. The amino acid sequence of FutA was used as a query in the search algorithm PSI-BLAST (Position Specific Iterated Basic Local Alignment Search Tool) in order to identify novel α(1,3) FTs. The PSI-BLAST program, using a given query protein sequence, generates a list of closely related proteins sequences based on a homology search of a database. These protein homolog hits are then used by the program to generate a profile reflecting their sequence similarities to the original query. The profile is then used by the algorithm to identify an expanded group of homolog proteins, and the process is iterated several times until the number of additional new candidates obtained after each iteration decreases. (Altschul et al., 1990, J. Mol. Bio. 215:403-410; Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402).

(52) The FutA amino acid sequence was used as a query for 3 iterations of the PSI-BLAST search algorithm. This approach yielded a group of 500 candidates with similarity to FutA, many of which were highly related to FutA (shared amino acid identity in the range of 50-90%) as well as a group that was more distantly related (shared amino acid identity less than 50%). Of note, FutA produces sub-optimal yields of 3FL when used in a metabolically engineered E. coli production strain. In addition, production of FutA appears to be moderately toxic in certain E. coli production strains, including the preferred strain for use herein. Therefore, candidates for further analysis were targeted from the more distantly related group identified via the PSI-BLAST search (shared amino acid identity to FutA of less than 50%) (Table 1). This group of candidates was similar to FutA, but primarily within the catalytic domain region of the respective proteins (Martin, S. L., et al. (1997). Lewis X biosynthesis in Helicobacter pylori. Molecular cloning of an alpha(1,3)-fucosyltransferase gene. J Biol Chem 272, 21349-356.; Breton, C., et al. (1998). Conserved structural features in eukaryotic and prokaryotic fucosyltransferases. Glycobiology 8, 87-94.; Rasko, A. (2000). Cloning and Characterization of the alpha (1,3/4) Fucosyltransferase of Helicobacter pylori. Journal of Biological Chemistry 275, 4988-994.). It is preferred that the α(1,3) fucosyltransferase of the invention, sharing 50% or less, preferably 45% or less, more preferably 42% or less overall sequence identity with FutA, at the same time possess a higher level of localized sequence identity to FutA within the catalytic domain (i.e. the regions covered by the thick black bars in FIG. 18). Without being bound by theory, it is believed that this group of candidates may include similar, better or distinct fucosyltransferase activities relative to FutA, but are different enough at the amino acid level to avoid the cryptic toxicity observed with FutA in production strains.

(53) These more distantly related (less than 50% sequence identity to FutA) predicted α(1,3) fucoysyl transferases (FTs) were further screened to identify predicted α(1,3) FTs from bacterial species that incorporate fucose into the 0-antigen of their lipopolysaccharide (LPS) or into the polysaccharide subunits that compose the cell surface capsule. Predicted α(1,3) FTs from these types of organisms are more likely to utilize fucose as a substrate, given the presence of fucose in their surface carbohydrate structures. Predicted α(1,3) FTs from known enteric bacterial species, either commensals or pathogens, were also identified. Such organisms sometimes display carbohydrate structures on their cell-surface that contain fucose and mimic various 3-fucosyl containing Lewis antigen structures found in higher organisms (Coyne, M. J., et al. (2005). Human symbionts use a host-like pathway for surface fucosylation. Science 307, 1778-781.; Appelmelk, B. J., et al. (1998). Phase variation in Helicobacter pylori lipopolysaccharide. Infect Immun 66, 70-76.; Ma, B., et al. (2006). Fucosylation in prokaryotes and eukaryotes. Glycobiology 16, 158R-184R.). Again, candidate α(1,3) FTs from these types of organisms are believed to be more likely to utilize fucose as a substrate and also to catalyze the linkage of fucose to useful acceptor oligosaccharides.

(54) 11 predicted α(1,3) FTs with homology to FutA ranging from 6-42% at the amino acid level were identified from PSI-BLAST. All of these candidates are found in bacteria that are known to interact with the gastrointestinal system of higher organisms. In addition, 3 of these candidates are found in bacteria that have been shown to incorporate fucose into their cell surface glycans. For ease of description, the genes encoding these proteins were named cafA-K for candidate alpha (1,3) fucosyltransferase. The caf genes were cloned by standard molecular biological techniques into an expression plasmid.

(55) This plasmid utilizes the strong leftwards promoter of bacteriophage λ (termed P.sub.L) to direct expression of the candidate genes (Sanger, F., 1982, J. Mol. Bio. 162:729-773). The promoter is controllable, e.g., a trp-cI construct is stably integrated the into the E. coli host's genome (at the ampC locus), and control is implemented by adding tryptophan to the growth media. Gradual induction of protein expression is accomplished using a temperature sensitive cI repressor. Another similar control strategy (temperature independent expression system) has been described (Mieschendahl et al., 1986, Bio/Technology 4:802-808). The plasmid also carries the E. coli rcsA gene to up-regulate GDP-fucose synthesis, a critical precursor for the synthesis of fucosyl-linked oligosaccharides. In addition, the plasmid carries a β-lactamase (bla) gene for maintaining the plasmid in host strains by ampicillin selection (for convenience in the laboratory) and a native thyA (thymidylate synthase) gene as an alternative means of selection in thyA.sup.− hosts. Alternative selectable markers include the proBA genes to complement proline auxotrophy (Stein et al., (1984), J Bacteriol 158:2, 696-700 (1984) or purA to complement adenine auxotrophy (S. A. Wolfe, J. M. Smith, J Biol Chem 263, 19147-53 (1988)). To act as plasmid selectable markers each of these genes are first inactivated in the host cell chromosome, then wild type copies of the genes are provided on the plasmid. Alternatively a drug resistance gene may be used on the plasmid, e.g. beta-lactamase (this gene is already on the expression plasmid described above, thereby permitting selection with ampicillin). Ampicillin selection is well known in the art and described in standard manuals such as Maniatis et al., (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring, N.Y.

(56) The expression constructs were transformed into a host strain useful for the production of fucosylated oligosaccharides and the ability to direct the production of 3FL using lactose as an acceptor sugar was assessed. Candidate α(1,3) FTs CafC (SEQ ID NO: 2), CafF (SEQ ID NO: 1), CafA (SEQ ID NO: 4) and CafB (SEQ ID NO: 5) were found to be lactose-utilizing α(1,3) fucosyltransferases. (See Table 1 and FIG. 4).

(57) TABLE-US-00003 TABLE 1 Summary of candidate α(1,3) fucosyltransferases analyzed in this study Gene Name Accession No. Organism 3FL synthesis LDFT synthesis.sup.1 LNF III synthesis.sup.2 futA NP_207177.1 H. pylori 26695 +++ ++ cafA CAH09151.1 B. fragilis NCTC ++ ++ nt 9343 cafB CAH09495.1 B. fragilis NCTC ++ nt nt 9343 cafC WP_007483358.1 B. nordii ++++ +++ nt CLO2T12CO5 cafD AAP76669.1 H. hepaticus ATCC − nt ++ 51449 cafE AAP78373.1 H. hepaticus ATCC − nt − 51449 cafF ACD04596.1 A. muciniphilia +++++ ++++ nt ATCC BAA-835 cafG WP_020995419.1 H. bilis ATCC − nt nt 43879 cafH WP_002956732.1 H. cinaedi ATCC − nt − 18818 cafI YP_004607881.1 H. bizzozeronii − nt − CIII-I cafJ YP_537673.1 R. bellii RML369-C − nt − wbfL/cafK BAA33600.1 V. cholerae MO45 − nt − nt = not tested .sup.1In combination with the α(1,2) fucosyltransferase WbgL (accession no. ADN43847.1) .sup.2In combination with the β(1,3) N-acetylglucosaminyltransferase LgtA (N. meningitidis MC58, accession no. NP_274923.1) and the β(1,4) galactosyltransferase HP0826 (H. pylori 26695, accession no. NP_207619.1)

(58) The second database screen to identify additional novel α(1,3)fucosyltransferases was then performed. A multiple sequence alignment was generated using the two strongest previously identified lactose-utilizing α(1,3)fucosyltransferase protein sequences from the first screen: i.e. CafC and CafF. The sequence alignment and percentage of sequence identity of these two sequences is shown in Table 2 below.

(59) TABLE-US-00004 TABLE 2 Akker- MKTLKISFLQSTPDFGREG--MLQLLKSRYHVVEDDSDFDYLVATPWFYVNREAFYDFLERAPGHITVMYGCHEAIAPDFMLFDYYI 85 mansia mucini- phila CafF Bacter- MKTIKVKFVDFWENFDPQHNFIANIISKKYRIELSDTP-DYLFFSVFGYEN----IDY-----HNCTKIFYSGENITPDFNICDYAI 77 oides nordii CafC Con- MKT.K..F......F....--........Y.....D..-DYL......Y.N----.D.-----...T......E.I.PDF...DY.I sensus Akker- GLDTVPGSDRTVKLP-YLRHHLEEVHGGKEGLDAHALLASKTGFCNFIYANRKSHPNRDAMFHKLSAFRFVNSLGPHLNNTPGDGHR 171 mansia mucini- phila CafF Bacter- GFNFLSFGDRYIRIPFYTAYGVQQLAAPKV-IVPEVVLNRK--FCSFVVSNAKGAPERERFFQLLSEYKQVDSGGRYKNNVGGP--- 158 oides nordii CafC Con- G.......DR....P-Y...........K.-......L..K--FC.F...N.K..P.R...F..LS....V.S.G...NN..G.--- sensus Akker- AEDWYASSIRMKKPYKFSIAFENAWYPGYTSEKIVTSMLAGTIPIYWGNPDISREFNSASFINCHDFPTLDDAAAYVKKVDEDDNLW 258 mansia mucini- phila CafF Bacter- ----VPDKTAFIKDYKFNIAFENSMCDGYTTEKIMEPMLVNSVPIYWGNKLIDRDFNPDSFINVSSYSSLEEAVEHIVRLDQNDDEY 241 oides nordii CafC Con- ----........K.YKF.IAFEN....GYT.EKI...ML....PIYWGN..I.R.FN..SFIN......L..A.......D..D... sensus Akker- CEIMSRPWKTPEQEARFLEETERETAKLYKIFDQSPEEARRKGDGTWVSYYQRFLKRGHRMQLAWRRLKNRLR----R 332 mansia mucini- phila CafF Bacter- LSLLSAPWFNEEN---YLNWEEQLITFFDNIFEKPLSESRYIPTHGYIQTYQYRLHRMMRDKLFRKRI-NPLKWFSSK 315 oides nordii CafC Con- ....S.PW...E.---.L...E........IF.....E.R..........YQ..L.R..R..L...R.-N.L.----. sensus Akker- (SEQ ID NO: 1) mansia mucini- phila CafF Bacter- (SEQ ID NO: 2) oides nordii CafC Con- (SEQ ID NO: 60) sesus

(60) A second iterative PSI-BLAST screen was then performed, this time using the FASTA-formatted CafC and CafF multiple sequence alignment as the query, with the NCBI PSI-BLAST program run on a local copy of NCBI BLAST+ version 2.2.29. An initial position-specific scoring matrix file (.pssm) was generated by PSI-BLAST, which the program then used to adjust the score of iterative homology search runs. The process is iterated to generate an even larger group of candidates, and the results of each run were used to further refine the matrix.

(61) This PSI-BLAST search resulted in an initial 2586 hits. There were 996 hits with greater than 25% sequence identity to CafF. 87 hits were of greater than 250 amino acids in length. Additional analysis of the hits was performed, including comparing the sequences by BLAST to the existing inventory of known α(1,3) fucosyltransferases, (i.e. FutA, CafC, CafF, CafA and CafB), and manual annotation of hit sequences to identify those hits originating from bacteria that naturally exist in the gastrointestinal tract, as well as to remove eukaryotic and “pylori” sequences and duplicates. An annotated list of the novel α(1,3) fucosyltransferases identified by this screen (and subsequent filtering) are listed in Table 5. Table 5 provides the bacterial species from which the candidate enzyme is found, the GenBank Accession Number, GI Identification Number, amino acid sequence, and % sequence identity to CafF.

(62) TABLE-US-00005 TABLE 5 % SEQ Bacteria GI Accession identity ID species number number Protein name to CafF Sequence NO Akkermansia 187735265 YP_001877377.1 hypothetical 100 CafF MKTLKISFLQSTPDFGREGMLQLLKSR 1 muciniphila; protein YHVVEDDSDFDYLVATPWFYVNREAFY Akkermansia Amuc_0760 DFLERAPGHITVMYGCHEAIAPDFMLF muciniphila [Akkermansia DYYIGLDTVPGSDRTVKLPYLRHHLEE ATCC BAA-835 muciniphila VHGGKEGLDAHALLASKTGFCNFIYAN ATCC BAA-835] RKSHPNRDAMFHKLSAFRFVNSLGPHL NNTPGDGHRAEDWYASSIRMKKPYKFS IAFENAWYPGYTSEKIVTSMLAGTIPI YWGNPDISREFNSASFINCHDFPTLDD AAAYVKKVDEDDNLWCEIMSRPWKTPE QEARFLEETERETAKLYKIFDQSPEEA RRKGDGTWVSYYQRFLKRGHRMQLAWR RLKNRLRR Bacteroides 494747950 WP_007483358.1 glycosyl- 30.24 CafC MKTIKVKFVDFWENFDPQHNFIANIIS 2 nordii; transferase KKYRIELSDTPDYLFFSVFGYENIDYH Bacteroides [Bacteroides NCTKIFYSGENITPDFNICDYAIGFNF nordii nordii] LSFGDRYIRIPFYTAYGVQQLAAPKVI CLO2T12C05 VPEVVLNRKFCSFVVSNAKGAPERERF FQLLSEYKQVDSGGRYKNNVGGPVPDK TAFIKDYKFNIAFENSMCDGYTTEKIM EPMLVNSVPIYWGNKLIDRDFNPDSFI NVSSYSSLEEAVEHIVRLDQNDDEYLS LLSAPWFNEENYLNWEEQLITFFDNIF EKPLSESRYIPTHGYIQTYQYRLHRMM RDKLFRKRINPLKWFSSK Bacteroides 60682921 YP_213065.1 LPS 26.49 CafA MCDCLSIILLVKMKKIYLKFVDFWDGF 3 fragilis; biosynthesis DTISNFIVDALSIQYEVVLSNEPDYLF Bacteroides related YSCFGTSHLEYDCIKIMFIGENIVPDF fragilis glycosyl- NVCDYAIGFNYIDFGDRYLRLPLYAIY NCTC 9343 transferase DGFSNLQNKKIDVNKALDRKFCSIVVS [Bacteroides NNKWADPIRETFFKLLSSYKKVDSGGR fragilis NCTC AWNNIGGPVDNKLDFISQYKFNIAFEN 9343] SRVLGYTTEKIMEPMQVNSIPVYWGNP LVGKDFNVDSFVNAHDFDSLERLVEYI IELDSSKDKYLEMLEKPWLLDKTYLDW KQLLLNFINNIMMKSYKDAKYLVNYGH AGKYRNEQRFWGRCERKFKLQRIIEYY SQLFDRK Bacteroides 60683260 YP_213404.1 putative 30.92 CafB MDILILFYNTMWGFPLEFRKEDLPGGC 4 fragilis; fucosyl- VITTDRNLIAKADAVVFHLPDLPSVME Bacteroides transferase DEIDKREGQLWVGWSLECEENYSWTKD fragilis [Bacteroides PEFRESFDLWMGYHQEDDIVYPYYGPD NCTC 9343 fragilis] YGKMLVTARREKPYKKKACMFISSDMN RSHRQEYLKELMQYTDIDSYGKLYRNC ELPVEDRGRDTLLSVIGDYQFVISFEN AIGKDYVTEKFFNPLLAGTVPVYLGAP NIREFAPGENCFLDICTFDSPEGVAAF MNQCYDDEALYERFYAWRKRPLLLSFT NKLEQVRSNPLIRLCQKIHELKLGGI Helicobacter 489046508 WP_002956732.1 alpha-1,3- 25.96 CafH MQKPIKKVYFCDGAVEGKIVKILKKHY 5 cinaedi; fucosyl NLIFTDRDPDYIFYSVMGEKHIEYDGI Helicobacter transferase RIFSTGENVRADFNFCDYAIGFDYIQF cinaedi [Helicobacter DDRYLRYPLYLHYTKDMQKAKNKHLAI CCUG 18818 cinaedi] NTQTLQNKDRFCTFVVSNGKADELRTQ FFDFLSQYKHIDSGGKYKNNIGKPIKD KSSFLAIGKFNIAFENSNTNGYTTEKL IQALSSQTVPIYWGDECVSKPLDSSGG GGGVNPKAFIHIKSVNDFDTALEKIQK LDNDDEAYLSMLKEPSFLDSNHEEIFD ERLENFLLHIFSQPIKKAYRRGFGQWR YNLEKRYKKFQRARKIANGFANIFKIP IQKLRTYIKY Butyrivibrio 551009204 WP_022753767.1 glycosyl- 36.25 CafK MRRVFAIHPSIKGIVDLSKYLGFKSCI 6 fibrisolvens transferase TEEIIWDSNSPEFIFVSERIYTDINEW [Butyrivibrio ELFKKMYNPQRIFIFVSGECMTPDLNI fibrisolvens] FDYAIVFDRKLKDLDRICRIPTNYIRH RSLIKKVNDMSFENALSRVKELDFCSF IYSNPKADQIREDIFWGLMNYKHVDSL GEYLNNSGVKTTRNDKHWRELSIEMKS HYKFSIAVENAQYEGYISEKLLTSFQS HSVPIYWGDPLVVDEYNPKAFINFNEM SSISELVNHVKEIDENDELWAEMVSAD WQTSEQVARVKKETEEYDLFIEHILSQ SVSDAIRRPRGCWPYIYTNRFFDEKWF LKSKAKRYIRKAIHCFEEQ Butyrivibrio 551028700 WP_022772782.1 glycosyl- 32.92 CafL MKVKFVDSFFAREQTMGVLNELFENVE 7 sp. AE2015 transferase ISDDPDFVFCSVDYKAEHMNYDCPRIM [Butyrivibrio VIGENIVPDFNCIDYAVGFNYMNFEDR sp. AE2015] YLRVPLYNFYLDDYKLAIRRHIDYKRD DNKKFCNFVYSNGRNAIPERDSFFADL SKYKQVDSGGRHLNNIGGPVDDKREFQ KQYKFSIAFENAVSRGYTTEKIIQAFS AGTIPIYYGNPLVAKEFNSKAFINCHE YRSFDEVIEKVKELDNDPDLYDSMMRE PIFTDIDERQDPLKDYRKFIYNICSQE SDKAIRRCDDCWGGKIQREKKRCYRFL TSTEGNGLKARVIRKLTEI Parabac- 494931837 WP_007657871.1 hypothetical 32.59 CafM MTVTMVRSLYFVHPKVHNVESFLNYVH 8 teroides protein ICELPQGLCLEWNDRNPELLFASEVIY goldsteinii; [Parabac- SDKKSSETFRRLYCEAKVVVYYGGEAS Parabac- teroides FTDFNIFDYGVGFDHTLKNQKYAQILS teroides goldsteinii] PIDFFDNFFYPDRTNLSEEVAQEKLRS goldsteinii GLKFCNFLYSNPVAHPYRDNLFYKLSE CLO2T12C30 YKKVDALGRHLNNTGIGGTGFAGHARE SVNLKENYKFSIASENCGFQGYTSEKI LTSLQAHTVPIYWGDPDVDLVVNPKCF INCNDFDTLDEVLQKVKEIDNNDDLWC EMVSQPWFTEKQLEERIQRNKNYHKFM LSLLCKSIDSLTTRPNGTFQYVYRAWF LNASVRNDILYRLKRKMNFRRLRNFSL SQNRKN Tannerella 547188760 WP_021930564.1 uncharac- 32.34 CafN MKTIKVKFVDFWKGFDPRNNFLMDILK 9 sp. CAG:118 terized QRYHIELSESPDYLIFSVFGFTNLNYE protein RCVKIFYTGENLTPDFNICDYAIGFDY [Tannerella LSFGDRYMRLPLYAVYGIEKLASPKVI sp. CAG:118] DKEKVLKRKFCSYVVSNNIGAPERSRF FHLLSEYKKVDSGGRWENNVGGPVPNK LDFIKDYKFNIAFENSMYDGYTTEKIM EPMLVNSLPIYWGNRLINKDFNPASFI NVSDFPSLEAAVEHIVMLDNNDDMYLS ILSKPWFNDENYLDWKARFFHFFDNIF NRPIDECKYLTPYGFCRHYRNQLRSAR LLKQRFRQLRNPLRWFR Lachno- 551037902 WP_022781636.1 glycosyl- 29.34 CafO MSKKKIKINYIDFWPGFKKEDNFFSRI 10 spiraceae transferase LDKYYDVEISDNPDYVFCSCFSRKHFK bacterium [Lachno- YADCVKIFYTGENIIPDFNLYDYSMGF NK4A136 spiraceae HYIDFEDRYLRLPHYALYDQCIKAAKE bacterium KHTHSDDYYLAKKKFCNYVISNPYAAP NK4A136] ERDLMIDALEKYMPVDSGGRYRNNVGG PVADKVEFASHYRFSMAFENSAMSGYT TEKIFDGFAACTIPIYWGSDRIKEEFN PESFVSARDFENFDQVVARVKEIYEND DLYLKMMKAPIAPEGFQAHECLKEDYA DAFLRNIFDQDIDKAKRRNMVYVGRDY QKKLKDANKVIEVLDVVKKPMHQFNKT KSQIASKFRKKK Methano- 288560783 YP_003424269.1 glycosyl 29.11 CafP MSEKKKIKVKFVDFQDSLKENDNFFID 11 brevibacter transferase SLKKNFDVEVSDDPDYLFFGAYGYKHL ruminantium; [Methano- DYDCIRIMWTIENYVPDFNICDYALAY Methano- brevibacter DIIEFGDRYLRFPFFLNRPEIENVRKT brevibacter ruminantium IERKPIDTSVKTDFCSFVVSNEWGDDY ruminantium M1] RIRLFHELSKYKKVDSGGRSLNNIGGP M1 IGMGLDKKFEFDVTHKFSFALENAQNR GYTTEKIFDAFAAGCIPIYWGDPNIEE EFNPKSFINCNDLTVEEAVEKIKEVDQ NDELYHAMLNEPTFLGDLDKYLQDFDD FLFNICNQPLEKAYRRDRIMKGKTQEH QYKLINRFYYKPYFFLIKVAQKLHIEF IGRKIYHFIRD Bacteroides 492718747 WP_005934126.1 hypothetical 28.94 CafQ MKKVKIKFVDFFDGFDKGRNEFLEVLK 12 salyersiae; protein QRYEIDISDEPDYVIYSGFGYEHLKYN Bacteroides [Bacteroides CIRIFFTGECQTPDFNECDYAIGFDRL salyersiae salyersiae] KFGDRYVRIPLYNMMQYKLDYKELLNR WAL 10018 = KSIISDDIKGRGFCSFVVSNCFANDTR DSM 18765 = AIFYELLNQYKYIASGGRYKNNIGGAI JCM 12988 KDKKTFLSKYKFNIAFENCSHDGYATE KIVEAFAAGVVPIYYGDPRIAEDFNPK AFINAHDYQSFEEMVERIKEIDADDRL YLTMLNEPIIQPNADVTELADFLYSIF DQPLAKAKRRSQSQPTQAMEAMKLRHE FFEMKIYKYYKKGMNQFTRLRKGVFLS SKRTK Lachno- 551037432 WP_022781173.1 hypothetical 27.08 CafR MKKEIKIAYVDFWNGFKPDSFFITKTI 13 spiraceae protein SKKYKVIIDNENPDFVICGTFGNTFLS bacterium [Lachno- YDCPRILYTGEANCPDFNIYDYAIGFE NK4A136 spiraceae RMVYEDRYLRYPLFLVNEDLLQDALNK bacterium HKKSDDYYLRRDGFCSFVVSASGGMDG NK4A136] LRNWYFDKISEYKQVASGGRFRNNLPD GKPVPDKKAFQENYRFSLCFENAGISG YATEKIVDAFAAGCIPIYYGDTNIEKD FNPKSFIHVKSREDLDSVLAWVKELEE NQNKYLEVIRQPAILPDSPIMGMLNNT YIEEFLFHIFDQEPQEAIRRHSKLTMW GQFYEYRLKKWNKIENNMFLKKARSIK RKYFGLKKIVK Parabac- 498502734 WP_010803436.1 hypothetical 26.82 CafS MKKKIYCNFVDFWLGFNYKTYFWYLSD 14 teroides; protein EYDLQIDKEHPDYLFYSCFGNEHLFYE Parabac- [Para DCIRIFWSDENIMPDLNICDYALSLSN teroides bacteroides] LQCDDRTFRKYSGFLYRKDSHLVLPVL goldsteinii KEEALLNRKFCNFVYSNNTCAVPYREL dnLKV18; FFKALSGYKRIDSGGAFLNNMGKKVGD Parabac- KRQFLHEYKFTLAIENSSMPGYVTEKI teroides LEPFMAQSLPLYWGSPTVSSDYNPNSF sp. ASF519 VNLMNYSSMEEAVEEVIRLDKDDAAYL DKMMTPFWLYGANFQEFRDSEIKKIKD FFSYIFEQPLDKAGRRVCYGRNRITIQ KQRRYYAPTFLELSKSMTKKLLKKK Clostridium 488634073 WP_002570751.1 hypothetical 26.43 Caff MKKIRLKYVDWWDGFQPEQYRFHQILT 15 bolteae; protein KHFDIEISDEPDYIIASVYSDEAKSYN Clostridium [Clostridium CVRILYTGENICPDFNIYDYAIGFEYL bolteae 90A9; bolteae] EFGDRYIRIPNFIMNPAYDIDIQKALS Clostridium KHLLSADDIKREKKFCSFVVSNGNAAP bolteae 90133; IREKMFEELNKYKRVDSGGRYLNNIGR Clostridium PEGVRDKFAFQSEHKFSLTFENSAHLG bolteae 90138 YTTEKLLQGFSAGTIPIYWGDPAVENC FNPKAFINISGNNVYDAIELVKEVDTQ DDLYFSMLREPAFLNNDYQTKLLEKLD NFLVHIFNQPLECAYRRNSFEHISNKS VLNEFVKEDRGRFSQWISNKARCFYGK RKNK Helicobacter 564725892 ETD25886.1 hypothetical 25.82 CafU MSKEKWKQEKRVHFVDCCDDGIRDKVC 16 canis NCTC protein PILEQHFTLIFDSVNPEYVFYSAYGEE 12740 HMPREF2087_ HLAYDCIRIFITGENITPNFTICDYAI 01720 GFDHLHFLDRYLRYPLYLFYEQDVKRA [Helicobacter SQKHKDIDEKLLASKSRFCNFVVSNGN canis NCTC ADPYREQVFYALNAYKRVDSGGRYLNN 12740] IGGSVADKFAFQSECRFSLCFENSSTP GYLTEKLIQAAAAQTIPIYWGDTLATK PLFDGGGGINAKAFINAHSFSSLESLI AHIAEIEADKTKQLAILQEPLFLDSNH IELFEKQFEQFLLSIVSQPYERSFRRG RVMWQSFVEQRYKRAMHLLALEDRIKA PYRKLRQFLRAFWDSLKEKRSHT Helicobacter 564725891 ETD25885.1 hypothetical 25.44 CafV MGDEVAMGKERKQIRVHFVDFSNMDNI 17 canis NCTC protein IEKICSILSRHFAVIIDGENPEYVFYS 12740 HMPREF2087_ AFGSEYLKYDCVRIFYTGENIVPDFNL 01719 CDYAIGFDHIKFLDRYLRYPLYLFYET [Helicobacter DVQKAARKHQNLSLEVVRNKKRFCNFV canis NCTC VTNGKGDPYREKVFHALCAYKRVDSAG 12740] KFLNNVGARVKDKFAFQSECRFSLCFE NSSTPGYLTEKLIQAAAAQTIPIYWGD PLATKPLFDGGGGINAKAFINAHEFAN IASLVRHIESIENDENKQLAILQEPLF LDSNHIELFEKQFEDFLVYIFSQPYER SFRRGKIMWQAHLEQIIKKGVQPTMLE IWLRRPLRNFERAIRIRVKKIIQKVKK PKDFM Akkermansia 548174855 WP_022396409.1 putative 92.15 MKTLKISFLQSTPDFGREGIYQLLKDR 18 sp. CAG:344 uncharac- YRVVEDDSDFDYLIATPWFYVNREAFY terized DFLERAPGHITVMYGCHEAIAPDFMLF protein DYYIGLDAVPGSDRTVKLPFLRHHLQE [Akkermansia VHGGKAGLDVRALLASKTGFCNFIYAN sp. CAG:344] RKSHPNRDAIFHKLSSVRFVNSLGPHL NNTPGDGHRSEDWYASSIRMKKPYKFS IAFENAWYPGYTSEKIVTSMLAGTIPI YWGNPDIGREFNSAAFINCHDFPTLDD AAAYVKKVDKDDGLWCEIMSRPWKTLE QEALFLEETERETAKLYRIFDQSPEEA RRKGDGTWIAYYQRFLKRGHRLRLAWR RLKNRLRH Gillisia 494045634 WP_006987752.1 putative LPS 33.24 MKTLKIWFTDFYPGFEPKDNLITQLLF 19 limnaea; biosynthesis KSYNIEFDKNKPDYLIYSCHGHEFLNY Gillisia related NCVRIFYTGENLKPDFNLCDYAIGFDY limnaea glycosyl- IHFNNRYLRFPNFAFYESQFQQLIISK DSM 15749 transferase NPGSLDLSAKKHFCNFIYANSNADPTR [Gillisia DNFFYLLNKYKKVASPGKHLNNISMDV limnaea] GERYAKDWMFTKIEFQSSCKFSIAFEN TSSPGYTTEKLLHAFITGTIPIYWGNP EVMKDFNPKAFINCHDFESFEDVVSKV KEIDNDDEMFLSMLNEPPFRNNIIPEN LKKEPLLVFLKNIFDQKREDAFQRSFY GTSAKYENDMKEMILFRKKYRSMIQFL GLLKKTLKIMKRNR Loktanella 518799211 WP_019955165.1 hypothetical 31.46 MKTIKLHYTDMWGTFDPLAPSQIDRIL 20 vestfoldensis protein RKHFHVVLTDQDPDYVICSVFGDGATR [Loktanella RRGVRLREHHLYPDAIKIMYSGENTLP vestfoldensis] DLNFCDYGIGFDHLVLGDRYQRVPLFA MNDGYQALLQPRAPLTRDDITSSVEFC NFTFTNNMAMPARDQFFHLLNDRKPVL STGRHLRNSDALDLHQQQTGLDPQQAK TDFLARFKFTIAFENSSHPGYTTEKVM DPLVARSVPIYLGNPRIADDFNTAAFI NGHDFPSLDALADEVMRIDADDAAYLA ILNAPPLPPGQREEPHLCALERFLLQI FTPPKAEAQRRQRYGWIGRIDDEYSAY RRRRTRRWRWF Azospirillum 392378323 YP_004985483.1 putative 30.77 MLDQRTSAFLEEFLAKPGGDPERLDRF 21 brasilense; glycosyl- LLHGPYRGRRGGRPRLKLAFHDFWPEF Azospirillum transferase DTGTNFFIEILSSRFDLSVVEDDSDLA brasilense [Azospirillum IVSVFGGRHREARSCRTLFFTGENVRP Sp245 brasilense PLDSFDMAVSFDRVDDPCHYRLPLYVM Sp245] HAYEHMREGAVPHFCSPVLPPVPPTRA AFAERGFCAFLYKNPNGERRNRFFPAL DGRRRVDSVGWHLNNTGSVVKMGWLSK IRVFERYRFAFAFENASHPGYLTEKIL DVFQAGAVPLYWGDPDLEREVAAGSFI DVSRFATDEEAVDHILAVDDDYDAYCA HRAVAPFLGTEEFYFDAYRLADWIESR L Lachno- 551039804 WP_022783468.1 glycosyl- 30.54 MLKTAATGNIFSKISDIFFILGILCEL 22 spiraceae transferase YVMPSGYAFGWYHEKTFIAAGMACFCV bacterium [Lachno- SIIFSMNLKKDFPVFALLAAYGAVCYR NK4A179 spiraceae YQGTALVLRIILALLAGRDKNRDRTVK bacterium MFFAGSMFVIVLAAVLSLLGIHNSVMQ NK4A179] TGNTRSFTETRLTLGFYNPNGFALFVF RTYVLAVFLLITALKDKKKGVFIAAAV SLPFLILILLSHSKMAAAAFVAVFILT MICIGVKGKAADITAYAASLGAVILQV VLLIVFRFQLLPKMRFGKNDTFFEKIN SLTTGRLMMTKALFKSAVPRPFGRPQG EMALTEMGFENSAFAQGYIFILLLLAC IFWLSIRFYRKKDRAGLVVLSATTLYA LAESYLAYFNKNSIWLMMIGICAAGAA CRERNEMGKDGKKKIRIDFAGFWPDFK KDDNYFYNRLKLYYDPEICDDPDYVFC SGFSDEHFKYMDCVKIFFTGENIMPDF NLFDYALGFHYIDFEDRYLRLPLYALY DKEKIIIPALKKHTHEDEYYLSKKKFC NRVVSNPFGAGERDEMFDKLSAYKQVD SGGRYRNNVGGPVDDKIAFERDYKFTL AFENSSMSGYTTEKILEAFAGDTIPVY FGSPRIKEEFNPESFIDASSFDSFDEV VEEIKKIDNDDELYLKMMKAPAVLPES QSKPVLEDDYIDAFLKNIFDQDLSTAK RRNMVYIGHDYQKKLKDANALKRVLDV VKRPVHLMHKIKWQITSKDK Butyrivibrio 551024122 WP_022768256.1 glycosyl- 29.08 MKKITIGYTDIYPGFDPTNNIIYNCLK 23 sp. NC2007 transferase DRYDVKIADTAALESSSEVQYLFYSAS [Butyrivibrio DNRYLDYNCIRIFVTGENLFPNFNLCD sp. NC2007] YAVGFEHMDVGDRFYRLPIYLWEQYRE DYDLLLQDRLELVGVSPEKRKFCGIVA TNNTFADPVREQFFHTLSRYRQVDSGG KAYNNIGLPEGVGDKRAFLKNYKFSIA FENSAYPGYCTEKLMQAFSAGTVPIYW GDETAIAEFNEKAFINCCGLSMEEAVA RVKEIDTNDELYLKMLGEQPLLDNELR VKVISGLSKWLYHIIDSDYESARRRPI HGKMAAYEENYKKRIRREEKLKSNKLI SAMVWVYKKIR Anaeromyxo- 220918351 YP_002493655.1 LPS 29.01 MKPVRVDFVDFWPGFDRRRNVLLDVLR 24 bacter biosynthesis ARFRVEVVDDPDFLFFANFGRRHRRYR dehalogenans; glycosyl- CTRVFFTGENVRPDFRRCDFALTFDHL Anaeromyxo- transferase PEEPRHLRWPLYNLYLDDPRFLLERRR bacter [Anaeromyxo- DVDALVAEKTRFCNLVCSNPAATERLR dehalogenans bacter FFEKLSRYKPVDSGGRVLNNVGGPVPD 2CP-1 dehalogenans KLAFIRQHRFTIAFENASYPGYTTEKI 2CP-1] VEPMRVGSIPIYWGNPLVHLDFDLRSI VSWHEHGNDEATIERVIQIDRDEELYR HMLLQPFLPDGRPTPYSDPGVLLNWLE RVFSTPRRDARPPRRWW Azospirillum 288957550 YP_003447891.1 alpha-(1,3)- 28.84 MLDRFLLHGPERGGRAARPRLKIAFFD 25 lipoferum; fucosyl- FWPEFDPSANFFVEILSSRFDVSVVDN Azospirillum transferase DSDLAILSVFGERHREARTARALFFTG sp. B510 [Azospirillum ENVRPPLDGVDMSVSFDRIDHPRHYRL sp. B510] PLYVMHAWDHRREGATPHFCHPVLPPV PPTREEAAKRKFCAFLYKNPHCARRND FFQMLCARRHVESVGWLLNNTGSVVKM GWLPKIRVFARYRFAFAFENAAHPGYL TEKILDAFQAGTVPLYWGDSGVLRDVA AGSFIDVSRYASDEEAIEAILAIDDDY DSYRRYRGTAPFLGTEDFYFDAYRLAE WIESRL Algoriphagus 495475427 WP_008200114.1 alpha (1,3)- 28.27 MVLIKIKFVDHYNGFNPESDRIFTFLK 26 sp. PR1 fucosyl- RHFPVVLTESDPDFIIYSSWGSEHLHY transferase DCPKIFYTGENHRPNFFLCDYALGFDF [Algoriphagus LNRTDYLRVPLYSILWYYDFSTLLFPK machipon- QQQILDQNPKTKFCCFVASNAGAMERN gonensis] NFFKKLSNYLPVDSGGKVLNNVGGPVP DKIQFMKPYKFCIAYENSSYPGYVTEK IMDCFIAGCIPIYWGSTCIEKDFNPKR ILNRLDYKSDEELIAEIKYLNENHSAY NEFIAQPIFTNNQFTEYFDESRLVKFF EKIFNGPSESRSKGIRKYIGLSLRFNK MIYSRIKKKLGYTGRVWY Helicobacter 564725553 ETD25547.1 hypothetical 28.03 MQSPHPNKSPIRIHFCDFGDMQGIAKA 27 canis NCTC protein ITALLQRHYTITLDSHSPQYLFYSVFG 12740 HMPREF2087_01 SEHIKYDCVRIFYTGENITPNFTICDY 375, partial AIGFDHLHFLDRYLRYPLYLFYEQDVK [Helicobacter RASQKHKDIDEKLLASKSRFCNFVVSN canis NCTC GNADPYREQVFYALNAYKRVDSGGRYL 12740] NNIGGSVADKFAFQSECRFSLCFENSS TPGYLTEKLIQAAAAQTIPIYWGDPLA TKPLFDGGGGINAKAFINAHSFSSLES LIEHIAEIEAD Anaeromyxo- 86159477 YP_466262.1 LPS 27.93 MNPVRLDFVDFWPGFDRRNNVLLDVLR 28 bacter biosynthesis TRFAVEVVDDPDFVFFANFGWRHWRYR dehalogenans; related CTRVFFTGENVRPDFRHCDFALTFDHL Anaeromyxo- glycosyl- PDEPRHLRWPLYNLYLGDPRFLLERRR bacter transferase DVNAIVAEKTRFCNLVCSNRAARERLR dehalogenans [Anaeromyxo- FFEKLSRYKPVDSGGRVRNNVGGPVKD 2CP-C bacter KLAFIRQHRFTIAFENASYPGYTTEKI dehalogenans VEPMRVGSIPIYWGNPLVHLDFDLRSI 2CP-C] VSWHEHGSDEAAIERVIQIDRDEELYR HMLLQPFLPEGRPTPYSDPGVLLDWLE RVFSTPRRDARPPRRWW 294056076 YP_003549734.1 alpha-1,3- 27.41 MKPTKRIAIVDAGRTPDIVHAVLPFIE 29 Coralio- fucosyl- ERYNLEITDDRDADYVFHSCLGHEVLK margarita transferase YSGIRIFVTGECVSPDFNISDYALAFD akajimensis; [Coralio- PIDFGDRYIRLPLIRLFTEAYESLCAP Coralio- margarita RAEPEQILAKKNGFCAYVMSNTKNSAP margarita akajimensis ERVELFEALSRYQPVASGGKWRNNVGG akajimensis DSM 45221] PVADKIAFQSTHKFVLALENESYPGYL DSM 45221 TEKFAQAAQSNAIPIYWGDPTITDIIN PRAFVNVRDFQSTDALVSHIQSLDQDD AAYLSMLSEPWFRGGKEPEEWRAQGYR DFLANIFEQPKERAYRRNRSRWGKKYE GRYYDMAFRPQRQFATLTKTALRRLRH SGQ Helicobacter 522684320 GAD18300.1 alpha (1,3)- 27.31 MDWWEQDTKENFYKNPFIQALSQKYNI 30 fennelliae Fucosyl- EYSNKPDFLLYGPFGQNNLQFPKEVVR MRY12-0050 transferase IFYTGENTRTDWNIADYGIDFDFMDFG [Helicobacter DRHLCMPLFFLPGECGISSRAITKHLR fennelliae AEQIFQEKREKFCAFLVSNGSNHIRNT MRY12-0050] AFKKLCAYKKVDSGGRYLNNIGGRIGD RFKDFEKSKYEWLLGYKFNLCFENSSY PGYVTEKILQAYEAGCIPIYWGDSTLC DVRYAKYRPTFNPKAFVNAHDFANLDE LVQEVRRIDNDNEAYLAMLKEPIFLDS TIDTHVLGGGASTS Prevotella 548234549 WP_022453039.1 uncharac- 26.9 MGNRTVTVKFVDFWQSFDWRDNRFVRA 31 sp. CAG:873 terized LRSQRQVTVLEPSSPEVPDILFYSRGP protein GCDHLRYDCLKVYFTGENDFPDFNECD [Prevotella YALSFYECDCGGRNLRYPLYMLYECDE sp. CAG:873] AACPPVLSDAEALDRGFCSLVMSNASN CHPRRLEIVDAIEAYRPLAYGGAFRNN VGSRVEDKISFISGYKFNLALENSVMP GYVTEKLLEPLAAATVPIYWGADAAKH DFNPESFVCVNDYATFDSLVAELRRLD NDSAAYLAMLRAPSHTGDTVARMDTRL AEFLNAIADRPERRISPYGEIHNLQRR NRALVPLWHSRVGRAAARLLGHIAK Flavobacterium 515556121 WP_016989022.1 hypothetical 26.5 RIFGLVFDKTNNYFYNLLVQKYIVNID 32 sp. ACAM 123 protein, ENPDFLFYSCYSNDYLNYNCTRIFFTG partial ENVRPDFLACDFAFSCDYNKQKNHFRL [Flavo- PLYSLYIDHHNLLDKLQSTLNKEEARR bacterium VWQAKSKFCCMVVSNPKCVERIEFFEN sp. ACAM 1231 LSKVKQVDSGGSVLNNVGGRVADKAEF IKDYKFVISFENESYDGYTTEKILEPI LMDCIPIYWGNKLVDKDFNAKRFINYN TFKTENKLIERLLEIDQNEELAIAMLL EQPFNKDKKTHEEEHQQVLDIISNMIE VDKKPIAQQLWKYVHKSKLFAAKFKKR FIKI Flavobacterium 515558176 WP_016991062.1 hypothetical 26.4 MKEIKINFVDFWPGFNKTNNYFYNLLI 33 sp. ACAM 123 protein QKYKVSIDANPDLLFYSCYNNDYLNFD [Flavo- CTRIFYTAENIRPDFSACDFAFSYGYN bacterium AKINHFRLPLYSMYIDLLNMKDKIEAT sp. ACAM 123] LSREEAQKIWKTKSKFCCMVVSNATGT KRLDFFKNLSKIKQVDSGGGIFNNIGG KVVDKLEFIKDYKFVISFENGQNDGYT TEKILEPIYKDCIPIYWGNKLVDKDFN SKRFLDYSKFECEKDLIDKLLEMELDD ELAISMLMQPAFGENKRPHEEERAEVL RILGRIIENPEKPIARQLWKYIHLLKR KYRKNKKRIKRILN Butyrivibrio 551033828 WP_022777675.1 hypothetical 26.38 MKKVKVKFVDTYGKQQKYLEKLLGDDI 34 sp. AE3009 protein ELEYSDEPDYLFYGVFGSGMEHYKYKN [Butyrivibrio CVKIFFASEGVIPDFNECDYAIAEYPM sp. AE3009] TVGDRYFCKPYMAPKEADFSVFDEKAD YLGRKFCNFVFSNETNGRGAVLRKQFC QKLMEYKHVDCPGKVLNNMKDAIEPRN GKWFHGKLDFIKDYKFTIAFENVNTPG MVSEKIYNAFQARTVPIYWGPDDVNKI YNPKSFINCSGLTIDEMVKKVAEVDSN DELYMDMLRQNPIAEGFNLNWEEDMAR FLRGIILENKDYYDKDPLGWDSGNKAA KELISLEDTMLYKLHKGREKVAKKLKR Helicobacter 537771103 WP_020982055.1 fucosyl- 26.38 MFQPLLDAFIESASIKKKLPLNLPPPL 35 pylori; transferase, KIAVANWFNGSKEFKASVLYFILKQRY Helicobacter partial KIILHSNPNEPSDLVFGNPLGQARKIL pylori [Helicobacter SYQNTKRVFYTGENEAPNFNLFDYAIG FD577 pylori] FDELDFNDRYLRMPLYYAYLHYKAEIV NDTTSPYKLKADSLYTLKKPSHKFKEN HPHLCALIHSESDPLKRGFASFVASNP NAPIRNAFYDALNSIEPVAGGGSVKNT LGYKVKNKNEFLSQYKFNLCFENSQGY GYVTEKILDAYFSHTIPIYWGSPSVAK DFNPKSFVNVHDFNNFDEAIDYIRYLH THQNAYLDMLYENPLNTLDGKASFYQD LSFEKILDFFKNILENDTIYHCNDAHY SALHRDLNEPLVSVDDLR Verrucomicrobia 518821759 WP_019977713.1 hypothetical 26.16 MLNQIKINYTDFYGDKNYERNPFHNFL 36 bacterium protein SSHFNLELSEEPDFLIHGVYGQDYLNY SCGC [Verruco- NCVRILYSAENMIPDFKTYDYSLTFCK AAA300-K03 microbia SSFQDRNWRVPLYAVWNDLSIQLDSHL bacterium GFRNATNLSQNRDVFCSFVVSNPYCSF SCGC RNNLFKRLEKYKFVHSGGGVFNNSGGK AAA300-K03] TGNKLHFIRNSKFNIACENQSYPGYTT EKILEAFLAGCIPVYWGNPEIAHEFNE KAFINCHNYKSINEVADRIIEIDQNKA LYLDYLSQPIFYNDTIPDDASHSRIVT IFNNIFYNTRPSRIACSKLPSKIFNIK KQLKKLAGKYSR Clostridium 495144632 WP_007869439.1 hypothetical 25.88 MEKIKTKIINKITKINLIGIALVFYTS 37 citroniae; protein VWRGYKEYCRLKKKHGNLPIITPTFKG Clostridium [Clostridium TGDFYMVAKYFPQWLKFKKIDKYMMIA citroniae citroniae] GGASEIRVLELFPQWFSNAQYEILSWE WAL-17108 HYTYLIHMRLFWGVEKSDIYVLNHIAN FGGEHTNYLWITWNLMGYKGLSLLDFY LIYGCKLSKLEKPLIPIFETDSNKIDK IFKYKKLKPGKTVMISPYSTGNGTFHV SFWNSIVKQLQLSGYSVCTNCFGSEKP LANTVKLGLDYRDLVPFMDKAGFAIGI RSGFFDIISSSTCKKIIIHTFKANHWP NGNSLPYTGLKHLGLCNDAIEYELNSN ESNFDVIRRSILGLFAIHVASSKKTIK IKYVDVPPDFNKEKIWITRVLREKYNV VFSDNPEFLFYSVFGLTFDQYKNCIKI FFTGEDTIPNFNECDYAMCHDRLELGD RYIRADVGERYGTPIGNLEPDWIEKGI SISGWINSSLIDIKDKIQNRSIVSEKL INRRFCNFIYSNESFGEGAVLRKKFCL ELMKYRRVDCPGRVLNNMKDGLGIRWS VKDGRDSIVDNWTSTKLEFIKNYKFTI AFENTAIPGHTTEKLIHPFYAYSIPIY WGNPDVVADFNPKAFINCNDYNNDWRA VCKRIKELDQDHEQYLEMLRQPPMQPD FDFGSEEKAKQFLYNIVEKGYKPYTKS SLAFTAPNVARNSYHELMEIKTSNSWK VARRIQAFLGTKWGWFPRQLCLALLNV RNRLVKKK Helicobacter 490187781 WP_004086382.1 hypothetical 25.84 MQKQQVKMRVLDWWNKDCEENFYNNFF 38 bilis; protein IQILQKKYDVVYSDKPDFILYGPFGYE Helicobacter [Helicobacter HLKYDCVRIFHTGENIRPDYNIADYSM bilis bilis] DFDYIEFEDRHLRLPHMFWVFCDEMRQ WiWa KEMDNRISLLDKKEKFCGFMVSNNALT DKRDMFFEALSKYKRVDSGGRWKNNMG GNVDDKIEWLKSYKFNLCFENSSYPGY LTEKLFDAFLAGCVPIYWGDTSLKIHK NTCADSKNSENINNQGGGSNDAFDMRI PNISHSLIDYEINPKAFINAHNFPTFQ DLIDEIKRIDNDSYAFESMLREPIFLN DFNPHEFYATKIAAFLNRIVSQGAIQA KRRGDGFLLKAYREFQSAIAENTQISS GFFSYCVKHGRVIQAIRDSSKLPKRFS RFIRRTRK Verrucomicrobia 518996369 WP_020152244.1 hypothetical 25.59 MVSNQIKIQFTDFYQIPNEEENYLYKY 39 bacterium protein LKQYFNLELSDDPDVVIYSNYGFEYKQ SCGC [Verruco- YECLRVLFCAEYAIPDIEDCDYCFSQH AAA300-N18 microbia HASYWGKNYRLPMYVFWQNFSLKFEEL bacterium LRPVDYEEIRKQDRGFCSFVVSSPLGS SCGC QTRVNFMHELSSYKKVDSGGKLLNNIG AAA300-N18] GPVANKRDFLKKYKFNIAFANGLADGY ADEKIVDPMFVDSIPIFWGNPRIAEDF NPASFVNCHDYDNFDSVIKEVIRIDKN EDVYRSYLEQPWFPENKLTRYVDLDHL QNRFRYIFSQIGKKVPAARSKRRFFYK LLKKLKPLTPIVQQWGDYQPSN Moumouvirus 451927149 AGF85027.1 family 10 25 MDKFKIVCINLARRQDRKDLITNKLIN 40 goulette protein QNMSNFEFFEAVDGSQIDPYDERLNLF [Moumouvirus KHSVSGLLRRGVTGCALSHYTIWKKLV goulette] NDPDYNTYLVIEDDINFGPDFKFGLEK ILEKKPNYGIILLGMTLELEKKAETKH LYQYDTSYTIHNLNRDLYCGGAFGYII SKSAAKYLVDYISHNGIRMVIDYLMFR SGVPMYESHPHLVFTDAVQHSIHYVDS DIQHDHEKIKYNKLPNDYQFDDYIFLS NRDSPRGDIREICADITTLKKAADMTS ECIAFNTYGWLKNILTDFDKFIVLHDK FYTHDGIYIKKSYFNLENKLKNLRLLE RPIRIFLNKNTINYSQHLVNIILKNIP NYNIVKDNNDADIIIDNINDSNLYYDQ TKINMIISGEPFNRKQKYDIAIDTKKN SNAECIIYHPFLFSSLHEHKKSINYLD YTNPKTKFCAYMFHMSYPHRINYFNIV SSYKHVDALGKCCNNVDIKNTRYVLNN KETYNDIAVEYFSQYKFVLAIENNMIP GYNTEKLINPMIANSIPIYWGDSElFK YINKRRLVYIPDFITNEDLINHIKYID EHDDVYENIIKESIFTDPDFTLDVIEQ NLSGEIDNLLGFNKN

(63) Of the identified hits, 12 novel α(1,3) fucosyltransferases were further analyzed for their functional capacity: Butyrivibrio fibrisolvens CafK, Butyrivibri sp. CafL, Parabacteroides goldsteinii CafM, Tannerella sp. CafN, Lachnospiracae bacterium CafO, Methanobrevibacter ruminantium CafP, Bacteroides salyersiae CafQ, Lachnospiraceae bacterium CafR, Parabacteroides goldsteinii CafS, Clostridium bolteae CafT, Helicobacter canis CafU and Helicobacter canis CafV. FIG. 6 demonstrates significant production of 3-FL by FutA, CafC, CafF and also by the new candidate α(1,3) fucosyltransferase enzymes derived from the second database screen; CafL, CafN, CafO, CafQ, CafU and CafV.

(64) The sequence identity between the 12 novel α(1,3) fucosyltransferases identified in this second screen, the previously identified lactose-utilizing α(1,3) fucosyltransferases from the first screen, and FutA is shown in Tables 2 and 3 below.

(65) TABLE-US-00006 TABLE 3 Sequence Identity 1 2 3 4 5 6 7 8 9 Bacteroides nordii CafC 1 25.00 23.50 66.88 49.25 39.64 38.10 38.32 36.31 Akkermansia muciniphila CafF 2 25.00 18.61 25.41 21.15 22.72 21.49 23.08 22.76 Helicobacter pylori FutA 3 23.50 18.61 25.81 24.77 25.23 23.11 23.97 20.96 Tannerella sp. CafN 4 66.88 25.41 25.81 53.45 39.23 35.82 35.82 36.42 Bacteroides fragilis CafA 5 49.25 21.15 24.77 53.45 36.86 35.06 34.10 35.77 Bacteroides salyersiae CafQ 6 39.64 22.72 25.23 39.23 36.86 35.36 42.43 40.75 Parabacteroides goldsteinii CafS 7 38.10 21.49 23.11 35.82 35.06 35.36 32.27 31.64 Clostridium bolteae CafT 8 38.32 23.08 23.97 35.82 34.10 42.43 32.27 41.91 Methanobrevibacter ruminantium CafP 9 36.31 22.76 20.96 36.42 35.77 40.75 31.64 41.91 Lachnospiraceae bacterium CafO 10 35.91 24.08 24.54 37.39 35.73 38.95 33.91 40.94 37.82 Lachnospiraceae bacterium CafR 11 35.10 23.53 23.97 33.92 30.95 35.23 30.46 36.89 33.71 Butyrivibrio sp. CafL 12 34.76 25.67 23.22 35.37 36.95 39.35 32.54 41.96 39.83 Helicobacter canis CafU 13 32.87 20.00 23.08 32.59 32.60 37.33 32.04 37.88 34.71 Helicobacter canis CafV 14 31.40 19.30 23.30 31.96 31.23 32.51 32.51 36.91 32.97 Butyrivibrio fibrisolvens CafK 15 25.53 24.25 16.63 22.49 23.18 24.43 22.98 25.39 22.92 Parabacteroides goldsteinii CafM 16 22.99 24.62 16.85 23.94 23.47 24.27 20.37 25.54 23.14 Bacteroides fragilis CafB 17 14.44 14.21 8.67 14.40 13.81 15.09 13.16 13.99 13.00 Helicobacter hepaticus CafD 18 12.11 7.81 8.68 9.49 11.05 7.44 10.26 8.82 7.41 10 11 12 13 14 15 16 17 18 Bacteroides nordii CafC 1 35.91 35.10 34.76 32.87 31.40 25.53 22.99 14.44 12.11 Akkermansia muciniphila CafF 2 24.08 23.53 25.67 20.00 19.30 24.25 24.62 14.21 7.81 Helicobacter pylori FutA 3 24.54 23.97 23.22 23.08 23.30 16.63 16.85 8.67 8.68 Tannerella sp. CafN 4 37.39 33.92 35.37 32.59 31.96 22.49 23.94 14.40 9.49 Bacteroides fragilis CafA 5 35.73 30.95 36.95 32.60 31.23 23.18 23.47 13.81 11.05 Bacteroides salyersiae CafQ 6 38.95 35.23 39.35 37.33 32.51 24.43 24.27 15.09 7.44 Parabacteroides goldsteinii CafS 7 33.91 30.46 32.54 32.04 32.51 22.98 20.37 13.16 10.26 Clostridium bolteae CafT 8 40.94 36.89 41.96 37.88 36.91 25.39 25.54 13.99 8.82 Methanobrevibacter ruminantium CafP 9 37.82 33.71 39.83 34.71 32.97 22.92 23.14 13.00 7.41 Lachnospiraceae bacterium CafO 10 38.57 37.90 30.66 31.69 22.82 24.34 13.74 8.96 Lachnospiraceae bacterium CafR 11 38.57 33.24 33.51 33.15 22.25 19.37 13.70 8.12 Butyrivibrio sp. CafL 12 37.90 33.24 35.85 33.80 26.98 26.33 14.78 9.49 Helicobacter canis CafU 13 30.66 33.51 35.85 64.33 22.14 22.31 14.07 9.38 Helicobacter canis CafV 14 31.69 33.15 33.80 64.33 22.96 21.26 14.25 7.90 Butyrivibrio fibrisolvens CafK 15 22.82 22.25 26.98 22.14 22.96 26.88 11.78 9.23 Parabacteroides goldsteinii CafM 16 24.34 19.37 26.33 22.31 21.26 26.88 13.12 7.43 Bacteroides fragilis CafB 17 13.74 13.70 14.78 14.07 14.25 11.78 13.12 8.13 Helicobacter hepaticus CafD 18 8.96 8.12 9.49 9.38 7.90 9.23 7.43 8.13

(66) TABLE-US-00007 TABLE 4 Sequence alignments Bacteroides nordii CafC M-----------------KTIK--VKF--------VDFWENFDPQHNF----IANIISKKYR-IELSDTPDY--LFFSV-FG-Y--ENIDY-HNC---TKIFY-SGENITPDFNICD 74 Akkermansia muciniphila M-----------------KTLK--ISF--------------LQSTPDFGREGMLQLLKSRYHVVE--DDSDFDYLVATPWFYVNREAFYDFLERAPGHITVMYGCHEAIAPDFMLFD 82 CafF Helicobacter pylori FutA MFQPLLDA-----FIESASIEKMASKSPPPPLKIAVANWWGDEEIKEFKKSVLYFILSQRYAITLHQNPNEFSDLVFSNPLG-AARKILSY-QNTK---RVFY-TGENESPNFNLFD 106 Tannerells sp. CafN M-----------------KTIK--VKF--------VDFWKGFDPRNNF----LMDILKQRYH-IELSESPDY--LIFSV-FG-F--TNLNY-ERC---VKIFY-TGENLTPDFNICD 74 Bacteroides fragilis MCDCLSII-----LLVKMKKIY--LKF--------VDFWDGFDTISNF----IVDALSIQYE-VVLSNEPDY--LFYSC-FG-T--SHLEY-D-C---IKIMF-IGENIVPDFNVCD 85 CafA Bacteroides salyersiae M-----------------KKVK--IKF--------VDFFDGFDKGRNE----FLEVLKQRYE-IDISDEPDY--VIYSG-FG-Y--EHLKY-N-C---IRIFF-TGECQTPDFNECD 73 CafQ Parabacteroides M----------------KKKIY--CNF--------VDFWLGFNYKTYF----WY--LSDEYDLQIDKEHPDY--LFYSC-FG-N--EHLFY-EDC---IRIFW-SDENIMPDLNICD 74 goldsteinii CafS Clostridium bolteae M-----------------KKIR--LKY--------VDWWDGFQPEQYR----FHQILTKHFD-DEISDEPDY--IIASV-YS-D--EAKSY-N-C---VRILY-TGENICPDFNIYD 73 CafT Methanobrevibacter MSE--------------KKKIK--VKF--------VDFQDSLKENDNF----FIDSLKKNFD-VEVSDDPDY--LFFGA-YG-Y--KHLDY-D-C---IRIMW-TIENYVPDFNICD 76 ruminantium CafP Lachnospiraceae MS---------------KKKIK--INY--------IDFWPGFKKEDNF----FSRILDKYYD-VEISDNPDY--VFSCS-FS-R--KHFKY-ADC---VKIFY-TGENIIPDFNLYD 76 bacterium CafO Lachnospiraceae M----------------KKEIK--IAY--------VDFWNGFKPDSFF----ITKTISKKYKVIIDNENPDF--VICGT-FG-N--TFLSY-D-C---PRILY-TGEANCPDFNIYD 75 bacterium CafR Butyrivibrio sp. CafL M--------------------K--VKF--------VDSFFAREQT--------MGVLNELFENVEISDDPDF--VFCSVDYK-A--EHMNY-D-C---PRIMV-IGENIVPDFNCID 68 Heliobacter canis CafU MSK--E---------KWKQEKR--VHF--------VDCCD--DGIRDK----VCPILEQHFTLIFDSVNPEY--VFYSA-YG-E--EHLAY-D-C---IRIFI-TGENITPNFTICD 78 Heliobacter canis CafV MGD--EVA-----MGKERKQIR--VHF--------VDFSN-MDNIIEK----ICSILSRHFAVIIDGENPEY--VFYSA-FG-S--EYLKY-D-C---VRIFY-TGENIVPDFNLCD 83 Butyrivibrio MRRVFAI----------HPSIKGIVD---------LSKYLGFK-----------SCITE--EIIWDSNSPEFIFVSERIYTDINEWELFKK-MYNPQRIFIFV-SGECMTPDLHIFD 83 fibrisolvens CafK Parabacteroides MTVTMVRS-----LYFVHPKVHNVESF--------LNYVHICELPQGL----CLEWNDRNPELLFASE------VIYSDKKS-S--ETFRR-LYCEAKVVVYY-GGEASFTDFNIFD 89 goldsteinii CafM Bacteroides fragilis M--------------------DILILFYNTMWGFPLEFRKE-DLPGGCVITTDRNLIAKADAVVF--HLPDLPSVMEDEIDKREGQLWVGWSLECEEN---YSWTKD---PEFRE-- 86 CafB Helicobacter hepaticus MKDDLVILHPDGGIASQIAFVALGLAFEQKGAKVKYDLSWFAEGAKGFWNP--SNGYDKVYDITW-----DISKAFPALHIEIANEEEIERYK------------SKYLIDNDRVID 98 CafD Consensus M-----------------KKIK--VKF--------VDFWDGFPDI.NF----.L.ILSKRY.-IEDSDNPDY--VFYSV-FG-N--EHLKY-D-C---IRIFY-TGENITPDFHICD Bacteroides nordii CafC YAIGFNF-LSFGDRYIR-IPFYTAY-GVQ-------------------QLAAPK------------VIVPEVVLNRK-FCSFVVSN--AKGA-PERERFFQLLSEYKQVDSG-GRYK 152 Akkermansia muciniphila YYIGLD-TVPGSDRTVK--------------------------------LPYLRHHLEEVHGGKEGLDAHALLASKTGFCNFIYAN--RK-SHPNRDAMFHKLSAFRFVNSL-GPHL 162 CafF Heliobacter pylori FutA YAIGFDE-LDFNDRYLR-MPLYYAHLHYKAELVNDTTAPYKLKDNSLYALKKPSHHFKENHPNLCAVVNDESDLLKRGFASFVASN--AN--APMRNAFYDALNSIEPVTGG-GSVR 216 Tannerella sp. CafN YAIGFDY-LSFGDRYMR-LPLYAVY-GIE-------------------KLASPK------------VIDKEKVLKRK-FCSYVVSN--NIGA-PERSRFFHLLSEYKKVDSG-GRWE 152 Bacteroides fragilis YAIGFNY-IDFGDRYLR-LPLYAIYDGFS-------------------NLQNKK-------------IDVNKALDRK-FCSIVVSN--NKWADPIRETFFKLLSSYKKVDSG-GRAW 164 CafA Bacteroides salyersiae YAIGFDR-LKFGDRYVR-IPLYNMMQ-YKLDYKE--------------LLNRKS------------IISDDIKG-RG-FCSFVVSN--C-FANDTRAIFYELLNQYKYIASK-GRYK 155 CafQ Parabacteroides YALSLSN-LQCDDRTFRKYSGFL----YRKDSHL--------------VL---P------------VLKEEALLNRK-FCNFVYSN--NTCAVPYRELFFKALSGYKRIDSG-GAFL 153 goldsteinii CafS Clostridium boltreae YAIGFEY-LEFGDRYIR-IPNFIMNPAYDIDIQK--------------ALSKHL------------LSADDIKREKK-FCSFVVSN--G-NAAPIREKMFEELNKYKRVDSG-GRYL 157 CafT Methanobrevibacter YALAYDI-IEFGDRYLR-FPFFLNRPEIE-NVRK--------------TIERKP------------I---DTSVKTD-FCSFVVSN--E-WGDDYRIRLFHELSKYKKVDSG-GRSL 156 ruminantium CafP Lachnospiraceae YSMGFHY-IDFEDRYLR-LPHYALYDQCI----K--------------AAKEKH------------THSDDYYLAKKKRCNYVISN--P-YAAPERDLMIDALEKYMPVDSG-GRYR 157 bacterium CafO Lachnospiraceae YAIGFER-MVYEDRYLR-YPLFLVNEDLLQD-----------------ALNKHK------------K-SDDYYLRRDGFCSFVVSA--SGGMDGLRNWYFDKISEYKQVASG-GRFR 157 bacterium CafR Butyrivibrio sp. CafL YAVGFNY-MNFEDRYLR-VPLYNF---YLDDYKL--------------AIRRHI------------DYKRDDN--KK-FCNFVYSN--GRNAIPERDSFFADLSKYKQVDSG-GRHL 148 Helicobacter canis CafU YAIGFDH-LHFLDRYLR-YPLYLF---YEQDVKR--------------ASQKHK------------DIDEKLLASKSRFCNFVVSN--GN-ADPYREQVFYALHAYKRVDSG-GRYL 160 Helicobacter canis CafV YAIGFDH-IKFLDRYLR-YPLYLF---YETDVQK--------------AARKHQ------------NLSLEVVRNKKRFCNFVVTN--GK-GDPYREKVFHALCAYKRVDSA-GKFL 165 Butyrivibrio YAIVFDRKLKDLDRICRIPTNY---------------------------IRHRSLIKKVNDMSFEMALSRVKELD---FCSFIYSN--PK-ADQIREDIFWGLMNYKHVDSL-GEYL 166 fibrisolvens CafK Parabacteroides YGVGFDH-TLKNQKYAQILSPIDFFDNFFYPDRT--------------NLSEE--------------VAQEKLRSGLKFCNFLYSN--PV-AHPYRDNLFYKLSEYKKVDAL-GRHL 173 goldsteinii CafM Bacteroides fragilis ---SFDLWMGYHQEDDIVYPYY------------------------------------GPDYGKMLVTARREKPYKKKACMFISSDMNR----SHRQEYLKELMQYTDIDS-YGKLY 159 CafB Helicobacter hepaticus YAPPL-YCYGYKGRIFHYL-----------------------------------------------------------YAPFFAQSFAPKEAQDSHTPFAALLQEIESSPSPCGVHI 155 CafD Consensus YAIGFDY-L.FGDRYLR-.PLYL----YE-------------------ALRKHK------------VISD.KLL.KK-FCSFVVSN--.K-ADPYRERFFH.LSEYK.VDSG-GRYL Bacteroides nordii CafC NN--VGGP-----VPD-KTAFIKDYKFHIAFENSMCDGYTTEKIMEPMLVNSVPIYWGNKLIDRDF-------NPDSFINVSSYSSLEEAVEHIVRLDQNDDEYLSLLS--A-PWFN 251 Akkermansia muciniphila NNTPGDGHRAEDWYAS-SIRMKKPYKFSIAFENAWYPGYTSEKIVTSMLAGTIPIYWGNPDISREF-------NSASFINCHDFPTLDDAAAYVKKVDEDDNLWCEIMS--R-PWKT 268 CafF Helicobacter pylori FutA NTLGY-------KVGN-KSEFLSQYKFNLCFENSQGVGYVTEKILDAYFSHTIPIYWGSPSVAKDF-------NPKSFVNVHDFNNFDEAIDYIKYLHTHPNAYLDMLY--ENPLNT 316 Tannerella sp. CafN NN--VGGP-----VPN-KLDFIKDYKFNIAFENSMYDGYTTEKIMEPMLVNSLPIYWGNRLINKDF-------NPASFINVSDFPSLEAAVEHIVMLDNNDDMYLSILS--K-PWFN 251 Bacteroides fragilis NN--IGGP-----VDN-KLDFISQYKFNIAFENSRVLGYTTEKIMEPMQVNSIPVYWGNPLVGKDF-------NVDSFVNAHDFDSLERLVEYIIELDSSKDKYLEMLE--K-FWLL 263 CafA Bacteroides salyersiae NN--IGG----A-IKD-KKTFLSKYKFNIAFENCSHDGYATEKIVEAFAAGVVPIYYGDPRIAEDF-------NPKAFINAHDYQSFEEMVERIKEIDADDRLYLTMLN--E-PIIQ 254 CafQ Parabacteroides NN--MGKK-----VGD-KRQFLHEYKFTIAIENSSMPGYVTEKILEPFMAQSLPLYWGSPTVSSDY-------NPNSFVNLMNYSSMEEAVEEVIRLDKDDAAYLDKMM--T-PFWL 252 goldsteinii CafS Clostridium bolteae CafT NN--IGRP--EG-VRD-KFAFQSEHKFSLTFENSAHLGYTTEKLLQGFSAGTIPIYWGDPAVENCF-------NPKAFINISGNNVYDA-IELVKEVDTQDDLYFSMLR--E-PAFL 257 Methanobrevibacter NN--IGGPIGMG-LDK-KFEFDVTHKFSFALENAQNRGYTTEKIFDAFAAGCIPIYWGDPNIEEEF-------NPKSFINCMDLTV-IIAVEKIKEVDQNDELYHAMLN--E-PTFL 258 ruminantium CafP Lachnospiraceae NN--VGGP-----VAD-KVEFASHYRFSMAFENSAMSGYTTEKIFDGFAACTIPIYWGSDRIKEEF-------NPESFVSARDFENFDQVVARVKEIYENDDLYLKMMK--A-PIAP 256 bacterium CafO Lachnospiraceae NN--L--PDGKP-VPD-KKAFQENYRFSLCFENAGISGYATEKIVDAFAAGCIPIYYGDTNIEKDF-------NPKSFIHVKSREDLDSVLAWVKELEENQNKYLEVIR--Q-PAIL 258 bacterium CafR Butyrivibrio sp. CafL NN--IGGP-----VDD-KREFQKQYKFSIAFENAVSRGYTTEKIIQAFSAGTIPIYYGNPLVAKEF-------NSKAFINCHEYRSFDEVIEKVKELDNDPDLYDSMMR--E-PIFT 247 Helicobacter canis CafU NN--IGGS-----VAD-KFAFQSECRFSLCFENSSTPGYLTEKLIQAAAAQTIPIYWGDTLATKPLFDGGGGINAKAFINAHSFSSLESLIAHIAEIEADKTKQLAILQ--E-PLFL 266 Helicobacter canis CafV NN--VGAR-----VKD-KFAFQSECRFSLCFENSSTPGYLTEKLIQAAAAQTIPIYWGDPLATKPLFDGGGGINAKAFINAHEFANIASLVRHIESIENDENKQLAILQ--E-PLFL 271 Butyrivibrio NNSGVKTTRNDKHWRELSIEMKSHYKFSIAVENAQYEGYISEKLLTSFQSHSVPIYWGDPLVVDEY-------NPKAFINFNEMSSISELVNHVKEIDENDELWAEMVS--AD---W 271 fibrosolvens CafK Parabacteroides NNTGIGGTGFAGHARE-SVNCKENYKFSIASENCGFQGYTSEKILTSLQAHTVPIYWGDPDVD-------LVVNPKCFINCNDFDTLDEVLQKVKEIDNNDDLWCEMVS--Q-PWFT 279 goldsteinii CafM Bacteroides fragilis RNCELP---VEDRGRDTLLSVIGDYQFVISFENAIGKDYVTEKFFNPLLAGTVPVYLGAPNIRE------FAPGENCFLDICTFDSPEGVAAFMNQCYDDEALYERFYAWRKRPLLL 267 CafB Helicobacter hepaticus RRGDLSQPHIVYGNPTSNEYFAKSIEL-MCLLHPQSSFYLFSDDLAFVKEQIVPLLKG-----KTYRICDVNNPSQGYLDLYLLSRCRNIIGSQGSMGE----FAKVLS-PHNPLLI 261 CafD Consensus NN--IGGP-----V.D-KF.FQSEYKFSIAFENS.YPGYTTEXILEAFAAGTIPIYWGDPLI.KDF-------NPKSFINAHDFSSLEEAVEH.KELDE.DDLYLEMLS--E-P.FL Bacteroides nordii CafC EENYL-NWEEQ----LITFFDNIFEKPLSESRYIPT--HGYIQ---------------------TY--QYRLHRMM--------------RDKLFRXRINPL--KWFSSK----- 315 Akkermansia muciniphila PEQEARFLEETERETAK--LYKIFDQSPEEARRKGDGTWV-------------------------------------------------SYYQRFLKRGHRMQLAWRRLKNRLRR 332 CafF Helicobacter pylori FutA LDGKAYFYQDLSFKKILDFFKTILEN--DTIYHKFSTSFMWEYDLHKPLVSIDDLRVNYDDLRVNYDRLLQNASPLLELSQNTTFKIYRKAYQKSLPLLRAV---RKLVK-KLGL 425 Tannerella sp. CafN DENYL-DWKAR----FFHFFDNIFNRPIDECKYLTP--YGFCR---------------------HYRNQLRSARLL--------------KQR-FRQLRNPL--RWF--R----- 314 Bacteroides fragilis DKTYL-DWKQL----LLNFINNIMMKSYKDAKYLVN--YGHAG---------------------KYRNEQRFWGRC--------------ERKFKLQRIIEYYSQLFDRK----- 331 CafA Bacteroides salyersiae PNADVTELAD--------FLYSIFDQPLAKAKRRSQ--SQPTQ---------------------AME-AMKLRHEFFEMKIY---KYYKKGMNQFTRLRKGVFLSSKRTK----- 329 CafQ Parabacteroides YGANFQEFRDSEIKKIKDFFSYIFEQPLDKAGRR----VCYGR---------------------NRITIQKQRRYYAPT---------------FLELSKSMTKKLLKKK----- 322 goldsteinii CafS Clostridium bolteae CafT NNDYQTKLLEK----LDNFLVHIFNQPLECAYRRNS--FEHIS---------------------NKS-VL---NEFV--------KEDRGRFSQWISNKARCFYGKRKNK----- 328 Methanobrevibacter --GDLDKYLQD----FDDFLFNICNQPLEKAYRRDR--IMKGK---------------------TQEHQYKLINHRYYKPYFFLIKVAQKLHIEFIGRKIYHFI----RD----- 335 ruminantium CafP Lachnospirceae EGFQAHECLXEDY--ADAFLRNIFDQDIDKAKRRNM--VYVGR---------------------DYQKKLKDANKVIEV-----LDVVKKPMHQFNKTKSQIASKFRKKK----- 336 bacterium CafO Lachnospirceae PDSPIMGMLNNTY--IEEFLFHIFDQEPQEAIRRHSKLTMWGQ---------------------FYEYRLKKWNKI--------------ENNMFLKKARSIKRKYFGLK-KIVK 335 bacterium CafR Butyrivibrio sp. CafL DIDERQDPLKD----YRKFIYNICSQESDKAIRRCD--DCWGG---------------------KIQREKKRCYRFL-------------TSTEGNGLKARV-IRKLTEI----- 316 Helicobacter canis CafU DSNKIELFEKQ----FEQFLLSIVSQPYERSFRRGR--VMWQS---------------------FVEQRYK--RAMHLLALEDRIKAPYRKLRQFLRA----FWDSLKEK-RSHT 347 Helicobacter canis CafV DSNHIELFEKQ----FEDFLVYIFSQPYERSFRRGK--IMWQA---------------------HLEQIIK--KGVQPTMLEIWLRRPLRNFERAIRIRVKKIIQKVKKP-KDFM 356 Butyrivibrio QTSEQVARVKKETEEYDLFIEHILSQSVSDAIRRPRGCWPYIYTNRFF------------------------------------------DEKWFLKSKAKRYIRKAIHC-FEEQ 343 fibrosolvens CafK Parabacteroides EKQLEERIQRN--KNYHKFMLSLLCKSIDSLTTRPNGTFQYVY---------------------RA--------WFLNASVRNDILYRLKRKMNFRRLRNFSLSQNRKN------ 357 goldsteinii CafM Bacteroides fragilis S--------------FTNKLEQVRSNPLIRL------------------------------------------------------------------------CQKIHELKLGGI 296 CafB Helicobacter hepaticus TPRRNIFKEVENVMCVNWGESVQHPPLVCSAPPP---------------------------------------------------------LVSQLKRNAPLNSRLYKEKDNASA 320 CafD Consensus DENYL-.FLKQ----FD.FL.NIFSQPLDKAKRRP.--.MWG.----------------------Y---LK----F--------------R.KQFLKLKA..-..K.KEK----- Bacteroides nordii CafC (SEQ ID NO: 2) Akkermansia muciniphila CafF (SEQ ID NO: 1) Helicobacter pylori FutA (SEQ ID NO: 54) Tannerrella sp. CafN (SEQ ID NO: 9) Bacteroides fragilis CafA (SEQ ID NO: 3) Bacteroides Salyersiae CafQ (SEQ ID NO: 12) Parabacteroides golsteinii CafS (SEQ ID NO: 14) Clostridium bolteae CafT (SEQ ID NO: 15) Methanobrevibacter ruminantium CafP (SEQ ID NO: 11) Lachnospiraceae bacterium CAfO (SEQ ID NO: 10) Lachnospiraceae bacterium CafR (SEQ ID NO: 13) Butyrivibrio sp. CafL (SEQ ID NO: 7) Helicobacter Canis CafU (SEQ ID NO: 16) Helicobacter Canis CafV (SEQ ID NO: 17) Butyrivibrio fibrosolvens CafK (SEQ ID NO: 6) Parabacteroides goldsteinii CafM (SEQ ID NO: 8) Bacteroides fragilis CafB (SEQ ID NO: 4) Helicobacter hepaticus CafD (SEQ ID NO: 53) Consensus (SEQ ID NO: 61)

(67) Based on the amino acid sequences of the identified α(1,3) fucosyltransferases (i.e., in Table 5), synthetic genes are designed and constructed by the skilled artisan using standard methods known in the art. For example, the synthetic genes include a ribosomal binding site, are codon-optimized for expression in a host bacterial production strain (i.e., E. coli), and have common 6-cutter restriction sites or sites recognized by endogenous restriction enzymes present in the host strain (i.e., EcoK restriction sites) removed to ease cloning and expression in the E. coli host strain. In a preferred embodiment, the synthetic genes are constructed with the following configuration: EcoRI site—T7g10 RBS—α(1,3) FT synthetic gene—XhoI site.

(68) The nucleic acid sequences of sample synthetic genes for the 12 identified α(1,3) fucosyltransferases are shown in Table 6. Start and stop codons are underlined and bolded

(69) TABLE-US-00008 TABLE 6 Nucleic acid sequences of 12 novel α(1,3) fucosyltransferase synthetic genes Gene SEQ Name Sequence ID NO CafK CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGCGTCGTGTGTTTGCGATCCACC 41 CATCTATTAAAGGCATCGTTGACCTGTCTAAATACCTGGGTTTCAAATCTTGCATCAC CGAAGAGATCATTTGGGATTCTAACAGCCCGGAGTTCATTTTCGTCTCTGAGCGTATT TACACTGACATCAACGAATGGGAACTGTTTAAGAAAATGTACAACCCGCAACGTATCT TTATTTTTGTTTCCGGTGAATGCATGACCCCGGACCTGAACATTTTCGACTACGCTAT TGTGTTCGACCGCAAACTGAAAGACCTGGACCGTATTTGCCGCATCCCGACCAATTAC ATCCGTCACCGTAGCCTGATCAAAAAAGTGAACGACATGAGCTTCGAAAACGCGCTGT CCCGTGTTAAAGAACTGGACTTCTGCTCTTTTATCTACAGCAATCCGAAGGCGGACCA GATCCGCGAAGACATTTTCTGGGGTCTGATGAACTACAAACACGTTGATTCTCTGGGC GAATACCTGAACAACTCTGGTGTAAAAACTACCCGTAATGACAAACATTGGCGTGAGC TGTCTATCGAAATGAAAAGCCACTACAAATTCAGCATCGCTGTTGAAAACGCTCAATA CGAAGGCTACATTTCCGAAAAACTGCTGACTTCCTTCCAGAGCCATTCTGTCCCTATC TACTGGGGCGACCCGCTGGTAGTGGATGAATACAACCCGAAAGCGTTCATCAACTTCA ACGAAATGTCCTCTATCTCTGAACTGGTTAATCACGTCAAAGAAATTGACGAAAATGA CGAACTGTGGGCAGAAATGGTTTCCGCCGACTGGCAGACCTCCGAACAGGTAGCTCGC GTCAAAAAGGAAACTGAAGAATATGATCTGTTTATCGAACACATCCTGTCTCAGAGCG TTTCCGATGCTATTCGTCGCCCGCGTGGCTGTTGGCCGTACATTTACACGAACCGTTT TTTCGATGAAAAATGGTTTCTGAAGTCCAAAGCAAAGCGTTATATTCGTAAAGCCATC CACTGTTTCGAGGAACAATAGTAGCTCGAGTGACTGACTG CafL AGTCAGTCAGAATTCAAGAAGGAGATATACATATGAAAGTTAAGTTTGTGGATAGCTT 42 TTTTGCACGTGAACAGACGATGGGCGTCCTGAACGAACTGTTCGAAAACGTTGAGATT TCCGACGACCCGGATTTCGTGTTTTGCTCCGTAGATTACAAAGCAGAACACATGAACT ACGACTGTCCGCGTATCATGGTGATCGGTGAAAACATTGTTCCAGACTTTAACTGCAT CGATTACGCTGTTGGTTTCAACTATATGAACTTCGAGGATCGCTATCTGCGTGTTCCG CTGTATAACTTCTACCTGGACGATTATAAACTGGCAATTCGCCGTCATATCGATTACA AACGTGACGACAACAAAAAATTCTGCAACTTCGTTTACTCCAACGGTCGTAACGCCAT TCCTGAACGTGATTCTTTCTTTGCGGACCTGAGCAAGTACAAGCAAGTTGATAGCGGT GGTCGTCACCTGAACAATATCGGCGGTCCGGTTGATGATAAACGCGAGTTCCAGAAAC AGTACAAGTTCTCCATTGCCTTCGAAAATGCTGTTTCCCGTGGTTACACCACCGAGAA AATCATCCAGGCTTTCAGCGCTGGCACTATCCCGATTTACTATGGCAACCCGCTGGTA GCTAAAGAATTTAACAGCAAAGCGTTCATTAATTGCCACGAATATCGTAGCTTCGACG AAGTTATCGAAAAAGTAAAAGAACTGGATAACGACCCAGACCTGTATGATTCTATGAT GCGTGAACCGATCTTCACTGACATCGACGAGCGTCAGGACCCGCTGAAGGATTATCGT AAATTCATCTACAACATTTGCTCTCAGGAGTCTGATAAAGCCATTCGTCGTTGTGACG ATTGCTGGGGTGGTAAAATCCAGCGTGAAAAGAAACGTTGTTACCGCTTCCTGACCTC TACCGAGGGTAACGGTCTGAAAGCACGTGTTATCCGTAAACTGACCGAAATTTAGTAG CTCGAGTGACTGACTG CafM CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGACCGTGACTATGGTACGCTCTC 43 TGTATTTTGTCCACCCTAAGGTTCACAACGTCGAATCCTTCCTGAATTATGTTCACAT CTGTGAACTGCCGCAGGGCCTGTGCCTGGAATGGAACGACCGTAACCCTGAACTGCTG TTCGCTTCTGAGGTAATCTATTCTGATAAAAAGTCCAGCGAAACGTTTCGCCGCCTGT ACTGCGAGGCCAAAGTAGTTGTTTATTATGGTGGTGAAGCATCTTTTACTGATTTTAA TATCTTCGACTATGGTGTCGGCTTCGACCATACCCTGAAAAACCAGAAATACGCGCAG ATCCTGTCTCCGATTGATTTTTTCGACAACTTCTTCTACCCAGACCGCACGAATCTGA GCGAAGAAGTAGCACAAGAAAAGCTGCGTTCTGGTCTGAAATTCTGCAACTTCCTGTA CTCCAACCCGGTTGCCCATCCGTACCGTGACAATCTGTTCTACAAGCTGTCTGAATAC AAGAAAGTTGACGCGCTGGGCCGTCACCTGAACAACACCGGCATCGGCGGCACTGGTT TCGCGGGCCACGCCCGTGAATCCGTGAACCTGAAGGAAAATTACAAATTTTCCATCGC GTCTGAAAACTGCGGTTTTCAGGGTTACACCTCTGAGAAAATCCTGACCTCCCTACAG GCCCACACTGTACCGATCTATTGGGGCGACCCGGACGTTGACCTGGTTGTAAATCCGA AATGCTTCATTAACTGTAACGACTTCGATACCCTGGATGAAGTACTACAGAAAGTGAA AGAGATTGACAACAACGACGATCTGTGGTGCGAAATGGTGTCTCAACCGTGGTTCACT GAAAAACAACTGGAAGAACGTATCCAGCGTAACAAAAACTATCATAAATTTATGCTGT CCCTGCTGTGTAAATCCATTGACAGCCTGACCACCCGTCCGAACGGCACGTTCCAGTA CGTATATCGTGCGTGGTTCCTGAACGCGAGCGTACGTAACGACATCCTGTACCGCCTG AAACGTAAAATGAACTTCCGCCGCCTGCGCAATTTTTCTCTGTCTCAAAACCGTAAAA ACTAGTAGCTCGAGTGACTGACTG CafN CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGAAGACCATCAAGGTAAAATTCG 44 TCGATTTCTGGAAAGGTTTCGACCCGCGCAACAACTTCCTGATGGACATCCTGAAACA GCGTTATCACATTGAACTGAGCGAAAGCCCGGACTACCTGATCTTCTCTGTCTTCGGT TTCACTAACCTGAACTACGAACGCTGCGTTAAAATCTTCTACACCGGTGAAAACCTGA CCCCGGATTTCAACATCTGCGACTACGCGATTGGTTTCGATTATCTGAGCTTCGGTGA TCGTTACATGCGTCTGCCACTGTACGCGGTCTATGGCATCGAGAAACTGGCTTCTCCG AAAGTTATCGACAAAGAAAAAGTTCTGAAGCGTAAATTCTGTTCTTACGTAGTAAGCA ATAACATCGGCGCGCCGGAACGTTCTCGTTTCTTCCATCTGCTGTCTGAATACAAAAA GGTTGACTCCGGTGGTCGTTGGGAAAACAACGTAGGCGGTCCGGTTCCGAATAAGCTG GACTTTATCAAAGACTACAAGTTCAACATCGCATTCGAAAACTCCATGTACGACGGCT ACACTACTGAAAAAATCATGGAACCGATGCTGGTGAACAGCCTGCCGATTTATTGGGG CAACCGCCTGATCAACAAAGACTTCAACCCAGCGTCTTTCATCAACGTTTCCGATTTC CCGTCTCTGGAAGCGGCGGTGGAGCACATTGTTATGCTGGACAATAACGATGATATGT ACCTGAGCATCCTGTCTAAACCGTGGTTTAACGATGAAAACTACCTGGACTGGAAAGC GCGCTTCTTCCACTTTTTCGATAACATCTTCAATCGTCCGATCGATGAATGCAAATAT CTGACCCCGTACGGCTTTTGTCGTCACTATCGTAACCAACTGCGTAGCGCTCGTCTGC TGAAACAGCGCTTTCGCCAGCTGCGTAACCCGCTGCGCTGGTTCCGCTAGTAGCTCGA GTGACTGACTG CafO CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGTCTAAAAAAAAAATCAAAATCA 45 ACTATATCGACTTTTGGCCGGGCTTCAAAAAGGAAGACAACTTCTTTTCCCGTATCCT GGACAAATACTACGATGTGGAAATTTCTGACAACCCGGACTATGTCTTTTGCAGCTGC TTCTCCCGCAAGCACTTCAAATATGCTGATTGCGTTAAAATCTTCTACACCGGTGAGA ACATCATCCCTGATTTTAACCTGTATGACTACTCTATGGGTTTCCACTACATCGATTT TGAAGATCGTTACCTGCGCCTGCCGCATTACGCGCTGTATGATCAGTGTATCAAGGCC GCGAAAGAAAAGCACACCCACTCTGATGACTATTACCTGGCTAAAAAAAAATTCTGTA ACTATGTTATTTCCAACCCGTACGCCGCCCCGGAACGTGACCTGATGATCGATGCGCT GGAGAAATACATGCCTGTTGATTCTGGCGGTCGTTATCGCAACAACGTCGGTGGTCCT GTAGCAGATAAAGTAGAATTTGCGTCCCACTATCGCTTCTCTATGGCGTTCGAGAATA GCGCGATGTCTGGTTACACCACTGAAAAAATCTTCGATGGTTTCGCCGCCTGTACCAT CCCGATCTACTGGGGCTCTGATCGCATTAAAGAGGAGTTCAATCCGGAGAGCTTTGTA AGCGCACGTGACTTCGAAAACTTCGATCAGGTGGTAGCGCGTGTCAAGGAAATCTACG AAAATGATGACCTGTACCTGAAAATGATGAAAGCGCCGATCGCGCCGGAAGGTTTCCA GGCCCACGAATGCCTGAAGGAGGATTATGCCGACGCGTTTCTGCGTAACATTTTTGAC CAGGACATCGACAAAGCTAAGCGCCGTAACATGGTTTACGTCGGTCGTGATTATCAGA AAAAGCTGAAGGATGCTAACAAAGTGATTGAGGTTCTGGATGTGGTGAAGAAACCGAT GCACCAGTTTAACAAAACTAAATCTCAGATCGCGTCTAAATTCCGTAAGAAAAAATAG TAGCTCGAGTGACTGACTG CafP CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGTCCGAAAAAAAAAAAATCAAAG 46 TTAAATTCGTAGATTTCCAGGACTCCCTGAAAGAAAACGACAACTTCTTTATTGACTC TCTGAAAAAAAACTTCGACGTTGAAGTTTCCGACGATCCGGACTATCTGTTTTTCGGT GCTTATGGCTACAAACACCTGGACTACGATTGTATCCGTATTATGTGGACCATCGAAA ACTATGTGCCGGATTTCAACATTTGCGACTATGCTCTGGCTTATGACATCATTGAGTT CGGTGACCGTTACCTGCGCTTCCCGTTCTTCCTGAACCGTCCGGAAATCGAAAACGTG CGTAAAACCATTGAACGTAAACCGATTGACACGTCCGTTAAAACGGACTTCTGTAGCT TTGTTGTAAGCAACGAATGGGGCGACGACTACCGTATTCGCCTGTTCCACGAACTGTC CAAATACAAAAAAGTGGACTCCGGCGGTCGTTCCCTGAACAACATTGGCGGTCCGATC GGCATGGGCCTGGATAAAAAATTCGAGTTCGATGTTACCCACAAATTCTCCTTTGCCC TGGAAAACGCGCAGAACCGCGGTTATACCACCGAAAAAATCTTCGATGCGTTCGCGGC GGGTTGCATTCCGATCTATTGGGGTGATCCGAATATTGAGGAAGAGTTCAACCCGAAA TCCTTCATCAACTGCAACGACCTGACCGTTGAGGAAGCCGTTGAGAAAATCAAAGAGG TTGACCAGAACGATGAACTGTACCACGCGATGCTGAACGAACCGACTTTTCTGGGCGA CCTGGACAAATATCTGCAAGACTTCGACGACTTCCTGTTCAACATTTGCAATCAGCCG CTGGAAAAAGCGTATCGTCGTGACCGCATCATGAAAGGCAAGACTCAGGAACACCAGT ACAAACTGATCAACCGTTTCTACTACAAGCCATATTTTTTCCTGATCAAAGTTGCTCA AAAACTGCACATCGAGTTTATCGGTCGTAAGATTTACCATTTTATCCGTGATTAGTAG CTCGAGTGACTGACTG CafQ CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGAAAAAAGTTAAGATCAAATTTG 47 TAGACTTCTTCGATGGTTTCGACAAAGGCCGTAACGAGTTTCTGGAAGTTCTGAAACA GCGCTATGAAATCGACATCTCTGATGAGCCTGATTATGTAATCTACAGCGGCTTCGGT TACGAACACCTGAAATACAACTGCATCCGTATCTTCTTCACCGGTGAGTGCCAGACCC CAGACTTCAACGAATGCGATTATGCAATCGGCTTTGATCGCCTGAAATTCGGTGACCG CTATGTCCGTATTCCGCTGTATAATATGATGCAATATAAACTGGACTATAAAGAACTG CTGAACCGTAAATCCATCATTTCCGACGATATTAAAGGTCGTGGCTTCTGCTCCTTTG TAGTGTCTAACTGTTTCGCGAATGATACCCGTGCGATCTTCTACGAACTGCTGAATCA GTATAAATATATCGCTAGCGGTGGCCGTTATAAAAACAATATCGGCGGTGCCATTAAA GATAAGAAGACGTTCCTGAGCAAATACAAATTCAACATCGCGTTCGAAAACTGTTCTC ATGATGGCTACGCCACCGAAAAAATCGTAGAGGCTTTTGCTGCCGGCGTAGTTCCGAT CTACTATGGCGACCCACGTATCGCAGAAGATTTCAACCCGAAGGCATTTATTAATGCA CACGATTATCAGAGCTTCGAAGAAATGGTGGAACGCATCAAAGAGATCGATGCCGATG ACCGTCTGTACCTGACCATGCTGAACGAACCGATCATTCAGCCGAACGCAGACGTGAC TGAACTGGCGGATTTCCTGTATAGCATCTTCGACCAGCCGCTGGCCAAGGCCAAACGC CGTTCCCAGTCCCAGCCGACTCAGGCTATGGAGGCAATGAAACTGCGCCACGAGTTCT TCGAAATGAAAATCTACAAATATTATAAAAAAGGTATGAACCAGTTCACGCGTCTGCG CAAGGGCGTGTTCCTAAGCTCTAAACGTACCAAATAGTAGCTCGAGTGACTGACTG CafR CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGAAAAAGGAAATCAAAATCGCGT 48 ACGTGGATTTCTGGAACGGCTTCAAGCCTGACTCCTTCTTCATCACCAAGACCATCAG CAAAAAATACAAGGTTATCATCGACAATGAAAACCCGGATTTCGTAATCTGTGGTACC TTCGGTAATACCTTCCTGTCCTATGACTGCCCGCGTATCCTGTATACCGGTGAAGCTA ACTGCCCGGATTTTAATATCTACGACTATGCAATTGGTTTCGAACGCATGGTTTACGA AGACCGCTATCTGCGCTACCCGCTGTTCCTGGTGAACGAAGACCTGCTACAGGATGCG CTGAACAAACACAAAAAATCTGATGACTACTATCTGCGTCGTGATGGCTTCTGTAGCT TCGTGGTGTCCGCGTCTGGCGGTATGGACGGTCTGCGTAACTGGTATTTTGATAAAAT CAGCGAATATAAGCAGGTAGCTTCCGGTGGCCGTTTTCGCAACAACCTGCCGGACGGC AAACCAGTTCCAGATAAAAAGGCATTCCAGGAAAACTACCGCTTCTCCCTGTGCTTCG AGAACGCTGGCATCAGCGGCTATGCTACCGAAAAAATTGTTGACGCATTCGCGGCTGG TTGCATCCCGATCTACTACGGTGACACCAACATCGAAAAAGACTTCAACCCGAAATCC TTTATTCACGTGAAATCTCGTGAAGACCTGGACTCCGTTCTGGCTTGGGTGAAGGAGC TGGAAGAAAACCAGAACAAATATCTGGAGGTGATCCGTCAACCTGCAATCCTGCCTGA CAGCCCGATCATGGGTATGCTGAACAACACGTACATCGAAGAGTTCCTGTTCCATATC TTCGACCAGGAACCTCAGGAGGCAATCCGTCGTCACAGCAAACTGACTATGTGGGGCC AGTTCTATGAATACCGTCTGAAAAAATGGAACAAGATCGAGAACAACATGTTTCTGAA GAAAGCACGTAGCATTAAACGTAAATACTTTGGCCTGAAAAAAATCGTTAAATAGTAG CTCGAGTGACTGACTG CafS CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGAAGAAAAAAATCTACTGCAACT 49 TCGTGGACTTTTGGCTGGGTTTTAACTATAAAACCTACTTCTGGTATCTGTCCGACGA GTACGATCTACAGATCGACAAAGAACATCCAGATTACCTGTTTTACTCCTGCTTCGGT AACGAACATCTGTTCTACGAAGACTGCATTCGCATTTTCTGGTCTGACGAGAACATCA TGCCGGACCTGAACATTTGCGACTACGCTCTGTCTCTGAGCAACCTACAGTGCGACGA CCGTACCTTCCGCAAGTACTCCGGTTTCCTGTACCGTAAGGATTCTCATCTGGTTCTG CCGGTACTGAAAGAAGAAGCGCTGCTGAATCGTAAATTTTGCAACTTCGTATACTCTA ACAACACCTGTGCTGTTCCGTACCGTGAACTGTTCTTTAAAGCGCTGTCTGGCTACAA ACGTATCGATTCTGGTGGTGCGTTTCTGAATAACATGGGTAAAAAAGTTGGCGATAAG CGCCAGTTTCTGCACGAATACAAATTTACTCTGGCTATCGAAAATTCCTCTATGCCGG GTTACGTGACCGAAAAAATCCTGGAGCCTTTTATGGCTCAGAGCCTGCCACTGTACTG GGGTTCTCCGACTGTTTCCTCTGACTATAACCCTAACTCCTTCGTAAATCTGATGAAC TACTCCTCTATGGAAGAAGCGGTAGAAGAAGTGATTCGCCTGGACAAAGACGACGCTG CGTATCTGGACAAAATGATGACGCCTTTCTGGCTGTACGGTGCAAACTTCCAAGAGTT CCGTGACTCCGAGATTAAAAAAATTAAAGATTTCTTCTCTTATATCTTCGAACAGCCG CTGGACAAAGCGGGCCGTCGCGTTTGTTACGGTCGTAATCGTATCACCATCCAAAAAC AGCGTCGTTACTACGCCCCGACTTTTCTGGAACTGTCTAAATCTATGACTAAGAAACT GCTGAAGAAAAAATAGTAGCTCGAGTGACTGACTG CafT CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGAAAAAAATCCGTCTGAAATACG 50 TTGATTGGTGGGATGGTTTCCAGCCGGAACAATATCGCTTTCATCAGATCCTGACTAA ACATTTCGACATCGAAATTAGCGATGAACCGGATTACATTATCGCTAGCGTGTACTCT GACGAAGCAAAAAGCTACAACTGTGTTCGCATCCTGTATACCGGTGAGAACATCTGCC CGGATTTCAACATCTATGACTATGCTATCGGCTTCGAATACCTGGAGTTCGGTGATCG CTATATCCGTATCCCGAACTTTATCATGAACCCGGCTTACGACATCGACATCCAGAAA GCGCTGTCTAAGCATCTGCTGTCTGCTGATGATATCAAACGCGAAAAAAAATTCTGCT CCTTCGTCGTTTCTAACGGCAACGCAGCGCCAATCCGTGAGAAGATGTTCGAAGAACT GAATAAATATAAGCGTGTGGACTCCGGCGGTCGCTACCTGAACAACATCGGTCGTCCA GAAGGCGTTCGTGACAAATTCGCTTTCCAATCTGAACACAAGTTTTCTCTGACCTTCG AGAACTCCGCGCACCTGGGTTACACTACGGAAAAACTGCTACAGGGCTTCTCTGCGGG CACGATTCCGATCTACTGGGGTGACCCGGCGGTGGAAAACTGCTTCAACCCGAAAGCG TTCATCAACATTTCCGGCAACAACGTTTACGACGCAATCGAACTGGTTAAAGAAGTTG ATACTCAGGACGACCTGTACTTTAGCATGTTGCGTGAACCGGCTTTTCTGAACAACGA TTACCAAACTAAACTGCTGGAGAAGCTGGATAACTTCCTGGTACACATCTTTAATCAG CCGCTGGAGTGCGCCTACCGTCGTAACAGCTTTGAGCATATCAGCAACAAATCTGTTC TGAATGAGTTCGTGAAAGAAGATCGTGGCCGTTTCTCCCAGTGGATCTCCAACAAGGC GCGTTGTTTCTATGGCAAACGTAAAAACAAGTAGTAGCTCGAGTGACTGACTG CafU CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGAGCAAAGAAAAGTGGAAACAGG 51 AAAAACGCGTTCATTTCGTAGATTGTTGCGACGACGGTATCCGTGACAAAGTTTGCCC GATCCTGGAACAACACTTTACTCTGATCTTCGACTCTGTAAACCCGGAATACGTGTTC TATTCTGCCTACGGTGAAGAACATCTGGCTTACGACTGCATCCGCATTTTTATCACTG GCGAAAACATCACCCCGAACTTCACGATTTGCGACTACGCTATCGGTTTCGACCACCT GCACTTTCTGGATCGTTACCTGCGCTACCCACTGTACCTGTTCTACGAACAGGATGTG AAACGCGCATCCCAGAAACACAAAGATATCGACGAAAAGCTGCTGGCTTCTAAATCCC GTTTTTGCAACTTTGTGGTGAGCAACGGCAACGCTGATCCGTACCGCGAACAGGTATT CTACGCGCTGAACGCCTACAAGCGTGTGGACAGCGGTGGTCGTTATCTGAACAACATT GGTGGTAGCGTGGCCGATAAATTCGCTTTCCAGTCTGAATGTCGTTTTAGCCTGTGCT TCGAAAACAGCTCTACGCCGGGTTACCTGACCGAGAAACTGATTCAGGCGGCGGCTGC TCAAACCATCCCAATTTATTGGGGCGACACTCTGGCGACTAAACCGCTGTTCGATGGC GGTGGCGGTATCAACGCCAAGGCATTCATCAACGCGCACTCCTTCTCTTCTCTGGAAT CTCTGATTGCTCACATCGCCGAGATTGAAGCGGATAAGACGAAACAGCTGGCCATTCT ACAGGAACCACTGTTCCTGGACTCTAATCACATCGAGCTGTTCGAAAAACAGTTCGAA CAATTTCTGCTGAGCATTGTGAGCCAGCCGTATGAACGTTCTTTCCGTCGTGGTCGTG TTATGTGGCAGTCTTTTGTTGAACAGCGCTACAAACGCGCCATGCATCTGCTGGCTCT GGAAGACCGCATCAAAGCTCCGTACCGTAAGCTGCGTCAGTTCCTGCGCGCGTTCTGG GACTCCCTGAAAGAAAAACGTTCCCACACTTAGTAGCTCGAGTGACTGACTG CafV CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGGGTGACGAAGTTGCTATGGGTA 52 AAGAGCGCAAGCAGATTCGCGTTCACTTCGTAGACTTCTCCAACATGGATAACATTAT TGAAAAAATTTGCTCTATTCTGTCCCGTCATTTCGCAGTTATCATTGACGGTGAAAAC CCGGAGTATGTATTCTACTCTGCTTTCGGTAGCGAATATCTGAAGTACGATTGTGTTC GTATCTTCTACACTGGCGAAAACATTGTACCGGATTTTAACCTGTGCGATTACGCTAT CGGTTTCGATCACATCAAGTTCCTGGACCGTTACCTGCGCTACCCTCTGTATCTGTTT TATGAAACCGATGTACAGAAAGCGGCTCGTAAACACCAGAACCTGTCTCTGGAAGTTG TCCGCAACAAAAAACGTTTTTGCAATTTCGTAGTTACCAACGGCAAAGGTGACCCGTA TCGTGAAAAAGTTTTTCATGCTCTGTGCGCTTACAAACGTGTAGATAGCGCTGGTAAG TTTCTGAACAACGTTGGTGCACGCGTTAAAGATAAATTTGCGTTCCAGAGCGAATGCC GTTTTTCCCTGTGCTTCGAGAACTCTAGCACCCCTGGTTATCTGACCGAAAAACTGAT CCAGGCAGCGGCTGCGCAAACTATCCCGATCTATTGGGGCGACCCGCTGGCGACCAAG CCGCTGTTTGATGGTGGCGGCGGTATCAACGCGAAAGCGTTCATCAACGCTCACGAGT TCGCCAACATCGCGTCCCTGGTGCGCCATATTGAGAGCATCGAAAACGACGAAAACAA ACAGCTGGCTATCCTGCAAGAACCGCTGTTTCTGGATTCCAATCATATTGAACTGTTC GAAAAACAGTTCGAGGATTTCCTGGTGTATATCTTTTCTCAGCCTTACGAGCGTAGCT TCCGTCGCGGTAAAATCATGTGGCAGGCGCATCTGGAACAGATCATCAAAAAAGGTGT TCAGCCGACCATGCTGGAAATTTGGCTGCGTCGTCCACTGCGCAACTTCGAGCGCGCG ATCCGCATCCGTGTGAAAAAAATTATTCAGAAAGTGAAAAAACCGAAAGATTTCATGT AGTAGCTCGAGTGACTGACTG

(70) In any of the methods described herein, the α(1,3) fucosyltransferase genes or gene products may be variants or functional fragments thereof. A variant of any of genes or gene products disclosed herein may have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid or amino acid sequences described herein.

(71) Variants as disclosed herein also include homolog, orthologs, or paralogs of the genes or gene products described herein that retain the same biological function as the genes or gene products specified herein. These variants can be used interchangeably with the genes recited in these methods. Such variants may demonstrate a percentage of homology or identity, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity conserved domains important for biological function, preferably in a functional domain, e.g. catalytic domain.

(72) The term “% identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For example, % identity is relative to the entire length of the coding regions of the sequences being compared, or the length of a particular fragment or functional domain thereof.

(73) 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 input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

(74) Percent identity is determined using search algorithms such as BLAST and PSI-BLAST (Altschul et al., 1990, J Mol Biol 215:3, 403-410; Altschul et al., 1997, Nucleic Acids Res 25:17, 3389-402). For the PSI-BLAST search, the following exemplary parameters are employed: (1) Expect threshold was 10; (2) Gap cost was Existence:11 and Extension:1;

(75) (3) The Matrix employed was BLOSUM62; (4) The filter for low complexity regions was “on”.

(76) The three dimensional structure of the lactose-utilizing α(1,3) fucosyltransferase Helicobacter pylori FutA (FucT) is described in H. Y. Sun, S. W. Lin, T. P. Ko, J. F. Pan, et al., J Biol Chem 282, 9973-82 (2007). Here the amino acid residues essential for the substrate binding and the catalytic mechanism of the enzyme are discussed—in particular the sequences lying between FutA residues 31-42 (substrate binding), 85-129 (active site region 1) and 180-266 (active site region 2), with specific amino acid residues E96, R196, E250 and K251 are involved in catalysis. FIG. 18 is a sequence alignment of FutA with 8 lactose-utilizing “Caf” α(1,3) fucosyltransferases (i.e. CafF, CafC, CafV, CafN, CafL, CafO, CafQ, and CafU) discovered in the computational screens of this invention. It can readily be seen that the FutA regions known to be involved in substrate binding are well conserved in all 8 novel sequences. Moreover each of the 4 residues known to be involved at the catalytic site is completely conserved across all 8 enzymes.

(77) Changes are introduced by mutation into the nucleic acid sequence or amino acid sequence of any of the genes or gene products described herein, leading to changes in the amino acid sequence of the encoded protein or enzyme, without altering the functional ability of the protein or enzyme. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence of any of sequences expressly disclosed herein. A “non-essential” amino acid residue is a residue at a position in the sequence that can be altered from the wild-type sequence of the polypeptide without altering the biological activity, whereas an “essential” amino acid residue is a residue at a position that is required for biological activity. For example, amino acid residues that are conserved among members of a family of proteins are not likely to be amenable to mutation. Other amino acid residues, however, (e.g., those that are poorly conserved among members of the protein family) may not be as essential for activity and thus are more likely to be amenable to alteration. Thus, another aspect of the invention pertains to nucleic acid molecules encoding the proteins or enzymes disclosed herein that contain changes in amino acid residues relative to the amino acid sequences disclosed herein that are not essential for activity (i.e., fucosyltransferase activity). Preferably, at least 0.1% of the activity of the reference enzyme is retained. In some embodiments, low α1,3 fucosyltransferase activity enzymes may be used in the production of large quantities of 3FL. For example, CafC is expressed very well in E. coli, leading to the easy generation of a vast excess of α1,3 fucosyltransferase enzymatic activity over that required for the production of large amounts of 3FL. Thus even variants of CafC enzyme with a relatively low level (e.g., 0.1, 1, 10%) of activity relative to the wildtype CafC enzyme, may produce useful levels of the product, 3FL.

(78) An isolated nucleic acid molecule encoding a protein essentially retaining the functional capability compared to any of the genes described herein can be created by introducing one or more nucleotide substitutions, additions or deletions into the corresponding nucleotide sequence, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.

(79) Mutations are introduced into a nucleic acid sequence by standard techniques such that the encoded amino acid sequence is altered, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Certain amino acids have side chains with more than one classifiable characteristic. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, tryptophan, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tyrosine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a given polypeptide is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a given coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for given polypeptide biological activity to identify mutants that retain activity. Conversely, the invention also provides for variants with mutations that enhance or increase the endogenous biological activity. Following mutagenesis of the nucleic acid sequence, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined. An increase, decrease, or elimination of a given biological activity of the variants disclosed herein can be readily measured by the ordinary person skilled in the art, i.e., by measuring the capability for mediating oligosaccharide modification, synthesis, or degradation (via detection of the products).

(80) The present invention includes functional fragments of the genes or gene products described herein, e.g., catalytic domain portions of the enzyme shown in FIGS. 18 and 19. A fragment, in the case of these sequences and all others provided herein, is defined as a part of the whole that is less than the whole. Moreover, a fragment ranges in size from a single nucleotide or amino acid within a polynucleotide or polypeptide sequence to one fewer nucleotide or amino acid than the entire polynucleotide or polypeptide sequence. Finally, a fragment is defined as any portion of a complete polynucleotide or polypeptide sequence that is intermediate between the extremes defined above.

(81) For example, fragments of any of the proteins or enzymes disclosed herein or encoded by any of the genes disclosed herein can be 10 to 20 amino acids, 10 to 30 amino acids, 10 to 40 amino acids, 10 to 50 amino acids, 10 to 60 amino acids, 10 to 70 amino acids, 10 to 80 amino acids, 10 to 90 amino acids, 10 to 100 amino acids, 50 to 100 amino acids, 75 to 125 amino acids, 100 to 150 amino acids, 150 to 200 amino acids, 200 to 250 amino acids, 250 to 300 amino acids, 300 to 350 amino acids, 350 to 400 amino acids, 400 to 450 amino acids, or 450 to 500 amino acids. The fragments encompassed in the present invention comprise fragments that retain functional fragments. As such, the fragments preferably retain the catalytic domains that are required or are important for functional activity. Fragments can be determined or generated by using the sequence information herein, and the fragments can be tested for functional activity using standard methods known in the art. For example, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined. The biological function of said fragment can be measured by measuring ability to synthesize or modify a substrate oligosaccharide, or conversely, to catabolize an oligosaccharide substrate.

(82) Within the context of the invention, “functionally equivalent”, as used herein, refers to a gene or the resulting encoded protein variant or fragment thereof capable of exhibiting a substantially similar activity as the wild-type fucosyltransferase. Specifically, the fucosyltransferase activity refers to the ability to transfer a fucose sugar to an acceptor substrate via an alpha-(1,3)-linkage. As used herein, “substantially similar activity” refers to an activity level within 5%, 10%, 20%, 30%, 40%, or 50% of the wild-type fucosyltransferase.

(83) To test for lactose-utilizing fucosyltransferase activity, the production of α(1,3) fucosylated oligosaccharides is evaluated in a host organism that expresses a candidate enzyme synthetic gene and which contains both cytoplasmic GDP-fucose and lactose pools. The production of fucosylated oligosaccharides indicates that the candidate enzyme-encoding sequence functions as a lactose-utilizing α(1,3)fucosyltransferase.

(84) The invention also provides nucleic acid constructs (i.e., a plasmid or vector) carrying the nucleic acid sequence of a novel α(1,3) fucosyltransferases for the expression of the novel α(1,3) fucosyltransferases in host bacterium.

(85) The invention also provides methods for producing fucosylated oligosaccharides by expressing the novel α(1,3) fucosyltransferases in suitable host production bacterium, as further described herein.

(86) Engineering of E. coli to Produce α(1,3) Fucosylated Human Milk Oligosaccharide

(87) Described herein is a gene screening approach, which was used to validate the novel α(1,3) fucosyltransferases (α(1,3) FTs) for the synthesis of fucosyl-linked oligosaccharides in metabolically engineered E. coli. Of particular interest are α(1,3) FTs that are capable of the synthesis of the HMOS 3-fucosyllactose (3-FL), lactodifucotetraose (LDFT), or lacto-N-fucopentaose III (LNF III). Of most interest are α(1,3) FTs that catalyze the synthesis of 3-FL. Preferably, the α(1,3) fucosyl-linked oligosaccharides are expressed in metabolically engineered E. coli.

(88) In particular, therefore, the invention provides α(1,3) FTs that are capable of the synthesis of the HMO (human milk oligosaccharide) 3-fucosyllactose (3FL). As explained above, 3FL is one of the most abundant fucosylated oligosaccharide present in human milk, and is thought to function with other HMOS to promote the growth of beneficial commensal bacteria in the infant gut.

(89) Production Host Strains

(90) A suitable production host strain is one that is not the same bacterial strain as the source bacterial strain from which the fucosyltransferase-encoding nucleic acid sequence was identified.

(91) E. coli K-12 is a well-studied bacterium which has been the subject of extensive research in microbial physiology and genetics and commercially exploited for a variety of industrial uses. The natural habitat of the parent species, E. coli, is the large bowel of mammals. E. coli K-12 has a history of safe use, and its derivatives are used in a large number of industrial applications, including the production of chemicals and drugs for human administration and consumption. E. coli K-12 was originally isolated from a convalescent diphtheria patient in 1922. Because it lacks virulence characteristics, grows readily on common laboratory media, and has been used extensively for microbial physiology and genetics research, it has become the standard bacteriological strain used in microbiological research, teaching, and production of products for industry and medicine. E. coli K-12 is now considered an enfeebled organism as a result of being maintained in the laboratory environment for over 70 years. As a result, K-12 strains are unable to colonize the intestines of humans and other animals under normal conditions. Additional information on this well-known strain is available at http://epa.gov/oppt/biotech/pubs/fra/fra004.htm. In addition to E. coli K-12, other bacterial strains are used as production host strains, e.g., a variety of bacterial species may be used in the oligosaccharide biosynthesis methods, e.g., Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Pantoea citrea, Pectobacterium carotovorum, or Xanthomonas campestris. Bacteria of the genus Bacillus may also be used, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans. Similarly, bacteria of the genera Lactobacillus and Lactococcus may be modified using the methods of this invention, including but not limited to Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species for the invention described herein. Also included as part of this invention are strains, modified as described here, from the genera Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium (e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa).

(92) Suitable host strains are amenable to genetic manipulation, e.g., they maintain expression constructs, accumulate precursors of the desired end product, e.g., they maintain pools of lactose and GDP-fucose, and accumulate end product, e.g., 3FL. Such strains grow well on defined minimal media that contains simple salts and generally a single carbon source.

(93) Biosynthesis of 3FL requires the generation of an enhanced cellular pool of both lactose and GDP-fucose (FIG. 2). Therefore, the host strain preferably has an enhanced cellular pool of lactose and/or GDP-fucose, preferably both lactose and gdp-fucose.

(94) In the Examples provided herein, the wild-type Escherichia coli K-12 prototrophic strain W3110 was selected as the parent background host to test the ability of the candidates to catalyze 3FL production (Bachmann, 1972). The particular W3110 derivative employed was one that previously had been modified by the introduction (at the ampC locus) of a tryptophan-inducible P.sub.trpB CI+ repressor cassette, generating an E. coli strain known as GI724 (LaVallie et al., 2000). Other features of GI724 include lacIq and lacPL8 promoter mutations. E. coli strain GI724 affords economical production of recombinant proteins from the phage X P.sub.L promoter following induction with low levels of exogenous tryptophan (LaVallie et al., 1993; Mieschendahl et al., 1986). Additional genetic alterations (described below) were made to this strain to promote the biosynthesis of 3FL. This was achieved in strain GI724 through several manipulations of the chromosome using λ Red recombineering (Court et al., 2002) and generalized P1 phage transduction.

(95) First: the ability of the E. coli host strain to accumulate intracellular lactose was engineered by simultaneous deletion of the endogenous β-galactosidase gene (lacZ) and the lactose operon repressor gene (lacI). During construction of this deletion the lacIq promoter was placed immediately upstream of the lactose permease gene, lacY. The strain thus modified maintains its ability to transport lactose from the culture medium (via LacY), but is deleted for the wild-type copy of the lacZ (β-galactosidase) gene responsible for lactose catabolism. An intracellular lactose pool is therefore created when the modified strain is cultured in the presence of exogenous lactose. In addition, the lacA gene was deleted in order to eliminate the production of acetyl-lactose from the enhanced pool of intracellular lactose.

(96) Second: the ability of the host E. coli strain to synthesize colanic acid, an extracellular capsular polysaccharide, was eliminated by the deletion of the wcaJ gene, encoding the UDP-glucose lipid carrier transferase (Stevenson et al., 1996). In a wcaJ null background GDP-fucose accumulates in the E. coli cytoplasm (Dumon, C., et al. (2001). In vivo fucosylation of lacto-N-neotetraose and lacto-N-neohexaose by heterologous expression of Helicobacter pylori alpha-1,3 fucosyltransferase in engineered Escherichia coli. Glycoconj J 18, 465-474.)

(97) The sequence of the chromosomal region of E. coli bearing the ΔwcaJ::FRT mutation is set forth below (SEQ ID NO: 55):

(98) TABLE-US-00009 (SEQ ID NO: 55) GTTCGGTTATATCAATGTCAAAAACCTCACGCCGCTCAAGCTGGTGATC AACTCCGGGAACGGCGCAGCGGGTCCGGTGGTGGACGCCATTGAAGCCC GCTTTAAAGCCCTCGGCGCGCCCGTGGAATTAATCAAAGTGCACAACAC GCCGGACGGCAATTTCCCCAACGGTATTCCTAACCCACTACTGCCGGAA TGCCGCGACGACACCCGCAATGCGGTCATCAAACACGGCGCGGATATGG GCATTGCTTTTGATGGCGATTTTGACCGCTGTTTCCTGTTTGACGAAAA AGGGCAGTTTATTGAGGGCTACTACATTGTCGGCCTGTTGGCAGAAGCA TTCCTCGAAAAAAATCCCGGCGCGAAGATCATCCACGATCCACGTCTCT CCTGGAACACCGTTGATGTGGTGACTGCCGCAGGTGGCACGCCGGTAAT GTCGAAAACCGGACACGCCTTTATTAAAGAACGTATGCGCAAGGAAGAC GCCATCTATGGTGGCGAAATGAGCGCCCACCATTACTTCCGTGATTTCG CTTACTGCGACAGCGGCATGATCCCGTGGCTGCTGGTCGCCGAACTGGT GTGCCTGAAAGATAAAACGCTGGGCGAACTGGTACGCGACCGGATGGCG GCGTTTCCGGCAAGCGGTGAGATCAACAGCAAACTGGCGCAACCCGTTG AGGCGATTAACCGCGTGGAACAGCATTTTAGCCGTGAGGCGCTGGCGGT GGATCGCACCGATGGCATCAGCATGACCTTTGCCGACTGGCGCTTTAAC CTGCGCACCTCCAATACCGAACCGGTGGTGCGCCTGAATGTGGAATCGC GCGGTGATGTGCCGCTGATGGAAGCGCGAACGCGAACTCTGCTGACGTT GCTGAACGAGTAATGTCGGATCTTCCCTTACCCCACTGCGGGTAAGGGG CTAATAACAGGAACAACGATGATTCCGGGGATCCGTCGACCTGCAGTTC GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGAAGCAGCTCCAGCC TACAGTTAACAAAGCGGCATATTGATATGAGCTTACGTGAAAAAACCAT CAGCGGCGCGAAGTGGTCGGCGATTGCCACGGTGATCATCATCGGCCTC GGGCTGGTGCAGATGACCGTGCTGGCGCGGATTATCGACAACCACCAGT TCGGCCTGCTTACCGTGTCGCTGGTGATTATCGCGCTGGCAGATACGCT TTCTGACTTCGGTATCGCTAACTCGATTATTCAGCGAAAAGAAATCAGT CACCTTGAACTCACCACGTTGTACTGGCTGAACGTCGGGCTGGGGATCG TGGTGTGCGTGGCGGTGTTTTTGTTGAGTGATCTCATCGGCGACGTGCT GAATAACCCGGACCTGGCACCGTTGATTAAAACATTATCGCTGGCGTTT GTGGTAATCCCCCACGGGCAACAGTTCCGCGCGTTGATGCAAAAAGAGC TGGAGTTCAACAAAATCGGCATGATCGAAACCAGCGCGGTGCTGGCGGG CTTCACTTGTACGGTGGTTAGCGCCCATTTCTGGCCGCTGGCGATGACC GCGATCCTCGGTTATCTGGTCAATAGTGCGGTGAGAACGCTGCTGTTTG GCTACTTTGGCCGCAAAATTTATCGCCCCGGTCTGCATTTCTCGCTGGC GTCGGTGGCACCGAACTTACGCTTTGGTGCCTGGCTGACGGCGGACAGC ATCATCAACTATCTCAATACCAACCTTTCAACGCTCGTGCTGGCGCGTA TTCTCGGCGCGGGCGTGGCAGGGGGATACAACCTGGCGTACAACGTGGC CGTTGTGCCACCGATGAAGCTGAACCCAATCATCACCCGCGTGTTGTTT CCGGCATTCGCCAAAATTCAGGACGATACCGAAAAGCTGCGTGTTAACT TCTACAAGCTGCTGTCGGTAGTGGGGATTATCAACTTTCCGGCGCTGCT CGGGCTAATGGTGGTGTCGAATAACTTTGTACCGCTGGTCTTTGGTGAG AAGTGGAACAGCATTATTCCGGTGCTGCAATTGCTGTGTGTGGTGGGTC TGCTGCGCTCCG

(99) Third: The magnitude of the cytoplasmic GDP-fucose pool was enhanced by the introduction of a null mutation into the lon gene. Lon is an ATP-dependent intracellular protease that has been shown to be responsible for degrading RcsA, a positive transcriptional regulator of colanic acid biosynthesis in E. coli (Gottesman and Stout, 1991). In a lon null background RcsA is stabilized, RcsA levels increase, the genes responsible for GDP-fucose synthesis are up-regulated, and intracellular GDP-fucose concentrations are enhanced. The lon gene was almost entirely deleted in our production strain (E638) and replaced by an inserted functional, wild-type, but promoter-less E. coli lacZ.sup.+ gene (Δlon::(kan, lacZ.sup.+). λ Red recombineering was used to perform the construction.

(100) Genomic DNA sequence surrounding the lacZ+ insertion into the lon region in the E. coli strain is set forth below (SEQ ID NO: 56):

(101) TABLE-US-00010 (SEQ ID NO: 56) GTGGATGGAAGAGGTGGAAAAAGTGGTTATGGAGGAGTGGGTAATTGAT GGTGAAAGGAAAGGGTTGGTGATTTATGGGAAGGGGGAAGGGGAAGAGG GATGTGGTGAATAATTAAGGATTGGGATAGAATTAGTTAAGGAAAAAGG GGGGATTTTATGTGGGGTTTAATTTTTGGTGTATTGTGGGGGTTGAATG TGGGGGAAAGATGGGGATATAGTGAGGTAGATGTTAATAGATGGGGTGA AGGAGAGTGGTGTGATGTGATTAGGTGGGGGAAATTAAAGTAAGAGAGA GGTGTATGATTGGGGGGATGGGTGGAGGTGGAGTTGGAAGTTGGTATTG TGTAGAAAGTATAGGAAGTTGAGAGGGGTTTTGAAGGTGAGGGTGGGGG AAGGAGTGAGGGGGGAAGGGGTGGTAAAGGAAGGGGAAGAGGTAGAAAG GGAGTGGGGAGAAAGGGTGGTGAGGGGGGATGAATGTGAGGTAGTGGGG TATGTGGAGAAGGGAAAAGGGAAGGGGAAAGAGAAAGGAGGTAGGTTGG AGTGGGGTTAGATGGGGATAGGTAGAGTGGGGGGTTTTATGGAGAGGAA GGGAAGGGGAATTGGGAGGTGGGGGGGGGTGTGGTAAGGTTGGGAAGGG GTGGAAAGTAAAGTGGATGGGTTTGTTGGGGGGAAGGATGTGATGGGGG AGGGGATGAAGATGTGATGAAGAGAGAGGATGAGGATGGTTTGGGATGA TTGAAGAAGATGGATTGGAGGGAGGTTGTGGGGGGGGTTGGGTGGAGAG GGTATTGGGGTATGAGTGGGGAGAAGAGAGAATGGGGTGGTGTGATGGG GGGGTGTTGGGGGTGTGAGGGGAGGGGGGGGGGGTTGTTTTTGTGAAGA GGGAGGTGTGGGGTGGGGTGAATGAAGTGGAGGAGGAGGGAGGGGGGGT ATGGTGGGTGGGGAGGAGGGGGGTTGGTTGGGGAGGTGTGGTGGAGGTT GTGAGTGAAGGGGGAAGGGAGTGGGTGGTATTGGGGGAAGTGGGGGGGG AGGATGTGGTGTGATGTGAGGTTGGTGGTGGGGAGAAAGTATGGATGAT GGGTGATGGAATGGGGGGGGTGGATAGGGTTGATGGGGGTAGGTGGGGA TTGGAGGAGGAAGGGAAAGATGGGATGGAGGGAGGAGGTAGTGGGATGG AAGGGGGTGTTGTGGATGAGGATGATGTGGAGGAAGAGGATGAGGGGGT GGGGGGAGGGGAAGTGTTGGGGAGGGTGAAGGGGGGATGGGGGAGGGGG AGGATGTGGTGGTGAGGGATGGGGATGGGTGGTTGGGGAATATGATGGT GGAAAATGGGGGGTTTTGTGGATTGATGGAGTGTGGGGGGGTGGGTGTG GGGGAGGGGTATGAGGAGATAGGGTTGGGTAGGGGTGATATTGGTGAAG AGGTTGGGGGGGAATGGGGTGAGGGGTTGGTGGTGGTTTAGGGTATGGG GGGTGGGGATTGGGAGGGGATGGGGTTGTATGGGGTTGTTGAGGAGTTG TTGTAATAAGGGGATGTTGAAGTTGGTATTGGGAAGTTGGTATTGTGTA GAAAGTATAGGAAGTTGGAAGGAGGTGGAGGGTAGATAAAGGGGGGGGT TATTTTTGAGAGGAGAGGAAGTGGTAATGGTAGGGAGGGGGGGTGAGGT GGAATTGGGGGGATAGTGAGGGGGTGGAGGAGTGGTGGGGAGGAATGGG GATATGGAAAGGGTGGATATTGAGGGATGTGGGTTGTTGGGGGTGGAGG AGATGGGGATGGGTGGTTTGGATGAGTTGGTGTTGAGTGTAGGGGGTGA TGTTGAAGTGGAAGTGGGGGGGGGAGTGGTGTGGGGGATAATTGAATTG GGGGGTGGGGGAGGGGAGAGGGTTTTGGGTGGGGAAGAGGTAGGGGGTA TAGATGTTGAGAATGGGAGATGGGAGGGGTGAAAAGAGGGGGGAGTAAG GGGGTGGGGATAGTTTTGTTGGGGGGGTAATGGGAGGGAGTTTAGGGGG TGTGGTAGGTGGGGGAGGTGGGAGTTGAGGGGAATGGGGGGGGGATGGG GTGTATGGGTGGGGAGTTGAAGATGAAGGGTAATGGGGATTTGAGGAGT AGGATGAATGGGGTAGGTTTTGGGGGTGATAAATAAGGTTTTGGGGTGA TGGTGGGAGGGGTGAGGGGTGGTAATGAGGAGGGGATGAGGAAGTGTAT GTGGGGTGGAGTGGAAGAAGGGTGGTTGGGGGTGGTAATGGGGGGGGGG GTTGGAGGGTTGGAGGGAGGGGTTAGGGTGAATGGGGGTGGGTTGAGTT AGGGGAATGTGGTTATGGAGGGGTGGAGGGGTGAAGTGATGGGGGAGGG GGGTGAGGAGTTGTTTTTTATGGGGAATGGAGATGTGTGAAAGAAAGGG TGAGTGGGGGTTAAATTGGGAAGGGTTATTAGGGAGGTGGATGGAAAAA TGGATTTGGGTGGTGGTGAGATGGGGGATGGGGTGGGAGGGGGGGGGGA GGGTGAGAGTGAGGTTTTGGGGGAGAGGGGAGTGGTGGGAGGGGGTGAT GTGGGGGGGTTGTGAGGATGGGGTGGGGTTGGGTTGGAGTAGGGGTAGT GTGAGGGAGAGTTGGGGGGGGGTGTGGGGGTGGGGTAGTTGAGGGAGTT GAATGAAGTGTTTAGGTTGTGGAGGGAGATGGAGAGGGAGTTGAGGGGT TGGGAGGGGGTTAGGATGGAGGGGGAGGATGGAGTGGAGGAGGTGGTTA TGGGTATGAGGGAAGAGGTATTGGGTGGTGAGTTGGATGGTTTGGGGGG ATAAAGGGAAGTGGAAAAAGTGGTGGTGGTGTTTTGGTTGGGTGAGGGG TGGATGGGGGGTGGGGTGGGGAAAGAGGAGAGGGTTGATAGAGAAGTGG GGATGGTTGGGGGTATGGGGAAAATGAGGGGGGTAAGGGGAGGAGGGGT TGGGGTTTTGATGATATTTAATGAGGGAGTGATGGAGGGAGTGGGAGAG GAAGGGGGGGTGTAAAGGGGGATAGTGAGGAAAGGGGTGGGAGTATTTA GGGAAAGGGGGAAGAGTGTTAGGGATGGGGTGGGGGTATTGGGAAAGGA TGAGGGGGGGGGTGTGTGGAGGTAGGGAAAGGGATTTTTTGATGGAGGA TTTGGGGAGAGGGGGGAAGGGGTGGTGTTGATGGAGGGGGGGGTAGATG GGGGAAATAATATGGGTGGGGGTGGTGTGGGGTGGGGGGGGTTGATAGT GGAGGGGGGGGGAAGGATGGAGAGATTTGATGGAGGGATAGAGGGGGTG GTGATTAGGGGGGTGGGGTGATTGATTGGGGAGGGAGGAGATGATGAGA GTGGGGTGATTAGGATGGGGGTGGAGGATTGGGGTTAGGGGTTGGGTGA TGGGGGGTAGGGAGGGGGGATGATGGGTGAGAGGATTGATTGGGAGGAT GGGGTGGGTTTGAATATTGGGTTGATGGAGGAGATAGAGGGGGTAGGGG TGGGAGAGGGTGTAGGAGAGGGGATGGTTGGGATAATGGGAAGAGGGGA GGGGGTTAAAGTTGTTGTGGTTGATGAGGAGGATATGGTGGAGGATGGT GTGGTGATGGATGAGGTGAGGATGGAGAGGATGATGGTGGTGAGGGTTA AGGGGTGGAATGAGGAAGGGGTTGGGGTTGAGGAGGAGGAGAGGATTTT GAATGGGGAGGTGGGGGAAAGGGAGATGGGAGGGTTGTGGTTGAATGAG GGTGGGGTGGGGGGTGTGGAGTTGAAGGAGGGGAGGATAGAGATTGGGG ATTTGGGGGGTGGAGAGTTTGGGGTTTTGGAGGTTGAGAGGTAGTGTGA GGGGATGGGGATAAGGAGGAGGGTGATGGATAATTTGAGGGGGGAAAGG GGGGGTGGGGGTGGGGAGGTGGGTTTGAGGGTGGGATAAAGAAAGTGTT AGGGGTAGGTAGTGAGGGAAGTGGGGGGAGATGTGAAGTTGAGGGTGGA GTAGAGGGGGGGTGAAATGATGATTAAAGGGAGTGGGAAGATGGAAATG GGTGATTTGTGTAGTGGGTTTATGGAGGAAGGAGAGGTGAGGGAAAATG GGGGTGATGGGGGAGATATGGTGATGTTGGAGATAAGTGGGGTGAGTGG AGGGGAGGAGGATGAGGGGGAGGGGGTTTTGTGGGGGGGGTAAAAATGG GGTGAGGTGAAATTGAGAGGGGAAAGGAGTGTGGTGGGGGTAAGGGAGG GAGGGGGGGTTGGAGGAGAGATGAAAGGGGGAGTTAAGGGGATGAAAAA TAATTGGGGTGTGGGGTTGGTGTAGGGAGGTTTGATGAAGATTAAATGT GAGGGAGTAAGAAGGGGTGGGATTGTGGGTGGGAAGAAAGGGGGGATTG AGGGTAATGGGATAGGTGAGGTTGGTGTAGATGGGGGGATGGTAAGGGT GGATGTGGGAGTTTGAGGGGAGGAGGAGAGTATGGGGGTGAGGAAGATG GGAGGGAGGGAGGTTTGGGGGAGGGGTTGTGGTGGGGGAAAGGAGGGAA AGGGGGATTGGGGATTGAGGGTGGGGAAGTGTTGGGAAGGGGGATGGGT GGGGGGGTGTTGGGTATTAGGGGAGGTGGGGAAAGGGGGATGTGGTGGA AGGGGATTAAGTTGGGTAAGGGGAGGGTTTTGGGAGTGAGGAGGTTGTA AAAGGAGGGGGAGTGAATGGGTAATGATGGTGATAGTAGGTTTGGTGAG GTTGTGAGTGGAAAATAGTGAGGTGGGGGAAAATGGAGTAATAAAAAGA GGGGTGGGAGGGTAATTGGGGGTTGGGAGGGTTTTTTTGTGTGGGTAAG TTAGATGGGGGATGGGGGTTGGGGTTATTAAGGGGTGTTGTAAGGGGAT GGGTGGGGTGATATAAGTGGTGGGGGTTGGTAGGTTGAAGGATTGAAGT GGGATATAAATTATAAAGAGGAAGAGAAGAGTGAATAAATGTGAATTGA TGGAGAAGATTGGTGGAGGGGGTGATATGTGTAAAGGTGGGGGTGGGGG TGGGTTAGATGGTATTATTGGTTGGGTAAGTGAATGTGTGAAAGAAGG

(102) The inserted lacZ.sup.+ cassette not only knocks out lon, but also converts the lacZ.sup.− host back to both a lacZ.sup.+ genotype and phenotype. The modified strain produces a minimal (albeit still readily detectable) level of β-galactosidase activity (1-2 units), which has very little impact on lactose consumption during production runs, but which is useful in removing residual lactose at the end of runs, and as an easily scorable phenotypic marker for moving the lon mutation into other lacZ.sup.− E. coli strains by P1 transduction.

(103) Fourth: A thyA (thymidylate synthase) mutation was introduced into the strain by P1 transduction. In the absence of exogenous thymidine, thyA strains are unable to make DNA and die. The defect can be complemented in trans by supplying a wild-type thyA gene on a multicopy plasmid (Belfort et al., 1983). This complementation is used here as a means of plasmid maintenance.

(104) An additional modification that is useful for increasing the cytoplasmic pool of free lactose (and hence the final yield of 3-FL) is the incorporation of a lacA mutation. LacA is a lactose acetyltransferase that is only active when high levels of lactose accumulate in the E. coli cytoplasm. High intracellular osmolarity (e.g., caused by a high intracellular lactose pool) can inhibit bacterial growth, and E. coli has evolved a mechanism for protecting itself from high intra cellular osmolarity caused by lactose by “tagging” excess intracellular lactose with an acetyl group using LacA, and then actively expelling the acetyl-lactose from the cell (Danchin, A. Bioessays 31, 769-773 (2009)). Production of acetyl-lactose in E. coli engineered to produce 3-FL or other human milk oligosaccharides is therefore undesirable: it reduces overall yield. Moreover, acetyl-lactose is a side product that complicates oligosaccharide purification schemes. The incorporation of a lacA mutation resolves these problems. Sub-optimal production of fucosylated oligosaccharides occurs in strains lacking either or both of the mutations in the colanic acid pathway and the lon protease. Diversion of lactose into a side product (acetyl-lactose) occurs in strains that do not contain the lacA mutation. A schematic of the lacA deletion and corresponding genomic sequence is provided above.

(105) The strain used in the Examples to test the different α(1,3) FT candidates incorporates all the above genetic modifications and has the following genotype:

(106) ΔampC::P.sub.trp.sup.B cI, Δ(lacI-lacZ)::FRT, P.sub.lacIqlacY.sup.+, ΔlacA, ΔwcaJ::FRT, thyA::Tn10, Δlon::(npt3, lacZ.sup.+)

(107) The strains engineered as described above to produce the desired fucosylated oligosaccharide(s) are grown in a minimal media. An exemplary minimal medium used in a bioreactor, minimal “FERM” medium, is detailed below.

(108) Ferm (10 liters): Minimal medium comprising:

(109) 40 g (NH.sub.4).sub.2HPO.sub.4

(110) 100 g KH.sub.2PO.sub.4

(111) 10 g MgSO.sub.4.7H.sub.2O

(112) 40 g NaOH

(113) 1× Trace elements:

(114) 1.3 g NTA (nitrilotriacetic acid)

(115) 5 g FeSO.sub.4.7H.sub.2O

(116) 0.09 g MnCl.sub.2.4H.sub.2O

(117) 0.09 g ZnSO.sub.4.7H.sub.2O

(118) 0.01 g CoCl.sub.2.6H.sub.2O

(119) 0.01 g CuCl.sub.2.2H.sub.2O

(120) 0.02 g H.sub.3BO.sub.3

(121) 0.01 g Na.sub.2MoO.sub.4.2H.sub.2O (pH 6.8)

(122) Water to 10 liters

(123) DF204 antifoam (0.1 ml/L)

(124) 150 g glycerol (initial batch growth), followed by fed batch mode with a 90% glycerol-1% MgSO.sub.4-1× trace elements feed, at various rates for various times.

(125) Bacteria comprising the characteristics described herein are cultured in the presence of lactose, and a fucosylated oligosaccharide is retrieved, either from the bacterium itself or from a culture supernatant of the bacterium. The fucosylated oligosaccharide is purified for use in therapeutic or nutritional products, or the bacteria are used directly in such products.

(126) Post-Fermentation Purification

(127) Fucosylated oligosaccharides produced by metabolically engineered E. coli cells are purified from culture broth post-fermentation. An exemplary procedure comprises five steps. (1) Clarification: Fermentation broth is harvested and cells removed by sedimentation in a preparative centrifuge at 6000×g for 30 min. Each bioreactor run yields about 5-7 L of partially clarified supernatant. (2) Product capture on coarse carbon: A column packed with coarse carbon (Calgon 12×40 TR) of ˜1000 ml volume (dimension 5 cm diameter×60 cm length) is equilibrated with 1 column volume (CV) of water and loaded with clarified culture supernatant at a flow rate of 40 ml/min. This column has a total capacity of about 120 g of sugar. Following loading and sugar capture, the column is washed with 1.5 CV of water, then eluted with 2.5 CV of 50% ethanol or 25% isopropanol (lower concentrations of ethanol at this step (25-30%) may be sufficient for product elution.) This solvent elution step releases about 95% of the total bound sugars on the column and a small portion of the color bodies. In this first step capture of the maximal amount of sugar is the primary objective. Resolution of contaminants is not an objective. (3) Evaporation: A volume of 2.5 L of ethanol or isopropanol eluate from the capture column is rotary-evaporated at 56 C.° and a sugar syrup in water is generated. Alternative methods that could be used for this step include lyophilization or spray-drying. (4) Flash chromatography on fine carbon and ion exchange media: A column (GE Healthcare HiScale50/40, 5×40 cm, max pressure 20 bar) connected to a Biotage Isolera One FLASH Chromatography System is packed with 750 ml of a Darco Activated Carbon G60 (100-mesh): Celite 535 (coarse) 1:1 mixture (both column packings were obtained from Sigma). The column is equilibrated with 5 CV of water and loaded with sugar from step 3 (10-50 g, depending on the ratio of 3-FL to contaminating lactose), using either a celite loading cartridge or direct injection. The column is connected to an evaporative light scattering (ELSD) detector to detect peaks of eluting sugars during the chromatography. A four-step gradient of isopropanol, ethanol or methanol is run in order to separate 3-FL from monosaccharides (if present), lactose and color bodies. Fractions corresponding to sugar peaks are collected automatically in 120-ml bottles, pooled and directed to step 5. In certain purification runs from longer-than-normal fermentations, passage of the 3-FL-containing fraction through anion-exchange and cation exchange columns can remove excess protein/DNA/caramel body contaminants. Resins tested successfully for this purpose include Dowex 22.

(128) The gene screening approach described herein was successfully utilized to identify new α(1,3) FTs for the efficient biosynthesis of 3FL and other α(1,3) fucosylated oligosaccharides in metabolically engineered E. coli host strains. The results of the screen are summarized in Tables 1 and 4.

(129) A directed screening approach was used to identify and characterize alternative bacterial α(1,3) FTs with different and desirable properties, (e.g. possessing higher specific activity, higher expression level, lower cellular toxicity, higher protease stability and/or different acceptor substrate specificity) that are useful for the large scale production of α(1,3)-linked fucosylated oligosaccharides. Specifically, the enzymes CafC, CafL, CafN, CafO, CafQ, CafU and CafV have utility for the production of 3FL and LDFT, two HMOS that are abundant in human milk that possess important and useful therapeutic properties. In addition, CafD is capable of promoting synthesis of LNF III, an HMOS that possesses the bona fide Le.sup.x epitope that is likely to possess therapeutic properties similar to that of 3FL and LDFT. The Le.sup.x epitope is involved in a myriad of biological recognition processes, and the ability to produce molecules containing this epitope on large-scale is useful as a tool to elucidate their modes of action (McEver et al., 1995; McEver and Cummings, 1997).

Example 1: α(1,3) Fucosyltransferase Expression in E. coli

(130) The strain used to test the different α(1,3) FT candidates incorporates all the above genetic modifications and has the following genotype:

(131) ΔampC::P.sub.trp.sup.BcI, Δ(lacI-lacZ)::FRT, P.sub.lacIqlacY.sup.+, ΔlacA, ΔwcaJ::FRT, thyA::Tn10, Δlon::(npt3, lacZ.sup.+)

(132) The E. coli strains harboring the different α(1,3) FT candidate expression plasmids were analyzed in small-scale experiments. Strains were grown in selective media (lacking thymidine) to early exponential phase. Lactose was then added to a final concentration of 1%, and tryptophan (200 μM) was added to induce expression of each candidate α(1,3) FT from the P.sub.L promoter. At the end of the induction period (˜20 h) equivalent OD 600 units of each strain were harvested. Lysates were prepared and analyzed for the presence of 3FL by thin layer chromatography (TLC). As shown in FIG. 4A-C, a control strain producing FutA was capable of the biosynthesis of 3FL and also produced a smaller amount of the tetrasaccharide lactodifucotetraose (LDFT). Interestingly, the strains producing CafA, CafC and CafF synthesized a significant amount of 3FL as compared to the control strain producing FutA. Specifically, the strain producing CafA synthesized approximately ˜50% as much 3FL compared to the control strain, but produced significantly more LDFT (FIG. 4A). Importantly, CafC and CafF reproducibly catalyzed the formation of greater levels of 3FL as compared to FutA (FIGS. 4A and 4B). Strains producing CafC and CafF also secreted a significant amount of 3FL into the culture supernatant. CafB was also able to catalyze the biosynthesis of 3FL, although at levels significantly less than that of the FutA control strain. Polypeptides of the predicted molecular weight for CafA, B, C and F were detected in protein lysates of the respective strains by SDS-PAGE analysis, indicating these proteins are robustly synthesized in our E. coli production strain (FIG. 5A-C). Thus, CafA, CafC and CafF are α(1,3) FTs that are useful for the large-scale production of fucosylated oligosaccharides. CafC and CafF are of particular interest, as strains synthesizing these enzymes routinely produced greater levels of 3FL as compared to the FutA control strain (Table 1). Of note, the remaining candidates (CafD, E, G, H, I, J and K) were unable to utilize lactose as an acceptor for the production of 3FL, despite the observation that most of these enzymes were robustly synthesized in E. coli. Therefore, the fact that only 3 of the 11 candidates tested were able to synthesize 3FL in the engineered E. coli strain indicates the uniqueness and surprising aspect of these findings.

(133) In a related aspect of the invention, the bacterial production strain may harbor an expression plasmid containing two or more different α(1,3) fucosyltransferases in a “tandem” or “stringed” arrangement under control of a promoter, e.g., a fortuitous promoter. A relatively low level of constitutive expression of 2 different α(1,3) fucosyltransferases was found to yield a net increase of enzyme activity without a drawback of undesirable or unacceptable cell toxicity has been observed with high, e.g., inducible/induced, expression of a single heterologous α(1,3) fucosyltransferase. An exemplary promoter comprises the P.sub.L promoter (e.g. pG420 shown in FIG. 21.) SEQ ID NO: 64 below provides the nucleic acid sequence for the pG420 expression plasmid.

(134) TABLE-US-00011 (SEQ ID NO: 64) caagaaggagatataCATATGAAGACCATCAAGGTAAAATTCGTCGATT TCTGGAAAGGTTTCGACCCGCGCAACAACTTCCTGATGGACATCCTGAA ACAGCGTTATCACATTGAACTGAGCGAAAGCCCGGACTACCTGATCTTC TCTGTCTTCGGTTTCACTAACCTGAACTACGAACGCTGCGTTAAAATCT TCTACACCGGTGAAAACCTGACCCCGGATTTCAACATCTGCGACTACGC GATTGGTTTCGATTATCTGAGCTTCGGTGATCGTTACATGCGTCTGCCA CTGTACGCGGTCTATGGCATCGAGAAACTGGCTTCTCCGAAAGTTATCG ACAAAGAAAAAGTTCTGAAGCGTAAATTCTGTTCTTACGTAGTAAGCAA TAACATCGGCGCGCCGGAACGTTCTCGTTTCTTCCATCTGCTGTCTGAA TACAAAAAGGTTGACTCCGGTGGTCGTTGGGAAAACAACGTAGGCGGTC CGGTTCCGAATAAGCTGGACTTTATCAAAGACTACAAGTTCAACATCGC ATTCGAAAACTCCATGTACGACGGCTACACTACTGAAAAAATCATGGAA CCGATGCTGGTGAACAGCCTGCCGATTTATTGGGGCAACCGCCTGATCA ACAAAGACTTCAACCCAGCGTCTTTCATCAACGTTTCCGATTTCCCGTC TCTGGAAGCGGCGGTGGAGCACATTGTTATGCTGGACAATAACGATGAT ATGTACCTGAGCATCCTGTCTAAACCGTGGTTTAACGATGAAAACTACC TGGACTGGAAAGCGCGCTTCTTCCACTTTTTCGATAACATCTTCAATCG TCCGATCGATGAATGCAAATATCTGACCCCGTACGGCTTTTGTCGTCAC TATCGTAACCAACTGCGTAGCGCTCGTCTGCTGAAACAGCGCTTTCGCC AGCTGCGTAACCCGCTGCGCTGGTTCCGCTAGtagcTCGAGCTGCAGTA ATCGTACAGGGTAGTACAAATAAAAAAGGCACGTCAGATGACGTGCCTT TTTTCTTGTGAGCAGTaagcttCTACGAACATCTTCCAGGATACTCCTG CAGCGAAATATTTGTTTTAAGCTCACTCACATATCGCAACATTTACTTT ACTTTAAGACAATTCCAGGCAAATTATACAACACTTTACGGGATAGTAA GTCCGCCTGAAAAATCGCGAGAGTGGCGCATTAGGTGACCCATGTTGTT CCGTTTAGTCATGATGAAATATTCAGGTAAGGGGAATTATCGTTACGCA TTGAGTGAGGGTATGCCATGTCAACGATTATTATGGATTTATGTAGTTA CACCCGACTAGGTTTAACCGGGTATCTGTTGAGTAGAGGGGTTAAAAAA AGAGAAATCAACGACATTGAAACCGTTGATGACCTTGCCATAGCTTGTG ATTCACAGCGCCCTTCAGTGGTGTTTATTAATGAGGACTGTTTCATCCA CGATGCTTCTAACAGTCAGCGTATCAAGCTCATCATTAATCAACATCCC AATACGTTATTTATCGTTTTTATGGCAATTGCCAATGTTCATTTTGATG AATATCTATTGGTCAGAAAAAATTTATTGATCAGTTCTAAATCGATTAA ACCGGAATCTCTCGACGATATCCTTGGCGATATTCTGAAAAAAGAGACA ACGATAACCTCGTTTTTAAATATGCCGACGTTATCATTGAGCCGAACCG AATCGAGTATGTTGCGAATGTGGATGGCAGGTCAGGGAACCATTCAAAT CTCTGACCAAATGAATATCAAAGCCAAGACCGTTTCATCGCATAAAGGT AATATTAAACGTAAGATCAAAACGCATAATAAACAGGTTATCTACCATG TCGTCCGACTGACGGATAATGTGACTAATGGTATTTTTGTCAACATGCG CTAACACATTCTGACTGGTGGTTTCCCACCAGTCAGGCTGAATAAGATT ACTCTGCTTTCTCCACAAAGATACCGTCCTGATGCCCTGCTTCATTAAA GAAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACC CTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAG CTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTG CGCAGCCTGAATGGCGAATGGCGCCTTCGGGAAGGCGTCTCGAAGAATT TAACGGAGGGTAAAAAAACCGACGCACACTGGCGTCGGCTCTGGCAGGA TGTTTCGTAATTAGATAGCCACCGGCGCTTTAATGCCCGGATGCGGATC GTAGCCTTCAATCTCAAAGTCTTCGAAACGGTAGTCGAAGATGGATTCG GGTTTACGTTTGATAATCAACTTCGGCAGCGGACGCGGTTCGCGGCTTA ATTGCAGATGAGTTTGATCCATATGGTTGCTGTACAGATGCGTGTCGCC ACCGGTCCAGACAAAATCACCCACTTCCAGATCGCACTGCTGCGCCATC ATATGCACCAATAACGCGTAGCTGGCAATGTTGAACGGCAGGCCGAGGA AGACGTCACAGGAGCGCTGATAAAGCTGGCAAGAGAGTTTGCCGTCTGC CACATAGAACTGGAAGAATGCATGGCACGGTGCCAGCGCCATTTTATCC AGTTCGCCTACGTTCCACGCTGAAACAATAATGCGGCGCGAATCCGGGT CGTTTTTCAGCTGGTTCAGTACCGTAGTGATCTGGTCAATATGACGACC ATCTGGCGTTGGCCAGGCGCGCCACTGTTTACCATACACTGGCCCGAGG TCGCCGTTTTCATCGGCCCATTCGTCCCAGATGGTGACATTGTTTTCGT GTAGATAAGCAATGTTAGTGTCGCCCTGCAGAAACCACAGCAGTTCATG GATGATGGAACGCAGGTGGCAACGTTTAGTTGTCACCAGCGGGAATCCA TCTTGCAGGTTAAAACGCATCTGATGACCAAAAATGGAAAGCGTTCCGG TTCCGGTACGGTCGTTTTTCTGTGTGCCTTCGTCGAGCACTTTTTGCAT CAGTTCTAAATACTGTTTCATGGTTCCTCAGGAAACGTGTTGCTGTGGG CTGCGACGATATGCCCAGACCATCATGATCACACCCGCGACAATCATCG GGATGGAAAGAATTTGCCCCATGCTGATGTACTGCACCCAGGCACCGGT AAACTGCGCGTCGGGCTGGCGGAAAAACTCAACAATGATGCGAAACGCG CCGTAACCAATCAGGAACAAACCTGAGACAGCTCCCATTGGGCGTGGTT TACGAATATACAGGTTGAGGAGGCGCCTGATGCGGTATTTTCTCCTTAC GCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAA TCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACC CGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGA CAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGT CATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTT ATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACT TTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATAC ATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCA ATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAG AAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGT GGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTT CGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTAT GTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCG CCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACA GAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTG CCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGAT CGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCAT GTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAA ACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCG CAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTA ATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGG CCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCG TGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCC CGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAAC GAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTA ACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTT CATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCA TGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCC CGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTA ATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTT TGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG CAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGC CACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAA TCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGG GTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGA ACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCG AACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGA AGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGA GAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTC CTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTC GTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGT TATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGA TACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAG GAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGC CGATTCATTAATGCAGAATTGATCTCTCACCTACCAAACAATGCCCCCC TGCAAAAAATAAATTCATATAAAAAACATACAGATAACCATCTGCGGTG ATAAATTATCTCTGGCGGTGTTGACATAAATACCACTGGCGGTGATACT GAGCACATCAGCAGGACGCACTGACCACCATGAAGGTGACGCTCTTAAA AATTAAGCCCTGAAGAAGGGCAGCATTCAAAGCAGAAGGCTTTGGGGTG TGTGATACGAAACGAAGCATTGGCCGTAAGTGCGATTCCGGATTAGCTG CCAATGTGCCAATCGCGGGGGGTTTTCGTTCAGGACTACAACTGCCACA CACCACCAAAGCTAACTGACAGGAGAATCCAGATGGATGCACAAACACG CCGCCGCGAACGTCGCGCAGAGAAACAGGCTCAATGGAAAGCAGCAAAT CCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCCGAAAGATTTTTTTA ACTATAAACGCTGATGGAAGCGTTTATGCGGAAGAGGTAAAGCCCTTCC CGAGTAACAAAAAAACAACAGCATAAATAACCCCGCTCTTACACATTCC AGCCCTGAAAAAGGGCATCAAATTAAACCACACCTATGGTGTATGCATT TATTTGCATACATTCAATCAATTtTTAGAAttcTAGaAAGAAGGAGATA TACATATGAAAACTATCAAAGTTAAATTCGTTGATTTCTGGGAAAACTT CGACCCGCAACACAACTTTATTGCAAACATTATCAGCAAAAAATACCGT ATCGAACTGTCCGATACCCCAGACTATCTGTTCTTTTCCGTGTTCGGTT ATGAAAACATCGACTACCATAACTGCACCAAAATCTTCTACTCTGGTGA AAACATTACTCCGGACTTCAACATTTGTGACTATGCAATTGGTTTCAAC TTCCTGTCCTTTGGTGACCGTTATATCCGTATCCCATTTTATACCGCGT ACGGTGTGCAGCAGCTGGCCGCGCCAAAAGTAATCGTTCCGGAAGTTGT TCTGAATCGTAAGTTCTGTAGCTTCGTTGTATCTAATGCCAAGGGCGCT CCGGAGCGCGAGCGTTTCTTCCAACTGCTGAGCGAATACAAACAGGTGG ACTCTGGCGGTCGTTACAAAAATAACGTTGGCGGTCCGGTACCAGATAA AACTGCATTTATCAAAGACTACAAATTCAACATTGCGTTCGAAAACTCC ATGTGCGACGGTTACACCACGGAAAAAATCATGGAACCTATGCTGGTCA ATTCCGTTCCAATTTACTGGGGTAACAAACTGATCGACCGTGACTTTAA CCCGGACTCCTTCATTAATGTATCCTCTTATTCTTCTCTGGAAGAAGCA GTTGAGCACATCGTCCGTCTGGATCAGAATGATGACGAATACCTGAGCC TGCTGTCCGCCCCGTGGTTCAACGAGGAAAACTACCTGAACTGGGAAGA ACAGCTGATCACTTTCTTCGACAACATCTTCGAAAAACCGCTGTCTGAA TCCCGTTATATCCCAACCCACGGTTACATCCAGACCTATCAGTACCGCC TGCATCGTATGATGCGTGATAAACTGTTCCGTAAACGTATCAACCCGCT GAAATGGTTTTCTTCTAAGTAA

Example 2: Synthesis of LDFT

(135) CafA, C and F were tested for utilization in combination with an α(1,2) fucosyltransferase produced in the same strain to catalyze the synthesis of Lactodifucotetraose (LDFT) (FIG. 7). The genes encoding CafA, C and F were inserted into plasmid pG297 (harboring wbgL encoding an α(1,2) fucosyltransferase from E. coli 0126) using standard molecular biology techniques. Thus, a series of “mini-operons” consisting of wbgL in combination with cafA, cafC or cafF under control of the P.sub.L promoter were constructed. The resulting plasmids were then transformed into an engineered E. coli production strain.

(136) The E. coli strains harboring the different LDFT expression plasmids were analyzed in small-scale experiments. Strains were grown in selective media (lacking thymidine) to early exponential phase. Lactose was then added to a final concentration of 1%, and tryptophan (200 μM) was added to induce expression of the α(1,2) and α(1,3) FTs from the P.sub.L promoter. At the end of the induction period (˜20 h) equivalent OD 600 units of each strain were harvested. Cell lysates were prepared and analyzed for the presence of intracellular LDFT by thin layer chromatography (TLC). As shown in FIG. 8, a control strain producing only the α(1,2) FT WbgL synthesized primarily 2′-FL and a relatively small amount of LDFT. In comparison, a strain producing WbgL in combination with the α(1,3) FT FutA or CafA synthesized an estimated 20-30% more LDFT. Strains producing WbgL in combination with CafC or CafF synthesized significantly more LDFT than strains producing WbgL alone or WbgL in combination with FutA. This effect was particularly pronounced for the WbgL plus CafF combination. Furthermore, we observed significant amounts of LDFT in the culture supernatant for the WbgL plus CafC and WbgL plus CafF combinations (data not shown) (Table 1). Therefore, these observations indicate that CafA, CafC and CafF will be useful for the large-scale synthesis of LDFT, another HMOS with high potential therapeutic value.

Example 3: Expression of LNF III

(137) The majority of the α(1,3) FT candidates tested from the first database screen (CafD, E, G, H, I, J, K) were unable to utilize lactose as a donor substrate and could not promote the synthesis of 3FL, despite the fact that most of these enzymes were well-expressed in E. coli (Table 1). One explanation for this observation is that some bacterial and higher eukaryotic α(1,3) FTs prefer N-acetylglucosamine (GlcNAc) rather than glucose (Glc) as an acceptor for the attachment of fucose (Breton, C., et al. (1998). Conserved structural features in eukaryotic and prokaryotic fucosyltransferases. Glycobiology 8, 87-94.; Ma, B., et al. (2003). C-terminal amino acids of Helicobacter pylori alpha 1,3/4 fucosyltransferases determine type I and type II transfer. J Biol Chem 278, 21893-1900.; Ma, B., et al. (2006). Fucosylation in prokaryotes and eukaryotes. Glycobiology 16, 158R-184R.). Therefore, studies were carried out to determine whether CafD, E, G, H, I, J or K catalyze the attachment of fucose to a GlcNAc moiety present within the HMOS LNnT (Lacto-N-neotetraose) to generate a fucosylated oligosaccharide found in human milk termed LNF III (Lacto-N-fucopentaose) (FIG. 9). To this end, these candidate α(1,3) FT genes were inserted into plasmid pG222 using standard molecular biology techniques. pG222 harbors genes encoding a β(1,3) N-acetylglucosaminyltransferase (lgtA) from N. meningitidis (Genbank Accession NP 274923.1) and a β(1,4) galactosyltransferase (JHP0765) from H. pylori (Genbank Accession NP_207619.1). In an alternative embodiment, Helicobacter pylori β(1,3) N-acetylglucosaminyltransferase JHP0563, (Genbank Accession YP_002301261.1) could be used. In another example, Neisserria meningitidis β(1,4) galactosyltransferase LgtB, (Genbank Accession NP_274922.1) could be used.

(138) LgtA catalyzes the attachment of GlcNAc to the galactose in lactose to produce Lacto-N-triose (LNT2), a precursor of many HMOS that has the structure GlcNAcβ1-3Galβ1-4Glc. JHP0765 (a β(1,4) galactosyltransferase) can then utilize LNT2 as an acceptor to generate LNnT, an abundant HMOS of human milk. LNnT has the structure Galβ1-4GlcNacβ1-3Galβ1-4Glc and is an important Bifidogenic prebiotic factor in human milk (Marcobal, A., et al. (2010). Consumption of human milk oligosaccharides by gut-related microbes. J Agric Food Chem 58, 5334-340.; Garrido, D., et al. (2012). A molecular basis for bifidobacterial enrichment in the infant gastrointestinal tract. Adv Nutr 3, 415S-421S.; Sela, D. A., et al. (2012). Bifidobacterium longum subsp. infantis ATCC 15697 α-fucosidases are active on fucosylated human milk oligosaccharides. Appl Environ Microbiol 78, 795-803.). Attachment of fucose in an α1,3 linkage to the GlcNAc in LNnT generates LNF III, another HMOS found in human milk.

(139) Derivatives of plasmid pG222 harboring each α(1,3) FT candidate were transformed into the E. coli production strain using standard techniques. The E. coli strains harboring the different LNF III expression plasmids were then analyzed in small-scale experiments. Strains were grown in selective media (lacking thymidine) to early exponential phase. Lactose was then added to a final concentration of 1%, and tryptophan (200 μM) was added to induce expression of the glycosyltransferases. At the end of the induction period (˜20 h) equivalent OD 600 units of each strain were harvested. Cell lysates were prepared and analyzed for the presence of intracellular LNF III by thin layer chromatography (TLC). As shown in FIG. 10, a strain producing both LgtA and JHP0765 synthesized LNnT as well as a larger oligosaccharide, e.g., having the structure Galβ1-4GlcNacβ1-3Galβ1-4GlcNacβ1-3Galβ1-4Glc (Lacto-N-neohexaose). Of the 7 α(1,3) FTs tested only CafD was capable of catalyzing the attachment of fucose to LNnT (FIG. 10, see lanes 3 and 4). Liquid chromatography coupled with mass spectrometry revealed that this fucosylated molecule possessed a mass consistent with that of LNF III indicating that CafD catalyzes the biosynthesis of bona fide LNF III in our E. coli production strain.

Example 4: α(1,3) Fucosyltransferases in Tandem or in a String Configuration

(140) Bacterial strains were constructed that harbor an expression plasmid containing two different α(1,3) fucosyltransferases in a “tandem” arrangement or in a string (three or more genes) configuration under control of the P.sub.L promoter. FIG. 21 provides a map of such a plasmid, pG420 (nucleic acid sequence SEQ ID NO: 64), that carries genes encoding two different α(1,3) fucosyltransferases; CafC (amino acid sequence SEQ ID NO: 2) and CafN (amino acid sequence SEQ ID NO: 44), arranged in an operon driven from the P.sub.L promoter.

(141) FIG. 22A-B demonstrates enhanced fermentor production of 3-fucosyllactose using an expression plasmid expressing dual α(1,3) fucosyltransferases. Specifically, FIG. 22A shows thin layer chromatography analysis of culture supernatants from fermentation run 126. In this experiment, an engineered E. coli production strain harboring plasmid pG366 (pEC2-P.sub.L-cafC-rcsA-thyA) was grown under fed-batch conditions with a defined linear lactose feed (50 g final lactose added per liter initial culture volume). A significant amount of 3-FL was produced under these conditions and exported to the culture medium. At the end of the process, the cells were heated at 65° C. for 20 minutes to release any remaining intracellular 3-FL to the culture medium. Analysis of product yield by HPLC in the final sample revealed that ˜7.5 g/L 3-FL was produced under these conditions. Surprisingly, the yield of 3-FL could be improved to ˜15 g/L when a second α(1,3) fucosyltransferase (cafN) was introduced into the parental plasmid pG366 to generate pG420 (pEC2-P.sub.L-cafC-cafN-rcsA-thyA, SEQ ID NO: 64) (FIG. 22B), and the cells were grown under the same fed-batch process regimen.

(142) Cellular toxicity and consequent lowered product yields were observed in 3-FL bioreactor runs such strains expressing high levels of α(1,3) fucosyltransferases driven by the fully-induced P.sub.L promoter. However, by keeping the P.sub.L promoter repressed (e.g. by eliminating the addition of tryptophan to the culture and relying on the low-level of constitutive transcription that originates from the promoter region) and by constructing a tandem arrangement of the α(1,3) fucosyltransferases CafC and CafN downstream of the promoter, the culture maintains good viability for the duration of the run and 3-FL yields are significantly improved.

Example 5: Enhanced Fermentor Production of 3-Fucosyllactose Using Casamino Acid Supplementation (CAA)

(143) High level expression (e.g. as driven from the induced P.sub.L promoter) of nearly all α(1,3) fucosyltransferases tested to date can be toxic to E. coli production strains, resulting in poor viability and low 3-FL yields in fermentation runs. One explanation is that many α(1,3) fucosyltransferases may possess an off-target activity in which an endogenous E. coli molecule essential for cell viability is inappropriately fucosylated rendering it non-functional and/or toxic. Of note, some α(1,3) fucosyltransferases have been shown to use N-acetylglucosamine as an acceptor. Therefore, the identity of the secondary endogenous E. coli target may be a molecule containing N-acetylglucosamine, such as the lipid II precursor for cell wall peptidoglycan. Thus, cells producing high levels of α(1,3) fucosyltransferase activity displayed aberrant cell envelope morphology (swelling, membrane blebbing), suggesting a defect in cell wall/membrane structure or biogenesis. Interestingly, supplementation of fermentation media with a nitrogen-rich additive such as casamino acids (CAA) or yeast extract (YE) protected against the toxic properties of α(1,3) fucosyltransferase activity, leading to significantly improved 3-FL production yields. In particular, CAA supplementation increased, e.g., doubled, the yield of 3FL obtained in fermentation runs. This yield-boosting activity is associated with any rich nutritional additive containing amino acids, peptides, minerals, vitamins, and other micronutrients. In addition to CAA and YE, such additives may include any protein hydrolysate (e.g., peptone) from a variety of sources, including but not limited to meat, casein, whey, gelatin, soybean, yeast and grains.

(144) FIG. 22C demonstrates enhanced fermentor production of 3-fucosyllactose using casamino acid supplementation (CAA). Specifically, an engineered E. coli production strain harboring plasmid pG420 (pEC2-P.sub.L-cafC-cafN-rcsA-thyA, SEQ ID NO: 64) was grown under identical conditions as described above in relation to FIGS. 22A-B, except 50 g final CAA was added per liter initial culture volume and delivered in a linear feed over the course of the run. The addition of CAA significantly boosted product formation, resulting in ˜30 g/L 3-FL as assessed by HPLC.

OTHER EMBODIMENTS

(145) While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

(146) The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

(147) While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Alpha (1,3) fucosyltransferases for use in the production of fucosylated oligosaccharides

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Abstract

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