# Patent

## Title
Bifidobacterial gene sequences and their use

## Abstract

This invention provides nucleic acids and proteins involved in oligosaccharide modification in the species Bifidobacteria. The invention provides methods for utilizing the proteins of the invention to generate human milk oligosaccharides or oligosaccharide mimics. The invention also provides compositions containing the human milk oligosaccharides or oligosaccharide mimics and methods for use.

## Background
CROSS-REFERENCES TO RELATED APPLICATIONS
The present application is the U.S. National Stage entry of International Application No. PCT/US2007/020032, filed Sep. 14, 2007, which claims priority to U.S. Application No. 60/845,130, filed Sep. 15, 2006, the disclosures of each are herein incorporated by reference in their entirety.


STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
NOT APPLICABLE
BACKGROUND OF THE INVENTION
The adult human has about 1014 microorganisms that normally reside in the digestive tract called the gut flora or intestinal bacteria. The bacteria that make up the normal flora of the digestive tract have a symbiotic or mutualistic relationship with their human hosts by performing a variety of beneficial functions such as breaking down and aiding in the absorption of otherwise undigestible food, stimulating cell growth, repressing the growth of harmful bacteria, training the immune system to respond to pathogens, and defending against some diseases.
As an important example, without the gut flora, humans would be unable to break down and use some of the carbohydrates they consume, because some species of intestinal bacteria have enzymes that human cells lack for breaking down polysaccharides. As another example, intestinal bacteria play an important role in promoting the early development and later functioning of the gut's mucosal immune system. These bacteria stimulate lymphoid tissue associated with the gut mucosa to produce antibodies to pathogens. During this process, the immune system is trained to recognize harmful bacteria, while leaving helpful species of bacteria unaffected, a tolerance developed in infancy.
The human infant is born with a digestive tract essentially free of bacteria. However, soon after birth, bacteria begin colonizing an infant's digestive tract. The first bacteria to be established in the gut affect the immune response making it more favorable to their own survival and less favorable to competing species; thus the first bacteria to colonize the gut are important in determining the person's lifelong gut flora makeup.
Among the bacterial genera found in the gut are Bacteroides, Clostridium, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Bifidobacterium, Escherichia, and Lactobacillus, among others. Among these species, Bifidobacteria are noteworthy because it has recently been found that this genera of bacteria is responsible for digesting unique oligosaccharides found in human milk. Estimates indicate that about 40-97% of these oligosaccharides pass through the human infant digestive tract undigested. It has been suggested that these oligosaccharides found in human milk serve as a prebiotic: a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or the activity of one or a limited number of bacteria in the gastrointestinal tract. Thus, the one function of the oligosaccharides in human milk is to promote the colonization of the infant gut by beneficial bacteria such as Bifidobacteria. 
Cow's milk and commercially available formulas, however, are lacking the oligosaccharides found in human breast milk. Because of the widespread use of cow's milk and commercially available formulas in the feeding of new born infants, it would be beneficial to provide infants with a source of human milk oligosaccharides which are lacking in these forms of nourishment. Other classes of individuals such as the elderly or patients treated with certain antibiotics and others who have compromised gut flora would also benefit.
BRIEF SUMMARY OF THE INVENTION
A first embodiment of the invention provides an isolated nucleic acid encoding a polypeptide involved in oligosaccharide modification that hybridizes under high stringency conditions to a sequence that can be SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. In an aspect of the first embodiment, the invention provides an expression vector comprising a nucleic acid, which can be SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 under the control of an expression control sequence. In another aspect of the first embodiment, a host cell comprising an expression vector as described above is provided, in which the host cell can be, e.g., E. coli. 
A second embodiment of the invention provides polypeptides involved in oligosaccharide modification that have an amino acid sequence at least 90% identical to the amino acid sequence encoded by a nucleic acid that can be SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.
In a third embodiment, the invention provides a method of synthesizing human milk oligosaccharides or oligosaccharide mimics by contacting an oligosaccharide containing precursor with a polypeptide of a sequence at least 90% identical to the amino acid sequence encoded by a nucleic acid that can be SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22 and then isolating a modified oligosaccharide containing precursor. In various aspects of the third embodiment, the oligosaccharide containing precursor can be a carbohydrate, a glycoprotein, or glycolipid. In other aspects of this embodiment, the oligosaccharide containing precursor has a terminal sialic acid, fucose, or N-acetyllactosamine. Furthermore, the oligosaccharide containing precursor can be plant, animal, or human derived.
A fourth embodiment of the invention provides a human milk oligosaccharide or oligosaccharide mimic derived by contacting an oligosaccharide containing precursor with a polypeptide of amino acid sequence at least 90% identical to the amino acid sequence encoded by a nucleic acid that can be SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, and isolating the resultant modified oligosaccharide precursor. In various aspects of the fourth embodiment, a baby formula, baby food, or supplemented milk product containing the human oligosaccharide or oligosaccharide mimic is provided. The milk product of this aspect can be bovine or human derived.
A fifth embodiment of the invention provides a milk product enriched for human milk oligosaccharides or oligosaccharide mimics derived by contacting a starting milk product with a polypeptide of amino acid sequence at least 90% identical to the amino acid sequence encoded by a nucleic acid that can be SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22. In an aspect of this embodiment, the starting milk product is animal or human derived.
In a sixth embodiment of the invention, a method of promoting growth of Bifidobacteria in the gastrointestinal tract of a human by administering to the human a human milk oligosaccharide or mimic made by the methods of this invention is provided.
In a seventh embodiment of the invention, a method of promoting growth of Bifidobacteria in the gastrointestinal tract of an infant by administering to the infant a baby formula made by the methods of this invention is provided.
In a eighth embodiment of the invention, a method of promoting growth of Bifidobacteria in the gastrointestinal tract of a human by administering to the human the supplemented milk product of made by the methods of this invention is provided.
In an ninth embodiment of the invention, a method of promoting growth of Bifidobacteria in the gastrointestinal tract of a human by administering to the human the enriched milk product made by the methods of this invention is provided.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates SEQ ID NO: 1 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 2 illustrates SEQ ID NO: 2 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 3 illustrates SEQ ID NO: 18 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 4 illustrates SEQ ID NO: 19 4 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 5 illustrates SEQ ID NO: 2 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 6 illustrates SEQ ID NO: 8 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 7 illustrates SEQ ID NO: 9 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 8 illustrates SEQ ID NO: 10 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 9 illustrates SEQ ID NO: 11 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 10 illustrates SEQ ID NO: 12 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 11 illustrates SEQ ID NO: 13 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 12 illustrates SEQ ID NO: 14 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 13 illustrates SEQ ID NO: 15 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 14 illustrates SEQ ID NO: 21 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 15 illustrates SEQ ID NO: 22 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 16 illustrates SEQ ID NO: 16 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 17 illustrates SEQ ID NO: 17 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 18 illustrates SEQ ID NO: 3 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 19 illustrates SEQ ID NO: 4 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 20 illustrates SEQ ID NO: 5 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 21 illustrates SEQ ID NO: 6 percent homology alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 22 illustrates SEQ ID NO: 7 percent homology from alignments across several different databases that represent protein sequence, protein domain and motif identifiers, as well as protein functional patterns.
FIG. 23 illustrates the alignment of the protein encoded by SEQ ID NO:10 (SEQ ID NO:23) with the amino acid sequence of beta-hexosaminidase A from Bifidobacterium longum (SEQ ID NO:24). Consensus peptides=SEQ ID NOS:25-59.
FIG. 24 illustrates the alignment of the protein encoded by SEQ ID NO: 12 (SEQ ID NO:60) with the amino acid sequence of beta-galactosidase from Bifidobacterium adolescentis (SEQ ID NO:61). Consensus peptides=SEQ ID NOS:62-94.
FIG. 25 illustrates the alignment of the protein encoded by SEQ ID NO: 17 (SEQ ID NO:95) with the amino acid sequence of N-acetylmannosamine-6-phosphate 2-epimerase from Propionibacterium acnes (SEQ ID NO:96). Consensus peptides=SEQ ID NOS:97-107.
FIG. 26 illustrates the alignment of the protein encoded by SEQ ID NO: 4 (SEQ ID NO:108) with the amino acid sequence of dihydrodipicolinate synthase from Bifidobacterium longum (SEQ ID NO:109). Consensus peptides=SEQ ID NOS:110-113.
FIG. 27 illustrates the alignment of the protein encoded by SEQ ID NO: 6 (SEQ ID NO:114) with the amino acid sequence of dihydrodipicolinate synthase from Propionibacterium acnes (SEQ ID NO:115). Consensus peptides=SEQ ID NOS:116-129.



DETAILED DESCRIPTION
Definitions
The term Bifidobacteria and its adjectives refers to a genus of anaerobic bacteria having beneficial properties for humans. Bifidobacteria is one of the major strains of bacteria that make up the gut flora, the bacteria that reside in the gastrointestinal tract and have health benefits for their hosts. See, e.g., Guarner F and Malagelada J R., Gut flora in health and disease. Lancet, 361, 512-519 (2003) for a further description of Bifidobacteria in the normal gut flora.
The term human milk oligosaccharide (HMO) refers generally to a number of complex carbohydrates found in human milk. Among the monomers of milk oligosaccharides are D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAC), L-fucose (Fuc), and sialic acid [N-acetylneuraminic acid (NeuAc)]. Elongation may be achieved by attachment of GlcNAc residues linked in (β1-3 or (β1-4 linkage to a Gal residue followed by further addition of Gal in a β-1-3 or β-1-4 bond. Most HMOs carry lactose at their reducing end. From these monomers, a large number of core structures may be formed. Further variations may occur due to the attachment of lactosamine, Fuc, and/or NeuAc. See, e.g., Kunz, C. et al., Annual. Rev. Nutri., 20:699-722 (2000) for a further description of HMOs.
The term oligosaccharide mimic refers generally to any compound that is able mimic the physical, chemical, or physiological effects of complex carbohydrates found in human milk. Such effects may include, but are not limited, to serving as a carbon source or growth factor for microorganisms such as Bifidobateria, binding to receptors on cells, inhibition of bacterial cell binding to the mucosal surface of the gastrointestinal tract, promotion of development of the intestinal flora in infants, the elderly, or others with impaired intestinal flora, serving as a prebiotic nutrient, among other functions for HMOs known in the art.
A prebiotic or prebiotic nutrient is generally a non-digestible food ingredient that beneficially affects a host when ingested by selectively stimulating the growth and/or the activity of one or a limited number of bacteria in the gastrointestinal tract.
The terms “identical” or percent “identity,” in the context of two or more nucleic acids 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 (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.
General
The inventors have identified gene sequences that are associated with a bifidobacterial strain, Bifidobacterium longum bv. infantis. The gene sequences disclosed herein encode activities related to the catabolism of HMOs. Thus, the sequences of this invention encode proteins with the capacity to cleave and metabolize complex milk carbohydrates. Accordingly, these genes would provide a significant growth advantage to the cognate bifidobacterial strain for growth on milk oligosaccharides, or milk oligosaccharide mimics, and thus provide a means for selective bifidobacterial strain enrichment within animal gastrointestinal tracts. Among other uses, these gene sequences enable rationale screens for new bifidobacterial strains that can be selectively enriched through growth on milk oligosaccharides, or milk oligosaccharide mimics.
The proteins encoded by these gene sequences can also be used in the construction of HMO mimics by promoting the reverse reactions catalyzed by these catabolic enzymes. In particular, these enzymes can be used to synthesize particular oligosaccharide structures. For instance, once a complex oligosaccharide structure present in a biological sample, such as human breast milk, has been identified as having a beneficial use, these enzymes can be used to synthesize these structures from a variety of starting materials including lactose or other milk derived materials including simpler oligosaccharide structures or by decorating plant derived oligosaccharides.
Embodiments of the Invention
Gene Sequences Encoding HMO Catabolic Proteins
In view of the above, in one aspect, the present invention provides nucleic acids and their encoded proteins involved in the catabolism of HMOs. As demonstrated in greater detail below, a bioinformatics approach was used to identify gene sequences homologous to the sequences of genes known to be involved with oligosaccharide catabolism by searching a draft Bifidobacterium longum bv. infantis UCD272 (ATCC15697) genome sequence using the ClustalW program using known oligosaccharide catabolic genes as the query sequence. Genes (SEQ ID NO: 1-22) were identified based on their extent of homology to the known genes used as query sequences.
A number of programs are known in the art to determine extents of homology. Optimal alignment of sequences for comparison can use any means to analyze sequence identity (homology) known in the art, e.g., by the progressive alignment method termed “PILEUP” (see below); by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981); by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity method of Pearson (1988) Proc. Natl. Acad. Sci. USA 85: 2444; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.); ClustalW (CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif., described by Higgins (1988) Gene, 73: 237-244; Corpet (1988) Nucleic Acids Res. 16:10881-90; Huang (1992) Computer Applications in the Biosciences 8:155-65, and Pearson (1994) Methods in Molec. Biol. 24:307-31), TreeAlign, MALIGN, and SAM sequence alignment computer programs; or, by inspection. See also Morrison (1997) Mol. Biol. Evol. 14:428-441, as an example of the use of PILEUP. PILEUP, creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method used is similar to the method described by Higgins & Sharp (1989) CABIOS 5: 151-153. The program can align up to 300 sequences of a maximum length of 5,000. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster can then be aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences can be aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program can also be used to plot a dendogram or tree representation of clustering relationships. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison.
Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
Other useful programs, as shown below, include the EasyGene 1.0 server which produces a list of predicted genes given a sequence of prokaryotic DNA. Each prediction is attributed with a significance score (R-value) indicating how likely it is to be just a non-coding open reading frame rather than a real gene. The user needs only to specify the organism hosting the query sequence. Two more parameters (start codon leniency and R-value cutoff) have default values which may be altered if needed.
Another program useful for the practice of this invention is the FGENESB gene prediction algorithm (SoftBerry), based on Markov chain models of coding regions and translation and termination sites. The FGENESB program performs the following features: finds all potential ribosomal'RNA genes using BLAST against bacterial and/or archaeal rRNA databases, and masks detected rRNA genes; predicts tRNA genes using tRNAscan-SE program (Washington University) and masks detected tRNA genes; provides initial predictions of long ORFs that are used as a starting point for calculating parameters for gene prediction; iterates until stabilizes; generates parameters such as 5th-order in-frame Markov chains for coding regions, 2nd-order Markov models for region around start codon and upstream RBS site, stop codon and probability distributions of ORF lengths; predicts operons based only on distances between predicted genes; runs BLASTP for predicted proteins against COG database, cog.pro; uses information about conservation of neighboring gene pairs in known genomes to improve operon prediction; runs BLASTP against NR for proteins having no COGs hits; predicts potential promoters (BPROM program) or terminators (BTERM) in upstream and downstream regions, correspondingly, of predicted genes (BTERM is the program predicting bacterial-independent terminators with energy scoring based on discriminant function of hairpin elements); and refines operon predictions using predicted promoters and terminators as additional evidences.
Synthesis of HMOs or Oligosaccharide Mimics
(a) Cloning and Expression of Recombinant Proteins Encoded by HMO Catabolic Genes
In another embodiment of this invention, methods for synthesizing human milk oligosaccharides or oligosaccharide mimics using SEQ ID NO: 1-22 are provided. Typically, the genes of this invention are cloned into expression vectors, and the proteins encoded by SEQ ID NO: 1-22 expressed, and the resultant proteins purified.
Standard methods in molecular biology and biochemistry can be employed to clone, express, and purify the proteins of this invention. Detailed descriptions of standard molecular biological methods techniques can be found, for example at Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)). In addition, kits for many molecular biological methods are commercially available.
To obtain high level expression of a cloned gene, the skilled artisan typically subclones the DNA sequence into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation. Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook et al., and Ausubel et al., supra. Bacterial expression systems are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.
Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the protein encoding nucleic acid in host cells. A typical expression cassette thus contains a promoter operably linked to the nucleic acid sequence encoding a monomeric subunit and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.
In addition to a promoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
The particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST, and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc.
Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
Expression of proteins from eukaryotic vectors can be also be regulated using inducible promoters. With inducible promoters, expression levels are tied to the concentration of inducing agents, such as tetracycline or ecdysone, by the incorporation of response elements for these agents into the promoter. Generally, high level expression is obtained from inducible promoters only in the presence of the inducing agent; basal expression levels are minimal. Inducible expression vectors are often chosen if expression of the protein of interest is detrimental to eukaryotic cells.
Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a monomeric subunit encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences. The particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable. The prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary.
(c) Purification of Proteins Encoded by HMO Catabolic Genes
Proteins and/or fragments thereof can be purified from any suitable expression system as described below. If desired, the protein may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook et al., supra., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)).
Recombinant proteins can be expressed by transformed bacteria in large amounts, typically after promoter induction; but expression can be constitutive. Promoter induction with IPTG is one example of an inducible promoter system. Bacteria are grown according to standard procedures in the art. Fresh or frozen bacteria cells are used for isolation of protein.
Proteins expressed in bacteria may form insoluble aggregates (“inclusion bodies”). Several protocols are suitable for purification of the expressed proteins from inclusion bodies. For example, purification of inclusion bodies typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells. The cell suspension can be lysed using 2-3 passages through a French Press; homogenized using a Polytron (Brinkman Instruments); disrupted enzymatically, e.g., by using lysozyme; or sonicated on ice. Alternate methods of lysing bacteria are apparent to those of skill in the art (see, e.g., Sambrook et al., supra; Ausubel et al., supra).
If necessary, the inclusion bodies are solubilized, and the lysed cell suspension is typically centrifuged to remove unwanted insoluble matter. Proteins that formed the inclusion bodies may be renatured by dilution or dialysis with a compatible buffer. Suitable solvents include, but are not limited to urea (from about 4 M to about 8 M), formamide (at least about 80%, volume/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M). Some solvents which are capable of solubilizing aggregate-forming proteins, for example SDS (sodium dodecyl sulfate), 70% formic acid, are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity.
Although guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of immunologically and/or biologically active protein. Other suitable buffers are known to those skilled in the art. One of skill in the art will recognize that optimal conditions for renaturation must be chosen for each protein. For example, if a protein is soluble only at low pH, renaturation can be done at low pH. Renaturation conditions can thus be adjusted for proteins with different solubility characteristics i.e., proteins that are soluble at neutral pH can be renatured at neutral pH. The expressed protein is separated from other bacterial proteins by standard separation techniques.
Often as an initial step, particularly if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest. The preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol includes adding saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This concentration will precipitate the most hydrophobic of proteins. The precipitate is then discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, either through dialysis or diafiltration. Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.
The molecular weight of a given protein can be used to isolate it from proteins of greater and lesser size using ultrafiltration through membranes of different pore size (for example, Amicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest. The retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be chromatographed as described below.
A protein can also be separated from other proteins on the basis of its size, net surface charge, hydrophobicity, and affinity for ligands. In addition, antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well known in the art. It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech).
After the proteins encoded by the genes of this invention are cloned, overexpressed, and purified, they are subjected to assays for their activity that vary according to the particular function of the given enzyme. (See, e.g., the references cited in Example 1.)
The kinetic parameters associated with catalysis by each enzyme is determined in order to favor catalysis by these catabolic enzymes in the reverse direction, namely in the direction of oligosaccharide synthesis in order to generate HMOs and oligosaccharide mimics of the present invention. Because enzymes, in general, catalyze both the forward and reverse reactions depending of the concentrations of reactants and products, the skilled artisan can drive the reverse reaction in most cases by supplying a large concentration of a reaction product. (See, e.g., Tzortzis et al., Appl. Microbiol. Biotechnol., 68: 412-416 (2005).) In the case of the enzymes of the present invention, the products used to drive the reverse reaction toward the synthesis of HMOs or oligosaccharide mimics include oligosaccharides containing end terminal sialic acid or fucose. The resulting oligosaccharides are used in a variety of applications as described below.
Uses of HMOS and Oligosaccharide Mimics of the Present Invention
The HMOs and oligosaccharide mimics of the present invention are used in a variety of applications. HMOs and oligosaccharide mimics can be administered directly to humans as a prebiotic food supplement to stimulate the growth of beneficial gut flora in individuals who have diminished numbers of beneficial bacteria in their gastrointestinal tracts. Examples of individuals in such need include infants, the elderly, persons with depleted gastrointestinal tract bacteria as a result of antibiotic therapy, chemotherapy, disease, or other causes.
One use of the HMOs or oligosaccharide mimics of the present invention is the supplementation of foods such as baby formula. Standard formulations for human baby formulas which can be supplemented can be found in the art, e.g., U.S. Pat. No. 5,902,617.
Other forms of administration of HMOs or oligosaccharide mimics includes the supplementation of animal milks, such as cow milk, which are normally lacking in HMOs.
EXAMPLES
The following examples are offered to illustrate, but not to limit the claimed invention.
Example 1
Identification of Genes Involved with HMO Catabolism
We received the Bifidobacterium longum bv. infantisUCD272 (ATCC15697) draft genome sequence from the Joint Genome Institute on Apr. 19, 2006 in 78 unannotated contigs. We began the in silico analysis by searching for genes whose presence we had predicted based on human milk oligosaccharide (HMO) utilization and known phenotypic traits exhibited by this strain. Specifically, we aligned previously determined sialidase genes from the phylum Actinobacteria (high G+C Gram-positive) of which Bifidobacteria are a member using the ClustalW program. This alignment yielded a nucleotide consensus sequence which was subsequently used to probe the 78 contigs for similarity utilizing b12seq (NCBI). Contigs 69 and 77 exhibited significant similarity to the consensus sialidase and were segregated for further analysis. Gene models were created for these two contigs by FgenesB (Softberry Inc.) and the EasyGene Server 1.0 (Center for Biological Sequences Analysis) which determined potential ORFs and transcriptional orientation. The two gene models were analyzed against the non-redundant protein database at NCBI (genbank), the Pfam database, as well as Cognitor which all confirmed the presence of putative sialidase homologues. Translational start and stop were determined through further sequence analysis using the VectorNTl (Invitrogen) suite of bioinformatic software.
Other genes of this invention were determined using methods analogous to the procedures described above. A gene model, trained on the previously sequenced B. longum bv. longum NC2705 genome, was predicted for all 78 contigs. BLASTP/Pfam was utilized to determine the potential function of each predicted ORF, in addition to known COGs in the Cognitor database (NCBI). Priority was given to identifying glycosyl hydrolases and enzymes that may be involved in the degradation and utilization of HMOs or constituents, as well as genes not found in known bifidobacterial sequences. These analyses yielded 22 genes involved in HMO degradation and utilization.
The extent of homology of the proteins encoded by the bifidiobacterial genes with known proteins from other species present in various databases known to involved with carbohydrate catabolism is shown in FIGS. 1-22 for each of the genes.
The functions of the proteins encoded by the gene sequences of this invention may be divided into 8 different classes which are described below.
Sialidase (EC:3.2.1.18 COG4409) is an enzyme which catalyzes the hydrolysis of alpha-(2->3)-, alpha-(2->6)-, alpha-(2->8)- glycosidic linkages of terminal sialic acid residues in oligosaccharides, glycoproteins, glycolipids, colominic acid, and synthetic substrates. Members of this family contain multiple BNR (bacterial neuraminidase repeat) repeats or Asp-boxes. The repeats are short, however the repeats are never found closer than 40 residues together suggesting that the repeat is structurally longer. These repeats are found in a variety of non-homologous proteins, including bacterial ribonucleases, sulphite oxidases, reelin, netrins, sialidases, neuraminidases, some lipoprotein receptors, and a variety of glycosyl hydrolases. See, e.g., Schauer, R. Sialic acids. Adv. Carbohydr. Chem. Biochem. 40 (1982)131-234.
Sialate O-acetylesterase (EC:3.1.1.53) acts on free and glycosidically bound N-acetyl- or N-glycoloyl-neuraminic acid; acts mainly on the 4-O- and 9-O-acetyl groups. This enzyme also acts on some other O-acetyl esters, both cyclic and acyclic compounds, which are not sialic acids. See, e.g., Shukla, A. K. and Schauer, R. High performance liquid chromatography of enzymes of sialic acid metabolism. Hoppe-Seyler's Z. Physiol. Chem. 363 (1982) 1039-1040.
N-acetylneuraminate lyase (EC:4.2.1.52 COG0329) acts on glycoloylneuraminate, and on O-acetylated sialic acids, other than 4-O-acetylated derivatives. N-acetylneuraminate lyase catalyzes the cleavage of N-acetylneuraminic acid (sialic acid) to form pyruvate and N-acetyl-D-mannosamine. The enzyme plays an important role in the regulation of sialic acid metabolism in bacteria. See, e.g., Comb, D. G. and Roseman, S. The sialic acids. I. The structure and enzymatic synthesis of N-acetylneuraminic acid. J. Biol. Chem. 235 (1960) 2529-2537.
N-acetylmannosamine-6-phosphate 2-epimerase (EC:5.1.3.9 COG3010) catalyzes the conversation of N-acyl-D-glucosamine 6-phosphate into N-acyl-D-mannosamine 6-phosphate. See, e.g., Ghosh, S, and Roseman, S. The sialic acids. IV. N-Acyl-D-glucosamine 6-phosphate 2-epimerase. J. Biol. Chem. 240 (1965) 1525-1530.
Beta-galactosidase (EC: 3.2.1.23 COG1874) catalyzes hydrolysis of terminal non-reducing beta-D-galactose residues in beta-D-galactosides. This class comprises a widespread group of enzymes that hydrolyze the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. A classification system for glycosyl hydrolases, based on sequence similarity, has led to the definition of 85 different families. See, e.g., Kuby, S. A. and Lardy, H. A. Purification and kinetics of beta-D-galactosidase from Escherichia coli, strain K-12. J. Am. Chem. Soc. 75 (1953) 890-896.
N-acetyl-beta-hexosaminidase (EC:3.2.1.52 COG3525) catalyzes the hydrolysis of terminal non-reducing N-acetyl-D-hexosamine residues in N-acetyl-beta-D-hexosaminides. This class comprises a widespread group of enzymes that hydrolyze the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. See, e.g., Isolation of beta-N-acetylhexosaminidase, beta-N-acetylglucosaminidase, and beta-N-acetylgalactosaminidase from calf brain. Biochemistry. 6 (1967) 2775-82.
Alpha-L-fucosidase (EC:3.2.1.51 COG3669) catalyzes the conversion of an alpha-L-fucoside+H2O=>L-fucose+an alcohol. See, e.g, Levvy, G. A. and McAllan, A. Mammalian fucosidases. 2. alpha-L-Fucosidase. Biochem. J. 80 (1961) 435-439.
Fucose dissimilation pathway protein (COG4154) is a protein linked to carbohydrate metabolism in E. coli. The gene for this protein is located in the fucose biosynthesis operon. See, e.g., Lu Z; Lin ECC., The nucleotide sequence of Escherichia coli genes for L-fucose dissimilation. (1989) Nucleic Acids Res., 17, 4883-4884.
Example 2
Glycosyl Hydrolase Activities in Strains of Bifidobacteria 
This example describes experiments showing the presence of enzymatic activities corresponding to the nucleic acids and polypeptides of the present invention in strains of Bifidobacteria. 
Methods and Materials
Glycosyl hydrolase assays. Assayed cells were incubated anaerobically at 37° C. on semi-synthetic MRS medium, with 1% (w/v) sterile filtered lactose or HMOs as the sole carbon source. Early stationary phase cells were harvested, and 200 mg of 0.1 mm diameter glass beads (BioSpec Products, Bartlesville, Okla.) were added to the suspensions which were subjected to two cycles of 45 s at a power of 6.0 m s−1 on a Fast Prep FP120 cell disruptor (Qbiogene, Morgan Irvine, Calif.). The beads and cell debris were removed by centrifugation. Cell-free extracts were collected and kept temporarily on ice until the start of the enzyme assays. Protein concentrations were determined according to the Bradford method using bovine serum albumin as the standard. Activity of α-L-fucosidase (EC 3.2.1.51) and sialidase (EC 3.2.1.18) was assayed with the fluorogenic substrates, 4-mtheylumbelliferyl α-L-fucopyranoside, and 2′-(4-methylumbelliferyl) α-D-N-acetylneuraminic acid, respectively. Fucoasidase activity was assayed with a fluorogenic substrate concentration of 1 mM in 0.2 M phosphate-citrate buffer (pH 5.2), while the sialidase assay employed 0.5 mM of substrate mixed in equal volume with 50 μL of 0.25 M sodium acetate-acetic acid buffer (pH 4.3). The reaction mixture containing 100 μL of cell-free extract and 100 μL of substrate solution was incubated for 1 h at 37° C., and the reaction was stopped with 1.3 mL of 0.17 M glycine-carbonate buffer, pH 9.8. The fluorescence of enzymatically liberated 4-methylumbelliferone was determined in a Bio-Rad VersaFluor fluorometer (Eureka, Calif.) by excitation at 360 nm and emission measured at 460 nm. Serially diluted 4-methylumbelliferone was used to calibrate relative fluorescence units with substrate concentration. The specific activity of the glycosyl hydrolase was expressed as nanomoles of hydrolyzed substrate per milligram of protein per hour. Assays were performed in duplicates, and the standard error was reported.
Results
The catabolic activity of bifidobacterial strains in HMO metabolism was measured by monitoring sialidase and fucosidase activities required to deconstruct complex glycan structures. Enzymatic assays showed that B. longum bv. infantis has a 16.6- and 33.7-fold higher sialidase activity when grown on lactose as compared to B. longum and B. breve, respectively. (See Table 1.) These data suggest that B. longum bv. infantis has an inherent and constitutive ability to process sialylated compounds. Furthermore, among the three strains tested, fucosidase activity was only present in B. longum bv. infantis and was only detected upon growth on HMO. As described below and elsewhere such catabolic activities may be reversed, thus assembling larger oligosaccharides from smaller ones (see, e.g., Tzortzis et al., Appl. Microbiol. Biotechnol., 68: 412-416 (2005)).










TABLE 1









Sialidase
Fucosidase



(nmol/min/mg protein)
(nmol/min/mg protein)












lactose
HMO
lactose
HMO















B. infantis ATCC
10.8 ± 1.0 
4.8 ± 0.7
ND
0.45 ± 0.04


15697


B. longum DJO10A
0.65 ± 0.03
NA
ND
NA


B. breve ATCC 15700
0.32 ± 0.03
NA
ND
NA





ND—Not detected


NA—Not available (not enough cell growth)






Example 3
Expression and Purification of the Proteins of the Invention and Synthesis of Oligosaccharide Mimics
In order to utilize the genes of the invention, any of SEQ ID NO: 1-22 is cloned into an expression vector downstream of an inducible promoter using methods well known to those of skill in the art, such as the methods described herein. The gene in an appropriate expression vector is overexpressed in a suitable E. coli host. The resulting recombinant protein is extracted and purified using any number of established purification schemes known in the art. Because the purified enzyme is required to be native, the expressed protein is refolded if solubilized from an inclusion body. Characterization of the native protein involves a determination of reaction kinetics, specificity, and other biochemical features on carbohydrate substrate(s). Moreover, the reverse reaction is characterized (i.e., transferase activity) by assaying for enzyme activity in the presence of high molar concentrations of hydrolysis products (e.g., sialic acid). In order to optimize conditions for the reverse reaction, protein engineering is used to remove superfluous or inhibiting enzyme domains. Oligosaccharide construction can proceed de novo from soluble monomers, or from incomplete oligosaccharides isolated from plant or mammalian sources.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

## Claims

1. A purified alpha-L-fucosidase polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence encoded by SEQ ID NO: 18.

2. A method of synthesizing human milk oligosaccharides or oligosaccharide mimics, the method comprising the steps of:
(a) contacting an oligosaccharide containing precursor with the polypeptide of claim 1; and
(b) isolating a modified oligosaccharide containing precursor.

3. The method of claim 2, wherein the oligosaccharide containing precursor is a carbohydrate.

4. The method of claim 2, wherein the oligosaccharide containing precursor is a glycoprotein.

5. The method of claim 2, wherein the oligosaccharide containing precursor is a glycolipid.

6. The method of claim 2, wherein the oligosaccharide containing precursor has a terminal sialic acid.

7. The method of claim 2, wherein the oligosaccharide containing precursor has a terminal fucose.

8. The method of claim 2, wherein the oligosaccharide containing precursor has a terminal N-acetyllactosamine.

9. The method of claim 2, wherein the oligosaccharide containing precursor is plant derived.

10. The method of claim 2, wherein the oligosaccharide containing precursor is human derived.

11. The method of claim 2, wherein the oligosaccharide containing precursor is animal derived.

12. The method of claim 11, wherein the animal is bovine.

13. The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence encoded by SEQ ID NO:18.